DOI: 10.5061/dryad.fbg79cns9
title: A green extraction design for enhancing flavonoid compounds from the Ixora javanica flowers using a deep eutectic solvent

description: In this study, an environmentally friendly extraction method for flavonoid
 compound from Ixora javanica as a new raw material candidate for herbal
 medicine and cosmetics, was developed. The objectives of the present work
 were to provide recommendations for the optimal extraction conditions and
 to investigate the effects of any extraction parameters on flavonoid
 yields from the I. javanica flower. The extraction process was performed
 using deep eutectic solvent (DES) (choline chloride and propylene glycol
 at molar ratio of 1:1) and ultrasound-assisted extraction (UAE) method.
 Both single-factor and response surface analyses using three-level and
 three-factor Box Behnken designs were conducted to obtain the optimum
 flavonoid concentrations. The results showed that the optimum extraction
 conditions for total flavonoids featured an extraction time of 40 min, 25%
 water content in DES, and a solid-to-liquid ratio of 1:25 g/mL. An extract
 obtained under optimum extraction conditions showed higher total flavonoid
 yields than an ethanolic extract which was used for comparison. Scanning
 electron microscope (SEM) images demonstrated that both of the solvents
 also showed different effects on the outer surface of the I. javanica
 flower during extraction process. In sum, our work succeeded in
 determining the optimum conditions for total flavonoids in the I. javanica
 flower using a green extraction method.
description: 1. Chemicals and materials
 Choline chloride was
 purchased from Xi'an Rongsheng Biotechnology Co, Ltd, China, while
 other solventsm such as propylene glycol and ethanol, were acquired from
 Merck, Germany. The quercetin standards used in this study were purchased
 from Sigma Aldrich, USA. 2. Plant materials The fresh red color
 <i>I. javanica</i> flower used in this study was authenticated
 by the Center for Traditional Medicine Information and Development,
 Faculty of Pharmacy, University of Surabaya. The flower was collected at
 full bloom from the Tenggilis Mejoyo district, Surabaya, East Java,
 Indonesia. After sortation, the flowers were washed, shade dried, and
 powdered. The dry powder was then sieved through a size 30 mesh sifter,
 stored in a sealed container, and kept under dry environmental
 conditions. 3. Preparation
 of DES The DES
 components used in this study were choline chloride (ChCl) as HBA and
 propylene glycol (Pg) as HBD. DESs were prepared by heating and stirring
 both components at certain molar ratios (1:1; 1:2; 2:1) in a flask at 50
 °C for 30 min constantly until a homogeneous clear mixture formed.
 Deionized water was added to the DES to prepare the DES solution
 containing various concentrations of water. The water content used in this
 study was 5%–35% w/w of water in the DES. All DES mixtures obtained in
 this study were stable in liquid form under room temperature storage. The
 DES mixtures were measured for their viscosity before being used for the
 extraction process (Brookfields<sup>TM</sup> cone plate
 viscometer, Ametek Brookfield, Middleboro, USA). 4. Extraction Procedures The extraction of <i>I.
 javanica</i> flowers was carried out using the UAE method. Dried
 flower powder was weighed and extracted using DES that was prepared at a
 certain solid-to-liquid ratio and then sonicated for many repetitions at
 room temperature. When investigating the effect of one variable on the
 flavonoid yields, the other variables were kept constant. To determine the
 effect of water content in DES on the flavonoid yields, 0.5 g dried powder
 was extracted with 10 mL DES (a combination of ChCl-Pg at a molar ratio of
 1:1), which contained different concentrations of water in DES (5%, 10%,
 15%, 20%, 25%, 30%, and 35% w/w) for 5 min. Extraction time: 0.5 g dried
 powder was mixed with 10 mL DES (a combination of ChCl-Pg at a molar ratio
 of 1:1 and water content of 20% w/w), and the extraction was carried out
 under different extraction times (10, 15, 20, 25, 30, 35, and 40 min). The
 solid-to-liquid ratio: 0.5 g dried powder was mixed with different volumes
 of DES (a combination of ChCl-Pg at a molar ratio of 1:1 and water content
 of 20% w/w) until reaching a certain solid-to-liquid ratio (1:23, 1:24,
 1:25, 1:26, 1:27, 1:28, and 1:29 g/ml) and was then sonicated for 5 min.
 The ultrasonic bath was set at room temperature with a fixed frequency
 radiation of 40 kHz. The extracts obtained were then centrifuged at 1500
 rpm for 15 min, and the filtrates were collected. The filtrate was then
 analyzed for its flavonoid levels. Extraction using a conventional
 extraction solvent was carried out by sonicating 0.5 g dried material
 using ethanol under the optimum solid-to-liquid ratio and extraction time
 obtained from the RSM analysis. The flavonoid levels in the ethanolic
 extract were compared with those in the DES extracts. All procedures of
 extraction in this study were conducted in triplicate. 5. Determination of total
 flavonoids The
 total levels of flavonoids in the extracts were determined using the
 method applied in the previous study with very slight modifications [24].
 About 1.0 mL of each extract’s filtrates was mixed with 1.5 mL of 0.33%
 AlCl3 and 1.5 mL of 10% acetic acid solution. The mixtures were added with
 96% ethanol until reaching a total volume of 10.0 ml. After 30 min
 incubation, the mixed solutions were analyzed spectrophotometrically
 (UV-1900, Shimadzu Corp, Kyoto, Japan) at 425.8 nm. Quercetin was
 used as a standard compound so that the total flavonoid levels in the
 dried flower powder could be expressed as their quercetin equivalent (mg
 QE/ g dried flowers). A calibration curve and regression
 equation were used to calculate the flavonoid levels in the extract sample
 and are shown in figure 1. All of the analysis procedures were conducted
 in triplicate.