A green extraction design for enhancing flavonoid compounds from the Ixora javanica flowers using a deep eutectic solvent

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.Funding provided by: Kementerian Riset Teknologi Dan Pendidikan Tinggi Republik IndonesiaCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100009509Award Number: PDUPT 2020 Grant Ref number NKB-86/UN2.RST/HKP.05.00/2020Funding provided by: Universitas SurabayaCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100012696Award Number: PhD program scholarship1. 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. javanica 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 (BrookfieldsTM cone plate viscometer, Ametek Brookfield, Middleboro, USA). 4. Extraction Procedures The extraction of I. javanica 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