Extraction optimization by using response surface methodology and purification of yellow pigment from Gardenia jasminoides var. radicans Makikno

Abstract Gardenia jasminoides var. radicans Makikno contains rich gardenia yellow pigment (GYP). In this study, the process of pigment extraction was optimized based on a Box–Behnken design (BBD) and response surface methodology (RSM). The absorbance and antioxidant activity (AA) were considered as responses. The result showed that the optimal extraction conditions were ethanol concentration 65.10%, liquid/solid ratio 10:1 ml/g, extraction time 59.85 min, and extraction temperature 60.04℃ for the maximal response values of absorbance (0.79) and AA (91.30%), respectively. Crude GYP was purified by the 13 different resins. The result showed that BJ‐7514 was suitable for purifying GYP with the absorption ratio of 95.4%. Moreover, the 80% of ethanol eluent is applicable on the BJ‐7514 with the desorption ratio of 91.93%. The major component of GYP (Crocin‐3) was isolated and identified from the purified GYP.

G. jasminoides var. radicans Makikno is a rare natural water-soluble carotenoid, which mainly composed of crocetin and crocins (Yin & Liu, 2018). It has been applied to foods, such as candy, noodles, and beverages, as a natural food colorant due to its good water solubility, low toxicity, and allergy (Bathaie et al., 2014;Xiao et al., 2017).
Besides, it is characterized by strong dyeing ability, high stability, abundant nutritional value compared with other natural colorants ). In addition, pigments responsible for yellow, orange are especially interested in food companies because they showed more widely applications than other colors of pigments (Hatzakis et al., 2019). Therefore, the demand for GYP production and purification is increasing in the international markets.
Nowadays, selecting an effective method to generate pigments is not easy, since the changes of temperature, pH, time et al may be led to degrading some compounds related with color (Gimenez et al., 2015;Li et al., 2020;Moller et al., 2020). Therefore, it is a key step to control these variables influencing the process efficiency. Combining with the variables that offer the maximum yield of the target compounds could provide an appropriate way to solve the extraction limitations of pigments based on feasible test conditions (Pinela et al., 2019). The response surface methodology (RSM) was commonly used in parameter testing and its interactive effects (Parra-Campos & Ordonez-Santos, 2019;Lin et al., 2018;Wang et al., 2020). To our best knowledge, there are few studies about the optimal extraction of compounds related to GYP. Sarfarazi et al. (2019) reported the extraction process of crocin pigment of saffron through subcritical water extraction (SWE). However, as one of the precious spices all over the world, saffron is too expensive to be widely applied as a colorant. Shang et al. (2019) extracted GYP from G. jasminoides Ellis by RSM.
Nevertheless, G. jasminoides Ellis, as traditional Chinese medicine, is mainly studied in the effect of its antioxidant components on human health.
It has been reported that G. jasminoides var. radicans Makikno contains a higher amount of GYP compared with G. jasminoides Ellis (Chen et al., 2012). This study aims to optimize the extraction process of GYP from G. jasminoides var. radicans Makikno through heataided solvent extraction and to assess its antioxidant activity (AA).
For that purpose, the RSM was used to optimize processing parameters (ethanol concentration, liquid/solid ratio, extraction time, and extraction temperature). Then, a suitable macroporous resin was selected to purify GYP. Meanwhile, the major component was isolated and identified from the purified GYP. Gardenia  Other reagents were of analytic grade.

| Extraction of GYP and solvent selection
The dried gardenia fruit was crushed by a flour-mixing machine and the powder granularity was 0-1 mm. 0.5 g gardenia powder and different solvents (distilled water, 60% methanol-water solution, 60% ethanol-water solution and 60% isopropanol-water solution) were added into a sealed test tube for extraction in designed extraction time, solvent concentration, liquid/solid ratio, and temperature.

| Single-factor experiment
The effect of every single factor on the extraction yield of GYP was evaluated by the single-factor experiment. The initial conditions were designed as followed ethanol concentration 60%, liquid/solid ratio 12:1 ml/mg, extraction time 40 min, extraction temperature 50℃. The effect of each single-factor (ethanol concentration, liquid/ solid ratio, extraction time, extraction temperature) was tested as follows: A factor was varied in defined ranges while the other factors were kept constant in each extraction experiment. Therefore, the effect of ethanol on extraction was tested at 50%, 60%, 70%, 80%, and 90% ethanol while the other factors were kept constant.
Similarly, the following effects of other factors were tested: liquid/ solid ratio from 10:1 to 16:1 ml/g; extraction time from 30 to 70 min; and extraction temperature from 30 to 70℃.

| Experimental design
The optimun conditions of the extraction process were evaluated by Box-Behnken design (BBD) of RSM. According to the results from the single-factor experiments, the levels of coded independent variables (ethanol concentration [X 1 ], liquid/solid ratio [X 2 ], extraction time [X 3 ], extraction temperature [X 4 ]) were selected to obtain optimistic extraction conditions as shown in Table S1. A total of 29 experiments with different combinations of four factors were performed based on BBD (Table S2). The absorbance and AA of GYP were selected as the responses. A second-order model was utilized in response surface methodology (Guo et al., 2019). The equation was expressed as follows: where Y is the predicted response; β 0, β i , β ii , β ij 0, i, ii, ij are constant coefficients of intercept, linear, quadratic, and interactive terms, respectively. X i, , X j X i X j are levels of the coded independent variables.

| Determination of GYP absorbance
After the extraction, the obtained solutions of GYP were centrifuged at 4,500 r/min for 5 min. 1 ml supernatant and same concentration aqueous ethanol were transferred into a 100 ml volumetric flask and then the final volume was adjusted to 100 ml. Aqueous ethanol was used as a contrast, and the absorbance of the sample solution was determined at 440 nm by UV-Vis spectrophotometer (Zhu et al., 2014).

| DPPH antioxidant assay
The DPPH scavenging activity for the tested sample was performed according to method detailed elsewhere with slight modifications (Sharmila et al., 2019). Briefly, 1 ml methanolic DPPH solution (0.0109 g in 100 ml methanol), 3 ml methanol, and 50 μl sample (pigment extraction) were mixed on the 10 ml tubes. 50 μl methanol was added to the test tube instead of the sample as the control. Then, the tubes were left in a dark place at room temperature for 30 min.
The absorbance was determined at 517 nm using UV-Vis spectrophotometer. The scavenging activity was evaluated by the following formula: where A control is the absorbance of the DPPH solution and A sample is the absorbance of the sample.  Table S3. The pretreatment of macroporous resin was described according to the previous method (Pan et al., 2017;Zhang et al., 2011) as follows: macroporous resins were first soaked in 95% ethanol for 24 hr, and then washed with distilled water until no alcohol taste.

| GYP purification by the macroporous resin
Next, the resins were pre-treated with 5% HCl and 2% NaOH solutions to remove salts and impurities. Finally, the resins were washed to neutrality with distilled water and dried in a vacuum at 60℃.

| Adsorption of macroporous resin on pigment
The specific operations about absorption and desorption texts of GYP were described as follows: thirteen different dry resins (1.0 g) were mixed with aliquots (50 ml) of diluted pigment solution in a 250-ml conical flask with a lid, respectively. Then, the flasks were put into a shaker and continued to shake (120 rpm) at 28℃ for 24 hr.
The absorbance of the solution at 440 nm was determined by UV/ Vis spectrophotometer. The adsorption rate of pigment was calculated using the following equations: where A 0 is the absorbance of pigment solution before adsorption, A 1 is the absorbance of pigment solution after absorption, Ar is the adsorption rate of sample (%).

| The effect of different ethanol concentrations on desorption
After the adsorption equilibrium of 1.00 g macroporous resin BJ-7514 was reached, the residual pigment on the surface of the resin was removed by washing with distilled water. The resins and 100 ml different concentrations of ethanol-water solution (10%, 30%, 70%, 80%, 90%, 100% respectively) were added in the 250-ml conical flask with a lid to shake (120 rpm) at 28 ℃ for 24 hr. Desorption rate was calculated by the following equation: where A 2 is the absorbance of the pigment-ethanol solution after desorption and Dr is the desorption rate of the test sample (%).
The structure of compound was identified by spectroscopic analysis. The NMR spectra were recorded at 25℃ with Agilent 600 MHz DD2 spectrometer NMR. The HR-ESI-MS spectra were recorded on Agilent-1260/6460 mass spectrometer.

| Comparison with different types of solvents
The solubility of GYP in different polar solvents was significantly different. Therefore, choosing a suitable solvent could improve the extraction amount of GYP (Rammuni et al., 2019). As shown in Fig. S1, the maximum absorbance was obtained with ethanol-water solution (0.835) followed by isopropanol-water solution (0.687),

| Single-factor experiment of GYP extraction
Single-factor experiments of GYP extraction were carried out with four selected parameters including ethanol concentration, liquid/ solid ratio, extraction time, extraction temperature, which provided a suitable range for the BBD (Gao et al., 2017).
In general, it is statistical significant because of p-value with <.05. As shown in Tables 1-2, the ANOVA presented that all the models were significant (p-value < .05), and lack of fit was not significant with p-value of .0544 (>.05). Therefore, the quadratic model was fitted well to the data of the experiment by ANOVA (Zhang et al., 2013). From Table 1, the linear effect of X 2 (liquid/solid ratio) , the square effect of X 2 1 (ethanol concentration), X 2 3 (extraction time), X 2 4 (extraction temperature) , and the interaction of X 2 X 3 (liquid/solid ratio versus extraction time) were significant for the absorbance of GYP. Similarly, the linear effect of X 2 (liquid/solid ratio), the square effect of X 2 3 (extraction time), X 2 4 (extraction temperature), and the interaction of X 3 X 4 (extraction time vs. extraction temperature) had significant effect for antioxidant activity (AA, Table 2).

| The interaction between the independent variables
The three-dimensional (3D) response surface curves denoted the interaction between the independent variables and ensured the optimal levels of each variable for the maximum absorbance and The significant negative linear effect of X 2 (liquid/solid ratio) and the negative square effect of X 2 3 (ethanol concentration), X 2 3 (extraction time), X 2 4 (extraction temperature) in the absorbance were presented in Figure 2, Part A. Therefore, the maximal absorbance was obtained at the low level of liquid/solid ratio (10 ml/g), the middle level of ethanol concentration (60%), the middle level of extraction time (50 min), the middle level of extraction temperature (60 ℃).
The effects of the four independent variables on the response value (AA) were visually described by the 3-D plot (Figure 2, Part   B, a-f). The results showed that the low level of liquid/solid ratio (10 ml/g), the middle level of ethanol concentration (60%), the middle level of extraction time (50 min), the middle level of extraction temperature (60℃), led to the optimal results.

| Verification of predictive model
Optimal conditions were predicted to get to the maximal value of absorbance (0.79) and AA (91.2%) by RSM with ethanol concentration 65.1%, liquid/solid ratio 10:1 ml/g, extraction time 59.8 min and extraction temperature 60.0℃. TA B L E 1 Analysis of mean square deviation of regress equation for the absorbance of GYP To verify the optimal values of extraction conditions predicted by RSM, the real experiment was operated under the optimal conditions. Ultimately, the real experimental values were absorbance (0.78) and AA (84.6%), which were well matched with values predicted by the regression model. Therefore, the extraction conditions acquired by RSM were practical.

| GYP purification by the macroporous resin
The crude GYP may contain other compounds that not related to the yellow pigment, such as iridoids, quinic acid derivatives, flavonoids, triterpenoids, and organic esters (Wang et al., 2016). Some of these compounds, especially the colorless geniposide and chlorogenic acid, could cause crude GYP to get green or darkened. Thus, it is a necessary step to select an effective method to purify crude GYP. The absorption method by macroporous resins is frequently considered to be low cost, easy to operate, and high purity ).

| Absorption effect of macroporous resin on the pigment
Thirteen different properties of macroporous resins were used to screen the most effective one for the purification of GYP. As showed in Figure 3, the highest absorption ratios of GYP was 95.4% on macroporous resin of BJ-7514. It may be attributed to acrylic acid of BJ-7514 possesses the stronger capacity to bond with GYP contained crocetin and crocins compared with other resins regarded polystyrene as a functional group. Therefore, BJ-7514 was thought of as a potential resin to purify GYP from G. jasminoides var. radicans Makikno.

| Effect of different concentration of ethanol solution on desorption
Macroporous resins are made of polymers that possess pores and larger surface areas. The principle of the target compound's surface adsorption could be achieved by the formation of physical and/or chemical bonds. And the compounds adsorbed onto the resin surface must be desorbed by using an eluting solvent that can destroy the bonds between them (Belwal et al., 2020).

| Structure elucidation of the major compound from GYP
The major compound of the purified GYP was identified as crocin-3 (2.74% yield rate) ( Fig S3)

| CON CLUS ION
The current work described the optimum extraction process and purification of GYP from G. jasminoides var. radicans Makikno. The optimum extraction conditions were determined by RSM based on single-factor experiments. The results showed that the optimal extraction conditions were ethanol concentration (65.10%), liquid/ solid ratio (10:1 ml/g), extraction time (59.85 min), and extraction temperature (60.04℃) for the maximal response values of absorbance (0.79) and AA (91.30%), respectively. BJ-7514 was screened out as the most suitable resin to purify the crude GYP. Moreover, the 80% of ethanol eluent was applicable on the BJ-7514 to obtain the purified GYP. In addition, the major component of GYP (Crocin-3) was isolated and identified from the purified GYP. This study will be a prospect for the application of GYP from G. jasminoides var. radicans Makikno.

This work was supported by Key R & D projects in Anhui Province
(201904a06020050) and Major Science and Technology Projects of Anhui Province (17030801018).

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that supports the findings of this study are available in the supplementary material of this article.