Optimization of Red Pigment Anthocyanin Recovery from Hibiscus sabdariffa by Subcritical Water Extraction

Abstract

The optimization of red pigment anthocyanin from roselle (Hibiscus sabdariffa) by subcritical water extraction (SWE) has not been the topic of a scientific investigation. Therefore, the objective of this paper was to establish the optimal parameters for obtaining the maximum yield, total anthocyanin compounds (TAC), total phenolic compounds (TPC), and total flavonoid compounds (TFC) by SWE utilizing a response surface methodology. The optimal conditions were 8.75 MPa, 393.54 K, and 4.89 mL/min, with a yield of 0.69 g/g, TAC of 927.74 mg/100 g, TPC of 39.54 mg/100 g, and TFC of 614.57 mg/100 g. High temperatures and flow rates are favorable for achieving a maximum yield. In contrast, a high temperature is suitable for obtaining high concentrations of anthocyanin, flavonoid, and phenolic compounds. This technique (SWE) recovers anthocyanin at a greater extraction rate than traditional methods; hence, SWE may be substituted for conventional methods for extracting anthocyanin.

1. Introduction

Since color provides the first impression of food quality to consumers, it is one of the most significant quality criteria influencing the consumer approval of food [1]. Natural additives, food colorants in particular, are increasingly being used in culinary applications on a global scale. Anthocyanins are among the many diverse plant compounds known as flavonoids, but they vary from other flavonoids in that they powerfully absorb visible light. The pink, magenta, red, violet, and blue hues found in higher plants’ petals, leaves, and fruits are all caused by these vividly colored water-soluble pigments [1].

Roselle has a long history of application in many different countries, including in medical sciences. Among other diseases, it is used to treat pyrexia, liver damage, hypertension, and leukemia. Additionally, roselle has several medicinal applications that have been researched worldwide [2]. Roselle extract may be used in the production of jam, drinks, and fruit juice [2]. Calyces are frequently used in a few industries to produce jam and fruit juices because of their unusual flavor and bright red color. Furthermore, the high contents of phenolic, flavonoid, and anthocyanin compounds in roselle contribute to the production of health and wealth goods [3,4].

Current research indicates that supercritical carbon dioxide is often used to extract important chemicals, including anthocyanin, phenolic, flavonoid, and antioxidant molecules and colorant compounds such as anthocyanin [5,6]. Supercritical carbon dioxide (ScCO₂) is an innovative method for maximizing the extraction of compounds of interest since it is a safe and environmentally friendly solvent. Mohd-Nasir et al. [7] reported that the key advantages of this technique over conventional extraction processes include the shortened extraction time, increased extract quality, lowered extraction agent costs, and environmentally friendly operation. However, pure ScCO₂ offers a low quantity of anthocyanin from roselle due to the different polarities of the solute and the solvent [6,8,9,10,11,12,13]. Typically, red pigment anthocyanin of roselle is also extracted using an organic solvent. The energy used during the solvent extraction amounts for about two thirds of the total energy consumed during the operation. The expenses of various extraction technologies for commercial use may be too expensive. In light of economic and environmental concerns, it is important to conduct basic research on the utilization of subcritical water for the recovery of anthocyanin [14].

Subcritical water extraction (SWE) is efficient and capable of attaining high extraction rates in a very short period of time. Depending on the operating conditions, SWE may have various effects on product yield and quality (pressure, temperature, flow rate, residence time, and particle size). The bioactive products that arise are a significant source of resources for the chemical, pharmaceutical, food, and energy industries. In addition, the SWE of roselle biomass has been shown to be an effective method for the generation of anthocyanin. Subcritical water is liquid water below its critical temperature and pressure (Tc = 374.15 C and Pc = 22.1 MPa). To maintain liquid water, the subcritical water pressure must be greater than the vapor pressure at a given temperature. The physical–chemical characteristics of subcritical water change substantially as the temperature rises. With a rising water temperature, its dielectric constant, viscosity, and surface tension all drop, but its diffusion coefficient increases. As the temperature rises, the viscosity, dielectric constant, and surface tension all decrease, but diffusivity increases. To keep the liquid at a certain temperature, the optimal pressure may be applied [15,16,17]. The significance of employing water as a solvent lies in the fact that water is regarded a green and nontoxic solvent, making it ideal for use in health and wellness products.

SWE has the benefit of being able to alter the dielectric constant over a wide temperature and pressure range [18,19]. Diffusion and convection mechanisms help SWE contribute to mass transportation. However, reducing the activation energy necessary for the desorption process might disturb the connection between adhesive (solute–matrix) and cohesive (solute–solute) interactions [20]. Furthermore, the higher pressure may enhance the process by pushing water to enter the matrix (pores) when normal pressure would not increase the pores. As temperature and pressure fluctuate, certain water characteristics change as well; for example, as temperature increases, the polarity of subcritical water diminishes. As a result, the polarity of anthocyanin may be distinguished.

Subcritical water extraction as a green extraction method for extracting red pigment anthocyanin, phenolic, and flavonoid compounds from roselle is the most significant innovation of this study. There were goals to accomplish in this study in order to establish this innovation, and the objective was to optimize the parameters with the maximum yield, total anthocyanin compounds (TAC), total phenolic compounds (TPC), and total flavonoid compounds (TFC) using subcritical water extraction.

2. Material and Methods

2.1. Preparation of Raw Material

The dried calyces of roselle originated from Ekomekar Resources in Terengganu. The roselle was ground and sieved with an Endecotts Octagon 2000 Digital Sieve Shaker. The moisture content of dried roselle was kept below 8%, and it was placed in a plastic bag and frozen at −7 °C.

2.2. Chemicals

Gallic acid and quercetin were provided by Sigma-Aldrich (St. Louis, MO, USA). All of the ethanol (analysis grade), Folin–Ciocalteu reagent, Al₂NO₃, CH₃COOK, HCL, KCl, Na₂SO₃, and Na₂CO₃ provided by Fisher Scientific (Atlanta, GA, USA) met or exceeded the ACS requirements. Using the distillated water, roselle was extracted.

2.3. Subcritical Water Extraction (SWE)

The equipment consisted of a 25 mL extraction vessel (internal diameter: 1.4 cm; length: 13 cm), a subcritical water pump (Eldex Opto Metering Pump, USA), a back pressure regulator (Swagelok, USA), and an oven (Memmert, Germany), constituting the SWE system, as shown in Figure 1. Extraction was performed under three independent parameters: pressure (2 to 10 MPa), temperature (373 to 413 K), and flow rate (3 to 7 mL/min), as stated in

Table 1. The parameters and responses of subcritical water extraction (SWE).

Run	A: Pressure	B: Temperature	C: Flow Rate	Yield, Y1	TAC, Y2	TPC, Y3	TFC, Y4
	MPa	K	mL/min	g/g	mg/100 g	mg/100 g	mg/100 g
1	2	373	5	0.58	698.81	14.11	290.18
2	6	413	7	0.7	956.77	8.64	672.22
3	6	393	5	0.71	850.57	33.35	762.91
4	2	393	3	0.58	805.55	27.66	182.18
5	6	393	5	0.69	898.93	39.51	671.12
6	10	373	5	0.48	974.41	21.18	197.45
7	6	373	3	0.62	772.69	7.79	514.72
8	6	393	5	0.68	861.79	33.1	667.36
9	6	393	3	0.56	778.43	20.83	484.73
10	6	393	5	0.67	857.92	32.61	663.45
11	6	393	5	0.67	859.12	32.76	668.91
12	10	393	7	0.58	907.21	37.16	269.92
13	2	393	7	0.31	749.9	18.91	571.73
14	10	393	3	0.54	906.86	37.11	571.64
15	10	413	5	0.67	912.56	39.54	571.72
16	2	413	5	0.49	882.1	37.37	289.73
17	6	373	7	0.39	656.06	10.38	290.18

. First, 200 ± 10 mg of roselle was weighed and placed in an extraction vessel. The extraction vessel and water were heated before the extraction process to make sure the temperature condition was achieved. Distilled water was pumped and controlled based on the variables of flow rate and pressure. The temperature of the oven was likewise set depending on the variable. The extraction length was set at 5 min. The extract was dried by a vacuum evaporator at 60 °C. To avoid any potential degradation, the extract was kept in a refrigerator (Liebherr EFL 3505, USA) at −10 °C.

2.4. Determination of Yield

As illustrated in Equation (1), the yield was estimated using the weight of the extract per unit weight of roselle:

$$\mathrm{Yield} (\mathrm{g}/\mathrm{g})=\left(\frac{{\mathrm{m}}_e}{{\mathrm{m}}_r}\right)$$

(1)

where ${\mathrm{m}}_e$ represents the weight of the extract (g) and ${\mathrm{m}}_r$ represents the weight of the dried roselle (g).

2.5. Analysis of Total Anthocyanin Compounds (TAC)

A pH differential technique was used to analyze the anthocyanin [11]. Two dilutions of the same material were prepared using KCl (0.025 M) and Na₂SO₃ (0.4 M). They were adjusted to pH 1.0 and 4.5, respectively, using HCL. The absorbance was determined using a UV-Vis spectrophotometer (Jasco, Japan) at 520 and 700 nm using Equation (2). TAC was represented as the milligram of cyanidin-3-glucoside equivalent per liter extract in Equation (3):

$$\mathrm{Absorbance}, \mathrm{A}: \left(A_{520}-A_{700}\right){ }_{p{H}_{1.0}-}\left(A_{520}-A_{700}\right){ }_{p{H}_{4.5} }$$

(2)

$$\mathrm{TAC} (\mathrm{mg}/\mathrm{L}): \frac{A\times Mr\times Df\times 1000\times L}{\mathsf{\epsilon }}$$

(3)

where $A$ is absorbance, $Mr$ is the molecular weight of cyanidin 3-glucoside (449.2 g/mol), $Df$ is a dilution factor, $\mathsf{\epsilon }$ is an extinction coefficient of cyanidin 3-glucoside (26,900 L/cm/mol), and $L$ is the cell path length (1 cm). The unit of TAC (mg/L) was converted to the mg extract of 100 g of dried roselle.

2.6. Analysis of Total Phenolic Compounds (TPC)

The TPC analysis was carried out. First, 5 mL of Folin–Ciocalteu reagent was diluted with 50 mL of distilled water. Then, 3 g of Na₂CO₃ was diluted in 40 mL of distilled water. Next, 1 mg of extract was dissolved in 1 mL of ethanol and mixed in 5 mL of Folin–Ciocalteu mixture. Then, 4 mL of Na₂CO₃ solution was added. Furthermore, the solution rested for 30 min. Using a UV-Vis spectrophotometer, the absorbance at 760 nm was measured (Jasco, Japan). The TPC was expressed as the gallic acid equivalent per 100 g of dried roselle (mg/100 g).

2.7. Analysis of Total Flavonoid Compounds (TFC)

The TFC analysis was carried out. First, 1 mg of extract was diluted with 1 mL of ethanol. Then, 1 mL of ethanoic extract was combined with 0.2 mL of Al₂NO₃ (10%) and 0.2 mL of CH₃COOK (1 M) and rested for 40 min. Using a UV-Vis spectrophotometer, the absorbance at 415 nm was measured (Jasco, Japan). The TFC was represented as the quercetin equivalent per 100 g of dried roselle (mg/100 g).

2.8. Experimental Design for Optimization

Utilizing the “Design Expert” software, a Box–Behnken design was created as the experiment’s design to analyze the impacts and best independent variables (Version 13.0.4, Stat-Ease Corporation, Minneapolis, MN, USA). The yield, TAC, TPC, and TFC responses as well as variables of pressure (2 to 10 MPa), temperature (353 to 393 K), and flow rate (3 to 7 mL/min) were all investigated. The model, which included linear and quadratic variables as well as interaction terms, was used to generate first- and second-order polynomial equations based on the experimental data as follows:

$$Y=B_0+{{\displaystyle \sum }}_{i=1}^k{B}_i{X}_i+{{\displaystyle \sum }}_i{{\displaystyle \sum }}_j{B}_{ij}{X}_i{X}_j+{{\displaystyle \sum }}_{i=1}^k{B}_{ii}{X}_i^2$$

(4)

where:

Y is an investigated response (yield, TAC, TPC, and TFC); B₀ is constant; B_i, B_ii, and B_ij are the coefficients of linear, quadratic, and interaction terms, respectively; and X_i and X_j are independent variables (Pressure, temperature and flow rate).

3. Results and Discussion

An extraction technique of SWE was utilized to recover anthocyanin, a naturally occurring red pigment, from the calyces of roselle. A response surface methodology was applied to maximize the yield, anthocyanin, phenolic, and flavonoid recovery. The constant parameters were chosen using preliminary data, an analysis of previous research, and the SWE system’s capabilities.

According to the limitation of the back pressure regulator (BPR) of SWE, the maximum operating pressure cannot exceed 10 MPa. Since water is an incompressible liquid, solvent densities are unaffected by high pressure. As a result, increasing the extraction efficiency does not need a substantial increase in the solvent solvation power. In order to avoid the degradation of anthocyanin during the short extraction time, the maximum temperature was increased to 413 K. [2]. Despite the thermostability of anthocyanins in extracts at temperatures between 80 and 120 °C, the maximum temperature condition was limited to 140 °C/413 K [21]. Due to the short extraction time, a temperature of 140 °C was still acceptable (5 min). Therefore, anthocyanin degradation could be avoided. In order to avoid the solvent channeling effect and minimize the residence duration, the flow rate of solvents was limited to 8 mL/min [18].

Table 1 presents the results of optimizing the extraction variables and the responses of SWE.

Table 2. ANOVA table for the responses of yield, TAC, TPC, and TFC.

Source	Sum of Squares	df	Mean Square	F-Value	p-Value	Sum of Squares	df	Mean Square	F-Value	p-Value	Sum of Squares	df	Mean Square	F-Value	p-Value	Sum of Squares	df	Mean Square	F-Value	p-Value
	Yield					TAC					TPC					TFC
Model	0.19	9	0.02	12.87	0.0014	1.16 × 105	9	12,863	11.36	0.002	1860.16	9	206.68	7.54	0.01	5.85 × 105	9	65,021.06	25.02	0.0002
A	0.002	1	0.002	1.4	0.27	46,361	1	46,361	40.94	0.0004	91.05	1	91.05	3.32	0.1	9304	1	9304.13	3.58	0.1
B	0.01	1	0.01	5.79	0.04	9948	1	9948	8.78	0.02	728.76	1	728.76	26.58	0.001	1.07 × 105	1	1.07 × 105	41.18	0.0004
C	0.04	1	0.04	24.97	0.0016	13,616	1	13,616	12.02	0.01	24.11	1	24.11	0.87	0.4	24,286	1	24,286.61	9.35	0.02
AB	0.02	1	0.02	11.37	0.01	15,023	1	15,023	13.27	0.01	6	1	6	0.22	0.65	35,105	1	35,105.13	13.51	0.01
AC	0.02	1	0.02	13.21	0.01	783	1	783	0.69	0.43	19.35	1	19.35	0.71	0.43	1.20 × 105	1	1.20 × 105	45.98	0.0003
BC	0.02	1	0.02	14.7	0.01	16,275	1	16,275	14.37	0.007	76.24	1	76.24	2.78	0.14	38,778	1	38,778.97	14.92	0.006
A²	0.03	1	0.03	15.77	0.01	4562	1	4562	4.03	0.08	117.24	1	117.24	4.28	0.08	1.47 × 105	1	1.47 × 105	56.4	0.0001
B²	0.004	1	0.004	2.27	0.17	2906	1	2906	2.57	0.15	367.03	1	367.03	13.39	0.008	61,955	1	61,955.52	23.84	0.0018
C²	0.04	1	0.04	22.32	0.002	13,297	1	13,297	11.74	0.01	371.3	1	371.3	13.54	0.008	33,343	1	33,343.95	12.83	0.0089
Residual	0.01	7	0.002			7927	7	1132			191.94	7	27.42			18,188	7	2598.39
Lack of Fit	0.01	3	0.0037	29.61	0.003	6475	3	2158	5.95	0.06	157.3	3	52.43	6.06	0.06	10,909	3	3636.45	2	0.26
Std. Dev.	0.04					33.65					5.24					50.97
Mean	0.5879					842.93					26.59					490.59
C.V.%	6.92					3.99					19.69					10.39
R2	0.94					0.94					0.91					0.97

further demonstrates the ANOVA table for all responses and parameters for SWE. The results of multiple optimizations for the extraction conditions of SWE are presented in

Table 3. The best value of each parameter and response for SWE.

Parameters	Values	Responses	Predicted	Observed	Error (%)
Pressure, MPa	8.75	Yield, g/g	0.69	0.733	5.73
Temperature, K	393.54	TAC, mg/100 g	927.74	924.28	3.79
Flow rate, mL/min	4.89	TPC, mg/100 g	39.54	42.27	6.43
		TFC, mg/100 g	614.57	678.82	9.46

. To validate the model, the ideal predicted value was then compared to the experimental result. Figure 2 depicts the predicted and actual yield, TAC, TPC, and TFC responses for the SWE extraction. The experimental data and the quadratic model data based on the RSM process are also presented in Figure 2. The p-value of subcritical water extraction was set to 0.05. In the multiple optimization processes, the parameter values of pressure, temperature, and flow rate were set within their ranges, and the responses were set to maximum. The optimal conditions were 8.75 MPa, 393.54 K, and 4.89 mL/min with a yield of 0.691 g/g, TAC of 927.74 mg/100 g, TPC of 39.54 mg/100 g, and TFC of 614.57 mg/100 g.

3.1. Effect of Process Parameters and Statistical Analysis on Yield

According to Table 2, the linear coefficient of temperature (B); linear coefficient of flow rate (C); interactions of AB, AC, and BC; quadratic temperature (B²); and quadratic flow rate (C²) had significant (p < 0.05) impacts on the yield. The quadratic model fit the experimental data well, where R² was 0.94 with a p-value of 0.05. This result also indicated that 94% of the response could be captured by the regression models. The model accurately represented the positive interactions between the response parameter values and accurately characterized the data variance.

The links between the process variables and the yield are revealed in Equation (5) by eliminating all insignificant regression coefficients. According to the p-values in Table 2, linear temperature and flow rate had a greater influence on yield recovery than any other variable. Therefore, the pressure effect did not predominate instead of the flow rate and temperature.

$$Y_1=0.61-0.03\mathrm{A}+0.1\mathrm{B}-0.13\mathrm{C}+0.07\mathrm{AB}+0.07 \mathrm{AC}+0.11\mathrm{BC}-0.08{ \mathrm{A}}^2-0.09{\mathrm{B}}^2-0.09{\mathrm{C}}^2$$

(5)

Figure 3a shows that, at a constant flow rate of 5 mL/min and a maximum pressure of 10 MPa, the extract yield rose when the temperature was raised from 373 to 414 K. As the temperature rises, the solute-solvent becomes more dominant, generating the observed pattern. Consequently, this enhances the diffusivity of subcritical water [22]. By increasing the solvent’s diffusivity, the solvent’s ability to dissolve the target chemicals is increased. In addition, an increase in temperature reduces water’s viscosity, increasing its diffusivity. As the viscosity of the solvent decreases, the solubility of the solute rises [16].

As seen in Figure 3b, the flow rates of subcritical water between 3 and 7 mL/min at constant pressure and temperature diminish the yield. Mass transfer resistance limited the amount of extract transported into the majority of the solvent, although a subcritical water flow rate of 7 mL/min shortened the residence time in the extraction vessel. Consequently, the subcritical water residence time was inadequate to rupture and open the surface pores of roselle in order to transport all the solute [23].

At a constant flow rate of 5 mL/min and a constant temperature of 373 to 414 K, the yield extract was not influenced by increasing and decreasing the pressure, as shown in Figure 3b. This was because water is categorized as a noncompressible solvent. Therefore, increasing of pressure does not increase/decrease the viscosity or density of water. Thus, the solvation power of water cannot be increased by pressure.

3.2. Effect of Process Parameters and Statistical Analysis on TAC

According to Table 2, the linear pressure (A), linear temperature (B), linear flow rate (C), interaction of pressure and temperature (AB), interaction of temperature and flow rate (BC), and quadratic flow rate (C²) had substantial (p < 0.05) effects on the TAC recovery. As a consequence, the quadratic model fit the experimental data satisfactorily compared to the linear and 2FI models due to the higher R² and lower p-value. The developed models accurately captured the actual relationships between the response parameters and appropriately characterized the variation in the data, as shown in Figure 4. This model’s F-value for lack of fit suggests that lack of fit is statistically significant compared to pure error. These values are consistent with the model. The repeatability (coefficient of variation) of the SWE extraction procedure was 5.89% (outstanding) when three design experiments were considered (center points). CV < 10 is an outstanding CV number, as described in previous subchapter. Therefore, 5.8% is desirable, showing that the TAC extraction experiments were conducted in a predictable pattern. The associations between the process factors and the anthocyanin concentration are shown in Equation (6) by eliminating all insignificant regression coefficients. Based on the lowest p-value in the ANOVA table (p-value < 0.05), as indicated in Table 2, and the greatest coefficient value in Equation (7), the pressure and temperature effects were more influential than the flow rate impact in enhancing TAC recovery. Therefore, the influence of flow rate slightly predominated in anthocyanin extraction.

$$Y_2=787.35+131.85\mathrm{A}+106.09\mathrm{B}-77.18\mathrm{C}-61.28\mathrm{AB}+13.99\mathrm{AC}+86.64\mathrm{BC}+34.77{ \mathrm{A}}^2-29.53{\mathrm{B}}^2-58.49{\mathrm{C}}^2$$

(6)

Figure 4a demonstrates that rising pressure conditions at a constant temperature and flow rate boosted TAC recovery. A higher pressure may accelerate the process by forcing water into the matrix’s pores, but normal pressure would not expand the holes. Therefore, the anthocyanin, as a red pigment, is easily extracted by subcritical water. High-pressure conditions also increase the extraction efficiency due to the high pressure of solvents increasing the diffusivity of water to recover the red pigment anthocyanin [15,16,24].

Increasing the temperature at a constant pressure of 10 MPa and a constant flow rate 5 mL/min increased the red pigment anthocyanin recovery. Certain water properties change when temperature and pressure vary; for instance, as the temperature rises, the polarity of subcritical water decreases [5]. Consequently, the polarity of anthocyanin may be distinguished [24]. High temperature water decreases the density and viscosity. Thus, the lighter water easily penetrates the matrices of roselle to extract the red pigment of anthocyanin. As seen in Figure 4b, an increase/decrease in the flow rate of water at a constant pressure and temperature does not significantly affect the red pigment anthocyanin recovery. A slower flow rate increases the residence time of water in the vessel; therefore, the extraction efficiency is increased. The mass-transfer process is also increased by increasing the residence time of solvents [17]. However, a slower flow rate of water decreases the time for the extraction to be completed [25].

3.3. Effect of Process Parameters and Statistical Analysis on TPC

As indicated in Table 2, an ANOVA was conducted to evaluate the quadratic effect, interactions, and coefficients of the treatment factors on the response variables. With a p-value of 0.05, suitable optimization coefficients were R² > 0.80 and p < 0.05. As a consequence, the quadratic model fit the experimental data satisfactorily. Hence, it was shown that the quadratic model corresponded to the experimental data. This result indicated that at least 80% of the variation in the response variables could be adequately accounted for by the regression models connecting independent variables and responses. The created models accurately reflected the real connections between the response parameters and accurately characterized the data variance. Figure 2c also demonstrates the expected and experimental response data for the TPC.

The F-value for lack of fit in this model implies that lack of fit is statistically significant relative to pure error, whereas these values correspond to the model. The repeatability (coefficient of variation) of the SWE extraction procedure was 49.70% (unacceptable) when three design experiments were considered (center points) for the TPC recovery. CV < 10 is an outstanding CV number, 10–20 is a decent CV number, 20–30 is acceptable, and CV > 30 is undesirable. In light of this, 5.7% is outstanding, showing that the extraction TPC experiments were conducted in a predictable pattern. The TPC is connected to the breaking of surface pores in roselle, whereas greater TPC recovery indicates that the pores of roselle are more damaged. The associations between the process factors and the TPC are shown in Equation (7) by eliminating all insignificant regression coefficients.

$$Y_3=15.47+5.84\mathrm{A}+28.71\mathrm{B}+1.88\mathrm{C}+3.25\mathrm{B}-1.22\mathrm{AB}+2.20 \mathrm{AC}-5.93\mathrm{BC}+5.57{ \mathrm{A}}^2-10.49{\mathrm{B}}^2-9.77{\mathrm{C}}^2$$

(7)

The linear coefficients of temperature (B), quadratic temperature (B²), and quadratic flow rate (C²) had significant (p < 0.05) effects on the TPC recovery, as shown in Table 2. The linear pressure (A) and linear flow rate (C) were not significant factors in the TPC recovery. As validated in Figure 4, increasing the pressure slightly increased the TPC. However, higher pressure increases the interaction energy of the solvent and solute [26]. Chemical solubility and solvent diffusivity increase proportionately with increasing pressure [27,28].

Figure 5a shows that the TPC of extract increased as the temperature was increased at a constant flow rate (5 mL/min) and pressure (2 to 10 MPa). The pattern was caused by the increasing prevalence of the vapor solute condition as the temperature increased. In turn, this enhanced the diffusivity of water [29]. By increasing the solvent’s diffusivity, the solvent’s ability to dissolve the target compounds increased. The reduction in the sugar content of the solution caused by the Maillard reaction at a high temperature can be used to explain the color change in the extracts [30]. The outcomes of the study on color intensity were consistent with those of earlier research by Singh and Saldaña [31]. In the extraction of phenolic chemicals from potato peel using subcritical water, they showed that the extraction temperature and the color of extracts are directly connected. They noticed that, while the production of phenolic compounds grew from 22.56 mg/100 g to 81.83 mg/100 g by raising the temperature from 100 °C to 180 °C, the color of the phenolic extracts at a pressure of 6 MPa became darker.

The effect of flow rate was not significant based on the p-value, as stated in Table 2 (p-value > 0.05). Thus, the flow rate did not significantly enhance the yield extract, as shown in Figure 5. The flow rate is mechanical factor in subcritical water extraction, whereas the flow rate is not affected by the density of water. Furthermore, a higher flow rate of water decreases the residence time for the solvent to penetrate the roselle particle in order to extract the TPC [32]. Therefore, a higher flow rate of water is not suggestable to extract the phenolic compounds from the roselle.

3.4. Effect of Process Parameters and Statistical Analysis of TFC

According to Table 2, the linear temperature, linear flow rate, interaction of pressure and temperature, interaction of pressure and flow rate, interaction of temperature and flow rate, quadratic pressure, quadratic temperature, and quadratic flow rate had substantial (p < 0.05) effects on the TFC. The linear pressure gave a significant impact to the TFC recovery using SWE from roselle. The satisfying optimization coefficients were R² = 0.97 with a p-value of 0.0002. As a consequence, the quadratic model fit the experimental data satisfactorily compared to the linear and 2FI models. This finding revealed that at least 97% of the variation in the response variables might be appropriately captured by the regression models linking independent variables and responses. The developed models accurately captured the actual relationships between the response parameters and appropriately characterized the variation in the data.

This model’s F-value for lack of fit suggests that lack of fit is statistically significant compared to pure error. These values were consistent with the model. The repeatability (coefficient of variation) of the SWE extraction procedure was 25.66% (outstanding) when three design experiments were considered (center points). CV 10 is an outstanding CV number, as described in the previous subchapter. Therefore, 25.66% is acceptable, showing that the TFC extraction experiments were conducted in a predictable pattern. The associations between the process factors and the TFC are shown in Equation (8) by eliminating all insignificant regression coefficients.

$$Y_4=467.98-59.07\mathrm{A}+347.93\mathrm{B}-103.08\mathrm{C}+93.68\mathrm{AB}-172.82\mathrm{AC}+133.74\mathrm{BC}-197.07{ \mathrm{A}}^2-136.01{\mathrm{B}}^2-92.62{\mathrm{C}}^2$$

(8)

Based on the p-values in the ANOVA table shown in Table 2, the effect of linear temperature was more dominant than another factors in enhancing the TFC recovery. Therefore, the effect of pressure was not dominant in enhancing/increasing the breaking/opening pore of the roselle surface to extract the flavonoid compounds. It is validated in Figure 6 that increasing the temperature increased the TFC recovery. On the other hand, increasing the pressure slightly increased the TFC recovery. Additionally, increasing the temperature improved the ionic product of water (K_w), which suggests that the pH changed from 7.0 at room temperature to around 5.7 at 180 C, resulting in a greater ionic strength of hydronium and hydroxide ions than at a level temperature. Consequently, the increased ionic strength and lower pH may stimulate the acid hydrolysis of the bonds between the bioactive compounds and the solid matrix, thereby facilitating their release. It is anticipated that the presence of compounds of interest resulting from Maillard reactions may also impact the increase in absorbance for samples obtained with subcritical water [33].

Furthermore, the increase in the flow rate of water slightly enhanced the TFC recovery. The mass transfer resistance limited the amount of flavonoid transported into the bulk of the solvent, whereas a water flow rate of 7 mL/min led to a reduced residence time of water in the extraction vessel. Therefore, the residence time of water was not enough to break and open the surface pores of roselle. Bai et al. [34] found that increasing the roughness or flow rate might enhance the heat transfer capability of a solvent. Moreover, in the same location of the fracture, the temperature of the fracture wall was found to always be higher compared to that of the solvent; the overall heat transfer capacity of the solvent rose with the increase in the injection flow rate.

As seen in Figure 6b, the effect of pressure did not give a significant effect of TFC recovery from roselle. Therefore, pressure was not significant factor to enhance the breaking pore of roselle in order to extract the flavonoid compounds. The high pressure maintained the liquid state of the solvent at temperatures above the atmospheric boiling point. Changes in pressure have a negligible impact on the extraction efficiency of quercetin compounds [35].

3.5. Multiple Response Optimization of Extraction of Roselle Using SWE

Multiple optimizations were performed to obtain the optimal conditions for multiple maximum responses (yield, anthocyanin, TPC, and TFC). The optimal conditions were 8.75 MPa, 393.54 K, and 4.89 mL/min with responses of 0.69 g/g yield, 927.74 mg/100 g TAC, 39.54 mg/100 g TPC, and 614.57 mg/100 g TFC. This result was compared with the previous studies related to the extraction of anthocyanin by other extraction methods, as shown in Figure 7. Table 3 shows the validation of the optimization of SWE extraction, where the error between the predicted and actual data was below the 10%. Therefore, this optimization data can be applied for the scale-up process.

According to Idham, Putra, Aziz, Zaini, Rasidek, Mili, and Yunus [6], the optimal conditions for supercritical carbon dioxide (S_CCO₂) extraction were 27 MPa, 58 °C, and a cosolvent ratio of 8.86% at a maximum anthocyanin concentration of 1197 mg/100 g. Additionally, Redzuan et al. [36] discovered optimizing anthocyanin extracts from roselle (Hibiscus sabdarifa) petals using the ultrasonic-assisted extraction (UAE) method. The results indicate that a particle size of 0.125 mm, a solvent concentration of 10:1 mL/g, and a 15 min extraction time resulted in the highest mass yield (64.72%), TAC (70.97 mg/100 g), and AA (90.05%).

The calyces of dried roselle were investigated for anthocyanin extraction in aqueous solution, according to Gartaula and Karki [37]. The TAC of roselle was determined to be 310.48 mg/100 g. The extraction temperature of 70 °C produced the maximum yield (66.69–70.61%). However, anthocyanins degraded at 90 °C. On the extraction of anthocyanin, the solid–liquid ratio had no significant impact (p > 0.05). However, particle size had a significant effect (p < 0.05). Zannou et al. [38] also discovered that a natural hydrophilic deep eutectic solvent (DES) comprised of sodium acetate (hydrogen bond acceptor) and formic acid (hydrogen bond donor) was used to assess the TPC, TFC, and TAC. As comparative solvents, distilled water, 70% ethanol, and methanol (99%) were employed. According to the data, DES produced in a molarity ratio was the most effective. The optimal extraction conditions for this significant DES were determined to be a 1:3.6 molarity ratio, zero percent added water, and 10 mL of solvent. The optimal concentrations of TPC, TFC, and TAC were 23,326 mg/100 g, 1014 mg/100 g, and 1062 mg/100 g, respectively.

The data suggest that SWE yields more anthocyanin than UAE and solid–liquid extraction but less than the ScCO₂ and DES methods, as shown in Figure 7. This is due to the supercritical carbon dioxide used as a higher-pressure solvent, which increases the density and diffusivity of the solvent in order to extract anthocyanin. However, additional production and safety expenses arise from heightened pressure circumstances. Additionally, DES is unsuitable for anthocyanin since methanol is considered a hazardous solvent. Due to water being a nontoxic and safe solvent for the anthocyanin recovery, this technique (SWE) may thus be employed in replacement of the conventional method for extracting anthocyanin.

4. Conclusions

Roselle has a lengthy history of use in several nations, including in medical sciences. It is used to treat, among other conditions, pyrexia, liver damage, hypertension, and leukemia. In addition, roselle has various medical uses that have been studied globally. The recovery of red pigment anthocyanins, phenolics, and flavonoids from roselle via subcritical water extraction (SWE) is also an unexplored field of study. The subcritical water extraction (SWE) technique is more straightforward and is capable of achieving rapid extraction rates. SWE may have varying impacts on product output and quality, depending on the operating circumstances (pressure, temperature, flow rate, residence time, and particle size). The best conditions, according to this study, were 8.75 MPa, 393.54 K, and 4.89 mL/min, with responses of 0.69 g/g yield, 927.74 mg/100 g TAC, 39.54 mg/100 g TPC, and 614.57 mg/100 g TFC. High-pressure and -temperature conditions improved the production, phenolic and flavonoid recovery, and anthocyanin yield. The water flow rate was slightly relevant for increasing the yield and its compounds of interest. Due to the increased anthocyanin recovery and the nontoxic solvent, this technique (SWE) may be used instead of the conventional method for extracting red pigment anthocyanin.