Separation of the active components from the residue of Schisandra chinensis via an ultrasound-assisted method

Abstract

A novel and efficient method, namely Solid-state fermentation combined with ionic liquid pretreatment and ultrasonic-assisted extraction (SSFIPU), was successfully established for the extraction of anthocyanins (ACs) from Schisandra chinensis residue samples. Additionally, an enzymatic combined with ionic liquid pretreatment and ultrasonic-assisted extraction technique (EIPU) was effectively developed to extract Schisandrin A (SDA) and Schizandrin B (SDB) from the residue after AC extraction. A comprehensive examination was conducted on ten crucial parameters of SSFIPU and eight essential parameters of EIPU. The Box–Behnken design was employed to optimize the influencing factors and precisely predict the optimal extraction conditions. The optimized conditions were as follows: For SSFIPU, with 4 mol/L [BMIM]BF₄, 7 % carbon, 5 % nitrogen, a material–reagent ratio of 1:3 g/mL, a bacterial solution concentration of 1 × 10⁶ cfu/mL, a pH of 4, a fermentation time of 4 days, an ultrasonic power of 350 W, and an ultrasonic time of 30 min, an AC extraction yield of 1.173 mg/g was achieved, which was remarkably 23.46 times higher than that of ethanol reflux extraction (ERE). For EIPU, under the conditions of 4.2 mol/L [BMIM]BF₄, a cellulase content of 20 %, an enzyme hydrolysis time of 2.5 h, a material–reagent ratio of 1:13 g/mL, a pH of 5, an ultrasonic power of 450 W, and an ultrasonication time of 30 min, an SDA extraction yield of 0.306 mg/g was obtained, which was 8.5 times greater than that of ERE, and an SDB extraction yield of 0.260 mg/g was achieved, which was 6.8 times higher than that of ERE. During the experiment, [BMIM]BF₄ was successfully recovered with a recovery rate of 91.21 %. Consequently, the proposed environmentally friendly SSFIPU and EIPU methods have laid a solid foundation for the recovery of active ingredients from plant residues.

1. Introduction

Schisandra chinensis (Schisandra chinensis (Turcz.) Baill), a member of the Magnoliaceae family. It is a traditional Chinese herbal medicine used for the treatment of coughs, palpitations, spermatorrhea, and insomnia [1]. It is a medicinal and food that contains active chemical components such as lignans, flavonoids, polysaccharides, volatile oils, etc. [2]. Among them, lignans are the basis of many pharmacological activities [3]. The medicinal components used for extraction and separation have attracted increasing attention; Schisandra chinensis is usually used to extract essential oils, and the residue of the extracted essential oils is often discarded as waste. However, the residue is still rich in many active components, such as anthocyanidin (AC), schizandrin A (SDA), and schizandrin B (SDB). Many studies have shown that AC effectively inhibits the proliferation of cancer cells, prevents inflammatory diseases and cardiovascular diseases, and has antioxidant and antiaging effects [4], [5]. It inhibits the proliferation of cervical cancer cells, liver cancer cells, breast cancer cells, and lung cancer cells [6], [7]. The pharmacological effects of SDA and SDB are also broad, and they have been shown to have good cardiovascular protection, antioxidant, and immune activity and have a wide range of prospects for application in cardiovascular disease, lung disease, antifatigue, and adjuvant treatment of tumors [8].

Naturally, ACs are present mostly in the plant body as glycosides called anthocyanidins, with very few free-state ACs present [9]. The commonly used methods cannot effectively extract these active components, reducing the bioavailability and accessibility of Schisandra chinensis. Therefore, the glycosidic bond must be broken for the bound state anthocyanin to enter a free state [10]. Both SDA and SDB are fat-soluble components that are normally insoluble in water and require organic reagents as solvents to be extracted [11]. The addition of cellulase at the time of extraction resulted in higher yields [12]. In previous studies, traditional methods such as ethanol reflux extraction (ERE) and maceration have been widely used in natural product extraction, but these methods suffer from long extraction times, low efficiency, high solvent consumption, high temperature, etc. [13]. In recent years, new methods, such as supercritical CO₂ extraction and microwave-assisted extraction, have emerged. Compared with traditional methods such as solvent extraction, although these methods have greater efficiency and shorter extraction times, some limitations, such as the use of organic solvents in extraction, which has a certain degree of danger, also do not adapt to today's green energy-saving concept. Therefore, it is necessary to seek an efficient, economical, safe, and environmentally friendly method for the graded extraction of residues from the extraction of essential oils from Schisandra chinensis.

The Aspergillus niger solid-state fermentation (SSF) method utilizes the growth of Aspergillus niger in the culture medium to produce metabolites for extraction. It is a common species of industrial microorganism, as it produces a rich variety of enzymes. The SSF of Aspergillus niger produces β-D-glucoside glycohydrolase, which belongs to the class of cellulases that can hydrolyze the nonreducing β-D-glucose bond bound to the terminal while releasing β-D-glucose and the corresponding ligand. SSF is a method of processing food products that utilizes microbial functional degradation of compounds contained in the material to produce simpler ingredients while breaking various binding chains between ingredients in the material. Through fermentation, the glycosidic bonds of anthocyanins are severed and released to form free compounds with functional properties [14]. This property is also effective in breaking the glycosidic bonds of anthocyanins during anthocyanin extraction, thus releasing AC [15]. Zhao [16] used this method to greatly increase the extraction yield of flavonoids.

The enzymatic hydrolysis extraction method uses mainly the substrate, and the enzyme can be a specific combination of the characteristics of the component dissolved, which has the advantages of time savings and high yield [17], [18]. Additionally, enzyme extraction is a more environmentally friendly method of extraction, as it generates less waste and uses milder extraction conditions [19]. Compared with traditional methods of extraction, such as solvent extraction, enzyme extraction has several advantages, including reduced extraction time, lower solvent consumption, and improved quality of the extracted compounds [20].

Ionic liquids, which consist of organic cations and inorganic or organic anions, are among the emerging research areas in green chemistry [21], [22], [23], [24]. Ionic liquids can dissolve part of the plant cell wall by dissolving cellulose for extraction, thus increasing the extraction rate and shortening the extraction time [25]. Moreover, ionic liquids can effectively stabilize the enzyme-substrate transition state and reduce the reaction activation energy so that the enzyme has high catalytic activity [26]. It also has the advantages of a low melting point; good chemical and thermal stability; nonflammability; good electrical and thermal conductivity; a wide range of soluble substances; high solubility of inorganic, organic, and other substances; and designability [27], [28]. By combining ionic liquids with SSF and enzymatic methods for the pretreatment of raw materials, the extraction rate can be improved, and the extraction time can be shortened.

Although solid-state fermentation (SSF) and enzymatic methods possess certain advantages, they are restricted by mass transfer resistance and sluggishness. However, these limitations can be overcome through the application of ultrasound-assisted extraction (UAE) process intensification techniques. UAE is a technique that uses high-frequency sound waves and solvents to extract target compounds from various plant materials [29]. Ultrasound generates cavitation bubbles, promoting the release of intracellular components [30]. It is praised for its ability to reduce extraction time, decrease solvent consumption, and maintain the integrity of heat-sensitive compounds, making it a leading green extraction technology [31]. UAE is frequently employed as a means to extract active ingredients from plant residues as well. Tan Phat Vo managed to recover bioactive compounds from yacon [32]. The UAE process disrupts the cell wall to a certain degree, enabling the rapid release of the active components from plants into the extraction solvent. UAE extraction also has the advantage of enhancing enzyme activity [33]. This represents an efficient and sustainable extraction approach.

The aim of this study was to develop two distinct pretreatment approaches in combination with ionic liquids for ultrasound-assisted extraction. Within an environmentally friendly framework, to comprehensively utilize Schisandra chinensis and extract a greater quantity of active substances. Through single-factor experiments, the impacts of diverse parameters on the yield of the target compound were explored. Three factors that notably influenced the yield of the target compound were singled out from those parameters that might potentially affect the yield. Subsequently, the Box-Behnken design (BBD) was employed to further optimize these three statistically significant factors so as to ascertain the optimal extraction conditions. Moreover, a comparison was made with the yield of the ethanol reflux extraction (ERE) method. The ionic liquid was recovered during the experiment. This study furnishes a scientific foundation for enhancing the bioavailability of Schisandra chinensis.

2. Materials and methods

2.1. Materials and reagents

Schisandra chinensis was purchased from a pharmacy in Mudanjiang city, Heilongjiang Province, China. It was identified as Schisandra chinensis by a botany professor from Mudanjiang Normal University. The residue of Fructus Schizandra chinensis after extraction of essential oil was collected into brown, sealed bottles for subsequent experiments. The drying oven was supplied by Yiheng Scientific Instrument Co., Ltd. (Shanghai, China). A high-performance liquid chromatograph (Waters e2695 column WondaSil C18 Superb 5 μm, 4.6 × 250 mm) from United States Waters and an ultrasonic cleaner (KQ-400DE) were obtained from Kunshan Ultrasonic Instruments Co., Ltd. (Jiangsu, China), and cellulase (100000 u/g) from Shandong Longkete Enzyme Preparations Co., Ltd. (Shandong, China). Aspergillus niger was provided by the Microbiology Laboratory of Mudanjiang Normal College. Vanillin, methanol (AR, HPLC), sucrose, NH₄Cl, and concentrated hydrochloric acid were obtained from Tianjin Tianxin Fine Chemical Development Centre. (Tianjin, China). Ionic liquids (1-butyl-3-methylimidazole acetate ([BMIM]Ac), 1-butyl-3-methylimidazole tetrafluoroborate ([BMIM]BF₄), 1-butyl-3-methylimidazole chloride ([BMIM]Cl), and 1-butyl-3-methylimidazole bromide ([BMIM]Br)), anthocyanins, Schizandrin A, and Schizandrin B (HPLC grade standard reagents) with purities > 97 % were obtained from Shanghai Yuan ye Biotechnology Co. (Shanghai, China). Before use, filter all solutions and samples with a 0.45 µm microporous nylon membrane.

Macroporous resin (HPD-300, HPD5000) was purchased from Guangfu Fine Chemical Research Institute (Tianjin, China). The macroporous resin was pretreated by soaking it in ethanol for 24 h and then washing it with circulating deionized water residue to completely remove the ethanol residue. The treated resin was stored in a desiccator with deionized water to keep the moisture constant, and the moisture content of the resin was determined before use. (HPD-300 moisture content of 64.52 % and HPD5000 moisture content of 68.77 %).

2.2. Preparation of standard solutions and quantitative analysis

A standard curve for AC was generated via vanillin colorimetry [34]: 3 mL of 40 mg/mL vanillin methanol solution and 1 mL of concentrated hydrochloric acid were added to 1 mL of sample mixture, mixed well, and then placed in a constant-temperature water bath at 20 ± 1°C for 15 min while keeping the mixture warm and avoiding light for colorimetric analysis. The blank control was replaced with 1 mL of methanol, and the absorbance was measured at 500 nm. The AC concentration is the horizontal axis, and the absorbance is the vertical axis.

Standard curves for SDA and SDB were generated via HPLC [35]. The chromatographic conditions were as follows: the mobile phase was methanol: water (78: 22, v/v), the flow rate was 1 mL/min, the detection wavelength was 254 nm, and the column temperature was 35 °C. A Shimadzu Wondasil C18 reversed-phase column (4.6 mm × 250 mm, 5 μm) was used.

The standard curves for [BMIM]BF₄ were generated via HPLC [36]. The chromatographic conditions were as follows: the mobile phase was acetic acid with a volume fraction of 1 %: acetonitrile (22:78, v/v), the flow rate was 1 mL/min, the column temperature was 25 °C, and the wavelength was 210 nm.

The standard solutions of SDA, SDB, [BMIM]BF_4, and extracts needed to be filtered through a 0.45 μm microporous membrane. The concentrations of the SDA, SDB, and [BMIM]BF₄ standards were taken as the horizontal axis, and the peak area was taken as the vertical axis.

Stability test: Determine the sample solutions of AC, SDA, and SDB after ultrasound-assisted extraction and 7 days later to test their stability.

Sample addition recovery experiment: 3 samples of each sample solution with measured AC, SDA, and SDB values were added to the AC, SDA, and SDB reference substances of a certain quality, the sample addition recovery rate was calculated to investigate the accuracy of the method.

Repeatability experiments: Six sample solutions were prepared in parallel, and the peak area was measured according to the HPLC conditions or the absorbance according to spectroscopic conditions to investigate the repeatability of the method.

2.3. Activation culture of Aspergillus niger

PDA (potato dextrose agar) (5 g of potato was added to 200 mL of deionized water, stirred, heated until completely dissolved, and then filtered after the pH was determined to be 6.0–6.2, 5 g of sucrose and 5 g of agar were added, after which the mixture was fixed to 250 mL) was added. The prepared medium was poured into conical flasks, autoclaved with Petri dishes at 121 °C for 20 min, and then placed on an ultraclean worktable after sterilization. The Aspergillus niger strains were isolated and cultured when the temperature was suitable, placed in a constant temperature box at 28 °C for 3 days, and then set aside.

2.4. Extraction

2.4.1. Extraction of AC via SSFIPU

After the essential oil was extracted, the residue of Schisandra chinensis was removed, dried (60 °C, 24 h), and passed through a 60-mesh sieve to obtain the dried residue. Before extraction, the Schisandra chinensis residue was pretreated to SSF pretreatment. Ionic liquids are used as extractants. 3 g of dried residue was weighed in a conical flask, then add 5 % carbon source (w/w) and 5 % nitrogen source (w/w), was inoculated with Aspergillus niger at 2 × 10⁶ cfu/mL, with a pH 3, and mix-ferment for 4 days. After 4 days, a material–reagent ratio of 1:4 g/mL, 2 mol/L [BMIM]BF₄ was added. After the pretreatment, place it under ultrasonic conditions at 50 °C, 400 W for 30 min to extract AC. The sample was centrifuged at 4000 rpm for 10 min, and the supernatant was collected for determination. The AC yield was examined at 500 nm.

2.4.2. Simultaneously extract SDA and SDB via EIPU

The residue after extraction of AC was removed and dried (60 °C, 24 h) to obtain the dried residue. Before extraction, the residue was pretreated with cellulase hydrolysis. Three grams of dried residue was weighed in a conical flask, and was added at a material–reagent ratio of 1:10 g/ml, 2 mol/L [BMIM]BF₄ was added, 5 % cellulase (w/w), and pH 4. Hydrolyze in a 50 °C water bath for 1 h. After the pretreatment, the mixture was placed under ultrasonic conditions at 40 °C, 400 W for 50 min, the mixture was centrifuged at 4000 rpm for 10 min, and the supernatant was collected for determination. The SDA and SDB yields were examined at 254 nm.

2.5. Single-factor experiments

2.5.1. Ten single-factor experiments on the extraction of AC from Schisandra chinensis by SSFIPU

Single-factor experiments were conducted to optimize carbon (sucrose) content (1 %, 5 %, 10 %, 15 %) (w/w), nitrogen source (NH₄Cl) content (1 %, 5 %, 10 %, 15 %) (w/w), fermentation time (2 days, 4 days, 6 days, 8 days), pH (3, 4, 5, 6, 7), and bacterial broth concentration (1 × 10 ⁵ cfu/mL, 5 × 10 ⁵ cfu/mL, 1 × 10⁶ cfu/mL, 2 × 10 ⁶ cfu/mL, 3 × 10⁶ cfu/mL), the ionic liquid type ([BMIM]Ac, [BMIM]BF₄, [BMIM]Cl, and [BMIM]Br), ionic liquid concentration (0 mol/L, 2 mol/L, 4 mol/L, and 6 mol/L), material–reagent ratio (1:1 g/mL, 1:2 g/mL, 1:3 g/mL, 1:4 g/mL, and 1:5/mL), ultrasound time (20 min, 30 min, 40 min, 50 min, 60 min), and ultrasound power (250 W, 300 W, 350 W, 400 W, 450 W) to investigate their effects on the AC extraction processes.

2.5.2. Eight single-factor experiments on the extraction of SDA and SDB by EIPU

Single-factor experiments were conducted to optimize the material–reagent ratio (1:5 g/mL, 1:10 g/mL, 1:20 g/mL, 1:30 g/mL, 1:40 g/mL), enzyme concentration (1 %, 5 %, 10 %, 15 %, 20 %, 25 %) (w/w), enzyme hydrolysis time (0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h), ionic liquid type ([BMIM]Ac, [BMIM]BF₄, [BMIM]Cl, [BMIM]Br), ionic liquid concentration (0 mol/L, 2 mol/L, 4 mol/L, 6 mol/L), enzymatic pH (3, 4, 5, 6, 7), ultrasound time(20 min, 30 min, 40 min, 50 min, 60 min), and ultrasound power (250 W, 300 W, 350 W, 400 W, 450 W) to investigate their effects on the SDA and SDB extraction processes.

2.6. Box–Behnken experimental design

A response surface methodology was used to investigate the interaction between conditions further and optimize the extraction of active components from Schisandra chinensis. The results of the single-factor test are combined, Box–Behnken's principle of central combinatorial experimental design is applied, and the factors that have a greater impact on the experimental results are optimized based on the results of the single-factor test. The following key factors are selected for optimization. The factors considered in the study of RSM to optimize the AC extraction process of Schisandra chinensis were X₁: inoculum of Aspergillus niger; X₂: time of fermentation; and X₃: content of the carbon source. The factors considered in the response surface methodology to optimize the ionic liquid-enzyme extraction of SDA and SDB were X₁: material-reagent ratio of SDA or material-reagent ratio of SDB; X₂: pH; and X₃: concentration of ionic liquid. The experiment was randomized as shown in Table 1. The aim was to maximize the effect of unexplained variability on extraction efficiency. Design Expert 8.0.7.1 was used to design a 3-factor, 3-level response surface test.

Table 1.

Box–Behnken experimental design.

	Box–Behnken experimental design (AC)			Box–Behnken experimental design (SDA, SDB)
Run	X 1	X 2	X 3	X 1	X 2	X 3
	A: inoculum of Aspergillus niger(cfu/mL)	B: time of fermentation(days)	C: content of carbon source (%)	A: material-reagent ratio (g/mL)	B: pH	C: concentration of ionic liquid (mol/L)
1	1 × 106	6	1	1:10	5	4
2	1 × 106	4	5	1:20	5	2
3	5 × 105	4	1	1:10	5	4
4	1 × 106	6	10	1:05	6	4
5	2 × 106	2	5	1:10	4	2
6	5 × 105	4	10	1:10	5	4
7	1 × 106	2	10	1:10	6	6
8	1 × 106	4	5	1:05	4	4
9	1 × 106	4	5	1:10	5	4
10	2 × 106	4	10	1:20	6	4
11	1 × 106	4	5	1:05	5	6
12	2 × 106	6	5	1:10	6	2
13	1 × 106	4	5	1:05	5	2
14	2 × 106	4	1	1:10	4	6
15	5 × 105	2	5	1:20	4	4
16	1 × 106	2	1	1:10	5	4
17	5 × 105	6	5	1:20	5	6

2.7. Comparison with the ethanol reflux method

Ethanol reflux method (ERE) [37] is the use of ethanol to extract raw material components. The powdered residue of Schisandra chinensis after essential oil extraction was weighed, and the solvent was a 90 % ethanol solution at a material–reagent ratio of 1:5 g/mL. The mixture was extracted for 1 h and centrifuged at 4000 rpm for 10 min, after which the supernatant was collected for determination.

2.8. Recovery of ionic liquids

50 g of the residue, which was used as the raw material, was sequentially extracted under optimized conditions. After extraction, the residue was separated by centrifugation at 1500 r/min for 15 min. The supernatant was collected as the sample mixture. The material residue was then washed twice with agitation via deionized water at material–liquid ratios of 1:8 g/mL and 1:6 g/mL (based on wet weight) to remove any remaining ionic liquids from the extracted residue. According to previous research [37], [38]. The macroporous resin HPD-300 was used to separate the AC and HPD5000 to separate the SDA and SDB.

A glass column (12 mm × 500 mm) packed with resin (15 g, dry weight basis) was used for separation. The bed volume (BV) was 20 mL, and the resin length was 25 cm. The ionic liquid extract of AC was passed through the column at a flow rate of 3 BV/h, with the effluent collected at 10 mL intervals. Once adsorption reached equilibrium, the column was washed with 3 BV of deionized water, and the effluent was collected. The ionic liquid concentration was determined via HPLC. The SDA and SDB ionic liquid extracts were passed through a glass column at a flow rate of 4 BV/h, with the effluent collected at 50 mL intervals. After adsorption equilibrium was reached, the column was washed with 4 BV of deionized water, and the effluent was collected. The ionic liquid concentration was again determined via HPLC. The two water-washing solutions of the extraction residue of the two materials were collected separately and combined with the wastewater discharge solution to form the ionic liquid to be recovered. The ionic liquid was then obtained by evaporating the water at 95 °C via a rotary evaporator under a vacuum of 0.09 MPa and drying it at 120 ± 2 °C for 2 h. After the separation of the target analytes, the macroporous resin-packed column was first washed with 90 % volume fraction ethanol to remove impurities and then washed with deionized water to thoroughly replace the ethanol to reuse the separation column packed with microporous resin.

2.9. Data processing

The single-factor experiment data were the average of three repeated measurements, and the experimental data were organized via Excel, Design Expert 8.0.7.1, Origin, and SPSS Statistics 25.

3. Results and discussion

3.1. Establishment of standard curves

Concentration gradients of 0.02, 0.04, 0.06, 0.08, 0.12, 0.16, and 0.2 mg/mL were prepared the standard solutions of AC via distilled water. The absorbance was measured via vanillin-hydrochloric acid color development method. The AC concentration is the horizontal axis, and absorbance is the vertical axis.

The standard solutions of SDA and SDB were prepared at gradient concentrations of 0.03125, 0.0625, 0.125, 0.25, and 0.5 mg/mL via chromatographic methanol. HPLC was used to determine the peak area of each standard. The concentration of the SDA and SDB standards were taken as the horizontal axis, and the peak area was taken as the vertical axis.

[BMIM]BF₄ standard solution was prepared at gradient concentrations of 0.03125, 0.0625, 0.125, 0.25, and 0.5 mg/mL via distilled water. HPLC was used to determine the peak area of each standard. The concentration of the standard was taken as the horizontal axis, and the peak area was taken as the vertical axis.

A total of four kinds of quantitative standard curves were obtained for each standard, the linearity of each standard was good, and the coefficients of the regression equations, R², were all greater than 0.9990, as shown in Table 2:

Table 2.

Regression equations for standard curves.

Name of standard product	Regression equation	R2	Linear range (ug/mL)
AC	Y= (3.91 ± 0.01) x +(0.0054 ± 0.0018)	R2 = 0.9992 (n = 7)	20–200
SDA	Y= (9638373.16 ± 210.78) x – (44524 ± 570.511)	R2 = 0.9996(n = 5)	31.25–500
SDB	Y= (15698219.57 ± 3677.2) x+(43411.96 ± 730.1)	R2 = 0.9996(n = 5)	31.25–500
[BMIM]BF4	Y= (10375686.60 ± 6435.77) x+(23632.04 ± 89.87)	R2 = 0.9993(n = 5)	31.25–500

3.2. Methodological investigation

3.2.1. Stability

Recovery was used as an indicator to assess the stability of AC SDA and SDB under ideal extraction circumstances. According to Table 3, AC, SDA, and SDB recovered on average 98.01 %, 98.62 %, and 98.67 %. The average recoveries were 97.62 %, 97.45 %, and 97.16 % after 7 days. The three extracts are shown to have good stability.

Table 3.

Stability studies of AC, SDA, and SDB.

Compounds	Initial concentration (mg/mL)	Recovered concentration (mg/mL)	RSD% (n = 3)	Average recovery (%)	Recovered concentration after 7 days (mg/mL)	RSD% (n = 3)	Average recovery (%)
AC	1.010	0.990	0.570	98.01	0.986	0.585	97.62
SDA	0.510	0.500	0.610	98.62	0.497	0.603	97.45
SDB	0.530	0.521	0.610	98.67	0.515	0.681	97.16

3.2.2. Recovery

The samples were mixed with control solutions of AC, SDA, and SDB, and HPLC was used to determine the outcomes. The results are shown in Table 4. With average recoveries of 98.98 %, 96.91 %, and 95.92 % for AC, SDA, and SDB, respectively, this approach produced good recoveries.

Table 4.

Recovery of AC, SDA, and SDB.

Sample	Contents of the sample (mg)			Mass of added reference substances (mg)			Mass of the sample analyzed with added reference substances (mg)			Recovery (%)
	AC	SDA	SDB	AC	SDA	SDB	AC	SDA	SDB	AC	SDA	SDB
1	1.1	0.3	0.2	0.55	0.12	0.12	1.64	0.41	0.31	99.39	97.67	96.8
2	1.1	0.3	0.2	0.54	0.14	0.14	1.62	0.43	0.33	98.78	97.72	97.05
3	1.1	0.3	0.2	0.53	0.13	0.13	1.61	0.41	0.31	98.77	95.34	93.93
Average										98.98	96.91	95.92

3.2.3. Repeatability

Parallel preparation of six sample solutions was carried out six times. Table 5 shows that the AC, SDA, and SDB RSD values were 0.472 %, 0.621 %, and 0.811 %, demonstrating the method's good repeatability.

Table 5.

Repeatability of AC, SDA, and SDB.

content（mg/g）			average（mg/g）			RSD（%）
AC	SDA	SDB	AC	SDA	SDB	AC	SDA	SDB
0.532	0.312	0.215
0.531	0.31	0.213
0.531	0.314	0.216	0.529	0.312	0.215	0.472	0.621	0.811
0.530	0.311	0.217
0.529	0.315	0218
0.525	0.311	0.215

3.3. Single-factor experiments and RSM analysis of AC extracted

3.3.1. Effect of type of ionic liquid

The effects of different ionic liquids on the yield of AC were investigated. The results are shown in Fig. 1(a). As shown in Fig. 1(a), the highest AC yield was obtained when the ionic liquid was [BMIM]BF₄. At this point, the yield of AC was 0.07548 mg/g. This may be due to the structure and nature of this type of ionic liquid, which is suitable for the extraction of this component [39]. This may be because BMIM]BF₄ tends to disrupt the cell membrane structure and improve the extraction rate [40].

Fig. 1.

The results of 10 single-factor experiments on AC extraction. Effect of type of ionic liquid (a), ionic liquid concentration (b), nitrogen source (c), carbon source (d), pH value (e), material–reagent ratio (f), fermentation time (g), and concentration of bacterial (h), ultrasonication power (i), and ultrasonication time (j) on the AC yield via SSFIPU.

3.3.2. Effect of ionic liquid concentration

The effects of different ionic liquid concentrations on the AC yield were investigated. The results are shown in Fig. 1(b). With increasing ionic liquid concentration, the yield increased and then decreased; when the ionic liquid concentration was 4 mol/L, the AC yield reached a maximum, and the yield of AC was 0.09561 mg/g. When the ionic liquid concentration continued to increase, the AC yield decreased, possibly because the increase in the volume of the ionic liquid was too high, the viscosity of the extract increased, and it was difficult for the ionic liquid to penetrate the cell wall of Schisandra chinensis [41].

3.3.3. Effect of the nitrogen source (NH₄Cl) content

The effects of different nitrogen source contents on the final yield of AC were investigated, with 1 %, 5 %, 10 %, and 15 % nitrogen source contents used as controls. The results are shown in Fig. 1 (c). Fig. 1 (c) shows that with increasing nitrogen source content, the yield increased but then decreased. When the nitrogen source content was 5 %, the maximum yield of AC was 0.46784 mg/g. This trend is consistent with previous findings that microbial activity decreases when the optimal nitrogen source concentration is exceeded [42]. This difference may be due to the dysregulation of the carbon-to-nitrogen ratio, which affects the growth of microorganisms and decreases the synthesis of enzymes, leading to a decrease in the extraction rate [43].

3.3.4. Effect of the carbon source (sucrose) content

The effects of different carbon source contents on the final yield of AC were investigated. The results are shown in Fig. 1 (d). As shown in Fig. 1 (d), with increasing carbon source content, the first yield increased and then decreased. When the carbon source content was 5 %, the maximum yield of AC was 0.65607 m g/g. This trend is consistent with previous findings that an adequate supply of carbon sources and their concentrations significantly promoted fungal growth and microbial activity decreased when the optimal carbon source concentration was exceeded [44]. This may be because too many carbon sources affect microbial growth, and a relatively high carbon-to-nitrogen ratio affects bacterial growth, leading to a decrease in enzyme production and a relatively low extraction rate [45].

3.3.5. Effect of pH

The effects of different pH values on the yield of AC were investigated. The results are shown in Fig. 1 (e). Fig. 1 (e) shows that with increasing pH, the yield increased but then decreased, and at pH 4, the maximum yield of AC was 0.64627 mg/g. This finding is similar to previous findings [46]. This is likely because Aspergillus niger produces the best enzyme at pH 3--5, generating more enzymes to break glycosidic bonds, which is favorable for anthocyanin extraction [47]. When the pH is less than 4 or when the pH continues to increase, the yield of AC decreases, and acidic or alkaline environments may change the permeability of the membrane, which in turn affects the normal growth of Aspergillus niger, leading to a reduction in enzyme production and enzyme activity [15]. The inability to fully break the glycosidic bond resulted in a lower AC yield.

3.3.6. Effect of the material–reagent ratio

The effects of different material–liquid ratios on the final yield of AC were investigated. The results are shown in Fig. 1 (f). Fig. 1 (f) shows that with increasing material–reagent ratio, the yield increased and then decreased. When the material–reagent ratio reached 1:3 g/mL, the maximum yield of AC was 0.67764 m g/g. When the material–reagent ratio was less than 1:3 g/mL, it might have been due to insufficient contact between the extracting liquid and the contact area of the materials, which resulted in poor AC dissolution. When the material–reagent ratio continued to increase, the yield decreased, possibly because an increase in the feed–to–liquid ratio promoted the production of nontargeted active products, which reduced the release of AC [48].

3.3.7. Effect of fermentation time

The effects of different fermentation times on the final yield of AC were investigated. The results are shown in Fig. 1 (g). As shown in Fig. 1 (g), with increasing fermentation time, the yield increased but then decreased. On the 4th day of fermentation, the maximum yield of AC was 0.72078 mg/g. The fermentation time was less than 4 days, possibly because of the insufficient number of days of fermentation and the small amount of enzyme production by Aspergillus niger, which led to poor AC solubilization. It is possible that when fermentation time is 4 days, the enzyme production of Aspergillus niger is maximal, which is favorable for the extraction of AC [49]. When the number of days of fermentation continued to increase, the decrease in the yield of AC with increasing fermentation time was possibly due to insufficient nutrients in the culture medium and decreased AC production with increasing fermentation duration. This finding is similar to the results of a previous study [50]. Therefore, a fermentation time of 4 days was chosen for subsequent experiments.

3.3.8. Effect of bacterial concentration

The effects of different inoculum amounts on the final yield of AC were investigated. The results are shown in Fig. 1 (h). As shown in Fig. 1 (h), with increasing bacterial mixture concentration, the yield first increased but then decreased. When the inoculum amount was 1 × 10⁶, the maximum yield of AC was 1.14823 mg/g. This trend is consistent with previous findings [51]. The inoculum amount is one of the important factors affecting the enzyme production of microbial fermentation; the inoculum amount is too low when the bacterial growth is slow, the inoculum content is small, the enzyme activity is low, and an inoculum amount that is too high will lead to insufficient nutrients to affect the proliferation of microorganisms, thus affecting the metabolism of enzymes [52].

3.3.9. Effect of the time of ultrasound

The effects of different ultrasonication times on the final yields of AC were investigated, and the results are shown in Fig. 1 (i). With increasing ultrasonication time, the yield increased and then decreased. At an ultrasonication time of 30 min, the maximum yield rate of AC was 0.09802 mg/g. When the ultrasonication time was prolonged, the yields of AC were reduced. It might be that the ultrasound time was too long, causing the structure of the AC to be damaged [53], so 30 min was chosen as the optimal condition.

3.3.10. Effect of the power of ultrasound

The effects of different ultrasonication powers on the final yields of AC were investigated, and the results are shown in Fig. 1 (j). With increasing ultrasonication power, the yield increased and then decreased. At an ultrasonication power of 350 W, the maximum yield rate of AC was 0.12160 mg/g. When the ultrasonication power was prolonged, the yields of AC were reduced. It might be that the ultrasound power is too high, which could cause other impurities in Schisandra to dissolve, interfering with the detection of the target substance [54]. Therefore, an ultrasonic power of 350 W is chosen as the optimal condition.

3.4. Single-factor experimental and RSM analysis of SDA and SDB extracted

3.4.1. Effect of type of ionic liquids

The effects of different types of ionic liquids on the final yields of SDA and SDB were investigated. The final yields of SDA and SDB results are shown in Fig. 2 (a). When the ionic liquid was 1-butyl-3-methylimidazolium borate, the maximum SDA yield was 0.00605 mg/g, and the maximum SDB yield was 0.00007 mg/g. This may be due to the structure and properties of this type of ionic liquid, which are suitable for the extraction of the component [39]. This may be because BMIM]BF₄ can easily disrupt the structure of the cell membrane and increase the extraction rate [40].

Fig. 2.

The results of 8 single-factor experiments on SDA and SDB extraction. Effect of the type of ionic liquid (a), material-reagent ratio (b), enzyme hydrolysis time (c), cellulase content (d), ionic liquid concentration (e), pH value (f), ultrasonication time (g), and ultrasonication power (h) on yield of EIPU.

3.4.2. Effect of the material–reagent ratio

The effects of different material–reagent ratios on the final yields of SDA and SDB were investigated. The results are shown in Fig. 2 (b). With increasing material–reagent ratio, the yield of SDA and SDB increased, which may be due to the greater contact between the extraction solution and the contact area of the material, resulting in better dissolution of SDA and SDB. When the material–reagent ratio reached 1:10 g/mL, the maximum yield of SDA was 0.00915 mg/g, and that of SDB was 0.00224 mg/g. As the material–liquid ratio continued to increase, the extraction rate decreased. This could be because the contact area between the solvent and the feedstock increases with the material–reagent ratios, and when the contact area reaches a certain value, the extraction rate saturates [55]. This could also be explained by the continuous increase in the solvent reducing the concentration of the enzyme, leading to a low substrate per unit volume [56].

3.4.3. Effect of enzyme hydrolysis time

The effects of time on the final yields of SDA and SDB were investigated, and the results are shown in Fig. 2 (c). As shown in the pictures, with prolonged enzyme hydrolysis time, the first yield increased and then decreased; at 2.5 h, the maximum yields of SDA and SDB were 0.01050 mg/g and 0.00295 mg/g, respectively. This may be because too little time, usually with incomplete destruction, can lead to incomplete extraction of the target component, whereas excessive time can lead to unnecessary energy consumption [42]. The yield decreased, possibly due to the initial extraction and increased degree of cellular fragmentation under enzymatic action [56].

3.4.4. Effect of cellulase content

The effects of different cellulase contents on the final yields of SDA and SDB results are shown in Fig. 2 (d). As shown in the figure, with increasing cellulase content, the first yield increased and then decreased; when the cellulase content was 20 %, the maximum yields of SDA and SDB were 0.01594 mg/g and 0.00446 mg/g, respectively. When the cellulase content was less than 20 %, the substrate and the enzyme did not combine sufficiently, which resulted in poor dissolution of SDA and SDB; when the cellulase content continued to increase, the yields of SDA and SDB gradually decreased, and the increase in the enzyme content likely increased the viscosity of the enzyme mixture, resulting in a decrease in its decomposition ability, which affected the progress of the enzymatic reaction [33].

3.4.5. Effect of ionic liquid concentration

The effects of different ionic liquid concentrations on the final yields of SDA and SDB were investigated. The effects of different ionic liquid concentrations on the final yields of SDA and SDB results are shown in Fig. 2 (e). With increasing ionic liquid concentration, the yield increased and then decreased; at 4 %, the maximum yields of SDA and SDB were 0.01742 mg/g and 0.00509 mg/g, respectively; when the ionic liquid concentration continued to increase, the yields of SDA and SDB gradually decreased, probably because the increase in the volume of the ionic liquid was too high, and the viscosity of the extraction solution increased, making it difficult for the ionic liquid to penetrate the cell wall of Schisandra chinensis [41].

3.4.6. Effect of enzymatic pH

The effects of different enzymatic pH values on the final yields of SDA and SDB were investigated, and the results are shown in Fig. 2 (f). With increasing enzyme hydrolysis pH, the yield increased and then decreased; at pH 5, the maximum yield of SDA was 0.02278 mg/g, and the maximum yield of SDB was 0.00755 mg/g. The above situation occurred because, on the one hand, cellulase could promote the decomposition of the cell wall to increase the release of natural active substances within the appropriate range [55], with increasing enzymatic pH, the yield gradually decreased, which may be due to the optimal pH maximizing cellulase activity and cell wall rupture. However, the conditions of the cellulase reaction changed with increasing pH. The cellulase activity decreased, possibly because pH affects cellulase conformation and substrate dissociation [33].

3.4.7. Effect of the time of ultrasound

The effects of different ultrasonication times on the final yields of SDA and SDB were investigated, and the results are shown in Fig. 2 (g). With increasing ultrasonication time, the yield increased and then decreased. At an ultrasonication time of 50 min, the maximum yield rate of SDA was 0.06820 mg/g, and the maximum yield rate of SDB was 0.01331 mg/g at an ultrasonication time of 30 min. When the ultrasonication time was prolonged, the yields of SDA and SDB were reduced. First, the ultrasonic shearing effect caused by an excessively long ultrasonication time resulted in the continuous destruction of the SDA and SDB structures [53]. Second, the extension of ultrasonication time promoted heat transfer in the whole system to a certain extent, and a change in temperature may affect enzyme activity or lead to partial degradation of SDA and SDB [57].

3.4.8. Effect of the power of ultrasound

The effects of different ultrasonication powers on the final yields of SDA and SDB were investigated, and the results are shown in Fig. 2 (h). With increasing ultrasonication power, the yield increased and then decreased. At an ultrasonication power of 450 W, the maximum yield of SDA was 0.01962 mg/g, and the yield of SDB was 0.0134 mg/g. When the ultrasonication power was prolonged, the yields of SDA and SDB were reduced. It might be that the ultrasound power is too high, which could cause other impurities in Schisandra to dissolve, interfering with the detection of the target substance [54]. Therefore, an ultrasonic power of 450 W is chosen as the optimal condition.

To further investigate the interaction between fermentation conditions and optimize the extraction of AC from Schisandra chinensis. RSM was used to further optimize AC extraction via SSFIPU. The experiments were randomized as shown in Table 6. Seventeen tests were conducted with five replications to estimate the pure sum of squares of the errors. The results of the experimental conditions are shown in Table 6, which fits the data via a quadratic polynomial model.

Table 6.

Box–Behnken experimental results (AC).

Run	X1 A: Number of Aspergillus niger (cfu/mL)	X 2 B: Time of fermentation(days)	X 3 C: Content of carbon source(%)	Response AC yield (mg/g)
1	1 × 106	6	1	0.360
2	1 × 106	4	5.5	1.160
3	5 × 105	4	1	0.412
4	1 × 106	6	10	0.920
5	2 × 106	2	5.5	0.781
6	5 × 105	4	10	0.542
7	1 × 106	2	10	0.652
8	1 × 106	4	5.5	1.021
9	1 × 106	4	5.5	1.055
10	2 × 106	4	10	0.996
11	1 × 106	4	5.5	1.122
12	2 × 106	6	5.5	0.990
13	1 × 106	4	5.5	1.185
14	2 × 106	4	1	0.473
15	5 × 105	2	5.5	0.620
16	1 × 106	2	1	0.710
17	5 × 105	6	5.5	0.560

By applying Design Expert 8.0.7.1 to fit a multiple regression to the data, the quadratic equation of Schisandra chinensis AC yield (Y) to each factor variable can be obtained as:

$$Y=1.11+0.14A+8.3\times {10}^{-3}B+0.14C+0.067AB+0.098AC+0.15BC-0.21A^2-0.16B^2-0.29C^2$$ (1)

The results of the regression model analysis shown in Table 7 indicate that this quadratic multinomial regression model had highly significant differences (P < 0.0001), indicating a highly significant correlation between the three independent variables involved and the response values of AC. The coefficient of determination R² = 0.8415 indicated that the model was well fitted with no abnormal terms and high reliability. The fitted regression equation can be used to analyze the optimum yield of AC from Schisandra chinensis under different conditions. The model terms that had a significant effect on the AC yield were A, C, AC, and BC. The 3D response surface plots and corresponding contour plots in Fig. 3 illustrate the effects of the variables and their interactions on the AC yield. The effects of the other variables on the AC yield were consistent with the previous analysis of the single-factor experiments when two variables were held constant. The optimum conditions for obtaining the highest AC yield were as follows: when the Aspergillus niger inoculum was 1605824.965, fermentation days were 4.663, and the carbon source was 7.377 %, the AC yield of Schisandra chinensis was as high as 1.173 mg/g. For ease of application, the optimized values are rounded to the nearest whole number. The yield of AC obtained is 1.170 mg/g, which is very close to the theoretical values of the model.

Table 7.

ANOVA of the quadratic response surface regression mode (AC).

Source	Sum of Squares	Df	Mean Square	F Value	P value
Model	1.19	9	0.13	51.52	< 0.0001
A-Number of nigrosinase inoculations	0.15	1	0.15	36.38	0.0005
B-Time of fermentation	5.561 × 104	1	5.561 × 104	0.13	0.7268
C-carbon source	0.17	1	0.17	39.65	0.0004
AB	0.018	1	0.018	4.29	0.0770
AC	0.039	1	0.039	9.18	0.0191
BC	0.095	1	1.562 × 104	22.71	0.0020
A2	0.19	1	0.19	45.33	0.0003
B2	0.11	1	0.11	25.00	0.0016
C2	0.35	1	0.35	84.26	<0.0001
residual	0.029	7	4.205 × 103
incoherent	0.010	3	3.410 × 103	0.71	0.5946
R	0.019	4	4.801 × 103
Sum	1.22	16

Fig. 3.

The 3D surface plots and contour plots show the effects of the number of inocula of Aspergillus niger (A), time of fermentation (B), and carbon source (C) on the AC yields by SSFIPU (a-b).

To further investigate the interactions between fermentation conditions and optimize the extraction of SDA and SDB. Further optimization of SDA and SDB extraction by EIPU via RSM. The experiment was randomized as shown in Table 8. The aim was to maximize the effect of unexplained variability on extraction efficiency. Seventeen tests were performed. The results of the experimental conditions are shown in the table. Fitting the data via a quadratic polynomial model.

Table 8.

Box–Behnken experimental results (SDA, SDB).

Run	X 1 A: Material-reagent ratio (g/mL)	X 2 B: pH	X 3 C: Concentration of ionic liquid (mol/L)	Response SDA yield (mg/g)	Response SDB yield (mg/g)
1	12.5	5	4	0.298	0.265
2	20	5	2	0.190	0.135
3	12.5	5	4	0.335	0.252
4	5	6	4	0.120	0.105
5	12.5	4	2	0.160	0.099
6	12.5	5	4	0.290	0.250
7	12.5	6	6	0.187	0.156
8	5	4	4	0.120	0.097
9	12.5	5	4	0.312	0.277
10	20	6	4	0.150	0.100
11	5	5	6	0.130	0.106
12	12.5	6	2	0.140	0.125
13	5	5	2	0.098	0.090
14	12.5	4	6	0.160	0.155
15	20	4	4	0.138	0.125
16	12.5	5	4	0.286	0.251
17	20	5	6	0.180	0.240

The quadratic equation for the yield of SDA (Y₁) for each factor variable can be obtained as:

$$Y_1=0.30+0.021A+5.3\times {10}^{-3}B+0.017C-3\times {10}^{-3}AB-7.9\times {10}^{-3}AC+8.8\times {10}^{-3}BC-0.092A^2-0.08B^2-0.035C^2$$ (2)

The quadratic equation for the yield of SDB (Y₂) concerning each factor variable is as follows:

$$Y_2=0.26+0.0253A+1.2\times {10}^{-3}B+0.026C-8.2\times {10}^{-3}AB-2.23\times {10}^{-3}AC-6.2\times {10}^{-3}BC-0.072A^2-0.081B^2-0.045C^2$$ (3)

The results of the regression model analysis are shown in Table 9. This quadratic multinomial regression model had highly significant differences (P < 0.01), indicating a highly significant correlation between the three independent variables involved and the response values of SDA and SDB. The coefficients of determination are R² = 0.9579 and R² = 0.9505, indicating that the models are well fitted, have no abnormal terms, and are highly reliable. The fitted regression equation can be used to analyze the optimum yield of SDA and SDB under different conditions. The model terms that have a significant effect on the yield of SDA and SDB are A and C. The 3D response surface plots and the corresponding contour plots in the Fig. 4 illustrate the effects of the variables and their interactions on the yield of SDA and SDB. The effects of the other variables on the SDA and SDB yield were consistent with the previous analysis of the single-factor experiment when two variables were held constant. The optimum conditions for simultaneously obtaining the highest yields of SDA and SDB were as follows: the yield of SDA could reach 0.306 mg/g and the yield of SDB could reach 0.260 mg/g at a material–reagent ratio of 1:13.44 g/mL, pH 5.039, and a 4.2 % concentration of ionic liquid. For ease of application, the optimized values are rounded to the nearest whole number. The yield of SDA obtained is 0.304 mg/g, and the yield of SDB is 0.253 mg/g, which is very close to the theoretical values of the model.

Table 9.

ANOVA of the quadratic response surface regression model (SDA, SDB).

Source	Sum of Squares		Df		Mean Square		F Value		P value
	SDA	SDB	SDA	SDB	SDA	SDB	SDA	SDB	SDA	SDB
Model	0.0944	0.0765	9	9	0.0105	0.0085	26.53	14.95	0.0001	0.0009
A-material-reagent ratio	0.0032	0.0051	1	1	0.0032	0.0051	8.03	8.97	0.0253	0.0201
B-pH	0.0002	0	1	1	0.0002	0	0.5078	0.022	0.4991	0.8863
C-Ionic liquid concentration	0.0028	0.0054	1	1	0.0028	0.0054	7.02	9.51	0.033	0.0177
AB	0	0.0003	1	1	0	0.0003	0.0911	0.4785	0.7716	0.5114
AC	0.0004	0.002	1	1	0.0004	0.002	1.12	3.48	0.3259	0.1043
BC	0.0006	0.0002	1	1	0.0006	0.0002	1.4	0.2746	0.2758	0.6164
A2	0.0358	0.0216	1	1	0.0358	0.0216	90.61	37.97	< 0.0001	0.0005
B2	0.0269	0.0274	1	1	0.0269	0.0274	68.14	48.11	< 0.0001	0.0002
C2	0.0164	0.0084	1	1	0.0164	0.0084	41.58	14.74	0.0004	0.0064
resiual	0.0028	0.004	7	7	0.0004	0.0006
Lack of Fit	0.0012	0.0034	3	3	0.0004	0.0011	1	8.25	0.4789	0.0346
Pure Error	0.0016	0.0006	4	4	0.0004	0.0001				0.0009
Cor Total	0.0971	0.0805	16	16				14.95		0.0201

Fig. 4.

The 3D surface plots and contour plots show the effects of the material-reagent ratio (A), pH (B), and ionic liquid concentration (C) on the SDA yields (c-d) and SDB yields (e-f) of the EIPU.

3.5. Comparison with the HRE

As shown in Table 10, compared with ERE, SSFIPU, and EIPU can greatly improve the extraction efficiency of the active ingredients.

Table 10.

Comparison with the ERE.

	SSFIPU/EIPU	ERE
AC yield	1.173 mg/g	0.050 mg/g
SDA yield	0.306 mg/g	0.036 mg/g
SDB yield	0.260 mg/g	0.038 mg/g

3.6. Recovery of ionic liquids

Because [BMIM]BF₄ is used in the extraction process, it is merged and recycled. After evaporating and drying the ionic liquid to be recovered, viscous [BMIM]BF₄ was obtained. The total recovery of the ionic liquids was 91.21 %. (Fig. 5).

Fig. 5.

Dynamic desorption curves of ionic liquids on a macroporous resin-packed column.

There are several innovations in this experiment. The residue left after extracting essential oil from Schisandra chinensis was used as the raw material for extracting AC. And the solid-state fermentation of Aspergillus niger combined with ionic liquid was employed to pretreat the raw material. This effectively breaks the glycosidic bonds of AC and increases the solubility of AC. Furthermore, the residue remaining after extracting AC was utilized to extract SDA and SDB. The combination of the ionic liquid and cellulolytic hydrolysis method for residue pretreatment effectively disrupted the cell walls and increased the amounts of released SDA and SDB. Then, ultrasound-assisted extraction, which is an economical and environmentally friendly approach, was adopted, providing an efficient and sustainable extraction method. Ultrasound-assisted extraction is a “green technology” that has the advantages of accelerating extraction efficiency, saving energy, and being environmentally friendly [58]. The ultrasound-assisted technique intensifies the disruption of cell walls and greatly promotes the release of AC, SDA, and SDB into the extraction solvent. Therefore, this method not only significantly reduces the cost of raw materials but also achieves the goals of environmental protection, high efficiency, and hierarchical extraction. In addition, it improves the bioavailability and accessibility of Schisandra chinensis. The efficient and environmentally friendly development and utilization of these bioactive components can realize the comprehensive utilization of medicinal and food products. It can also be used to recover active ingredients from agricultural waste and then conduct comprehensive utilization and the development of new ideas, thus achieving the full utilization of resources.

However, ionic liquids have unique advantages, such as good stability, nonvolatility, designable structure, and tunable properties [18]. It has shown good results in the field of natural product extraction and is also widely used in various fields. Research on the extraction and separation of ionic liquids has made some progress. However, the promotion of ionic liquids is still somewhat difficult [59]. The economic benefits of using ionic liquids in natural extraction processes are challenging, which is a solid barrier to the commercialization of this technology. Currently, fewer manufacturers sell reagent-grade ionic liquids, and the prices are high. Ionic liquids prepared by people are somewhat difficult to purify and therefore costly [60]. Therefore, the application of ionic liquids for natural product separation should be carried out in terms of cost reduction and easy recovery [61]. The production costs decrease as the demand for ionic liquids increases, and these costs are likely to decrease further [62]. Several current methods for recovering ionic liquids include distillation, extraction, adsorption, membrane separation, and aqueous solutions [63]. The recovery rate of ionic liquids is approximately 90 %. [36]. These conditions are believed to gradually improve as people continue to study ionic liquids. If these economic issues are resolved, the potential for large-scale applications of ionic liquid extraction methods will be enormous [64]. The application of ionic liquids in the extraction of natural products is bound to become more mature, efficient, and perfect and is also bound to become an important research and development direction in the field of scientific research.

4. Conclusions

In this work, a hierarchical extraction method of AC, SDA, and SDB from the essential oil residue of Schisandra chinensis extract was established through SSFIPU (Solid-State Fermentation with Ionic Liquid Pretreatment and Ultrasound-Assisted Extraction) and EIPU (Enzymatic Hydrolysis with Ionic Liquid Pretreatment and Ultrasound-Assisted Extraction). Via single-factor experiments and Box–Behnken experimental design, the optimal conditions obtained were as follows: for AC extraction, 4 mol/L [BMIM]BF₄, 7 % carbon, 5 % nitrogen, a material–reagent ratio of 1:3 g/mL, a bacterial solution concentration of 1 × 10⁶ cfu/mL, a pH of 4.0, and a fermentation period of 4 days, an ultrasound time of 30 min, and an ultrasound power of 350 W, with an AC yield of 1.173 mg/g. For SDA and SDB extraction, 4.2 mol/L [BMIM]BF₄, a cellulase content of 20 %, an enzyme hydrolysis time of 2.5 h, a material–reagent ratio of 1:13 g/mL, an enzyme hydrolysis pH of 5, an ultrasound time of 30 min, and an ultrasound power of 450 W, with SDA and SDB yields of 0.306 mg/g and 0.260 mg/g respectively. Compared with the ethanol extraction method, these two methods achieved significantly higher extraction rates, which were 23.46, 8.5, and 6.8 times higher than the ethanol reflux extraction (ERE). Additionally, the separation of the extract from the ionic liquid was accomplished by macroporous resin, and [BMIM]BF₄ was recovered through dehydration and evaporation, with a recovery rate of 91.21 %, leading to favorable cost savings of ionic liquids. In conclusion, this study offers valuable scientific support for the recovery of active components from plant residues.

CRediT authorship contribution statement

Jingwei Hao: Writing – review & editing, Writing – original draft, Project administration, Methodology, Investigation, Data curation. Yingying Pei: Writing – original draft, Software, Investigation, Formal analysis, Data curation. Nan Dong: Validation, Formal analysis. Yifan Sun: Validation, Formal analysis. Yi Zhou: Validation, Data curation. Qiuxuan Li: Validation, Software. Heming Liu: Validation, Formal analysis.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.