How To Seperate Acteoside From Cistanche Tubulosa

Helin Xu a, Xueqin Li a,⇑ , Yanyan Hao a, Xiaobin Zhao a, Yun Cheng a, Jinli Zhang a,b

a School of Chemistry and Chemical Engineering/Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, Shihezi University, Shihezi 832003, China

b Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

Contact: joanna.jia@wecistanche.com / WhatsApp: 008618081934791

Abstract

To achieve simple and efficient separation of acteoside (ACT), extraction of ACT from crude extracts of Cistanche tubulosa via ionic liquid (IL) based on an aqueous two-phase system (ATPS) is investigated. The IL–ATPS comprising [C4mim]BF4 and (NH4)2SO4 exhibits excellent extraction performance, extracting almost all ACT (Acteoside) in the [C4mim]BF4-rich phase. The high polarity of [C4mim]BF4 having multi-hydrogen bond receptors is conducive to extracting ACT (Acteoside) in the IL-rich phase, and (NH4)2SO4 not only provides a weak acidic microenvironment for increasing ACT stability in the IL-rich phase but also reduces the solubility of ACT (Acteoside) in the salt-rich phase via the salting-out effect. In addition, molecular simulation results show that is extracted in the [C4mim]BF4-rich phase via multi-interactions, including hydrogen bonding, van der Waals forces, and p–p stacking. This study is expected to provide a valuable reference for the separation of bioactive constituents from natural products.

Keywords: Ionic liquids, Salt, Aqueous two-phase system, Extraction, Acteoside

Acteoside in Cistanche tubulosa

Introduction

Cistanche tubulosa (Orobanchaceae) is a parasitic plant that grows on the roots of Tamarix and Salvadora and is mainly distributed in the arid lands and deserts of northwestern China [1–3]. The stems of Cistanche tubulosa, known as 'ginseng of the deserts', are officially recorded in the Chinese Pharmacopoeia (2015 edition) [4,5], and their main components have been reported to be phenylethanoid glycosides (PhG), polysaccharides, and iridoids.

PhG compounds, a group of water-soluble bioactive constituents in Cistanche tubulosa, possess a common structure composed of cinnamic acid and hydroxyphenylethyl moieties that are attached to a b-glucopyranose group through ester and glycosidic linkages, respectively [6]. Acteoside (ACT) is the main component of PhG compounds and is the index component for the content determination of Cistanche tubulosa [7]. Modern chemical and pharmacological studies have demonstrated that ACT (Acteoside) exhibits multiple pharmacological activities, such as antioxidation, antifatigue, neuroprotection, and hepatoprotection [8–10]. Thus, ACT has a broad application prospect in the fields of medicine, health care, food, etc. However, the separation and purification of ACT (Acteoside) are difficult due to the complex composition of PhG compounds, which have a similar structure, polarity, and solubility. At present, the main methods for the separation and purification of ACT include molecular imprinting, macroporous adsorption resin separation, high–speed countercurrent chromatography, and membrane separation. For example, Liu et al used macroporous resins to separate PhG from Cistanche deserticola, and Xu et al. used mesoporous carbon adsorbents to adsorb PhG, and the adsorption capacity reached 358.09 mg/g. However, these methods have numerous disadvantages, such as time consumption, complex procedure, and low selectivity. Therefore, the development of a simple and effective method for the selective separation of bioactive constituents from natural products is highly desired.

micronized purified flavonoid fraction

In recent years, the aqueous two-phase system (ATPS), which can be performed in a mild environment and is easily scalable [11–15], has emerged as a novel liquid-liquid extraction technique for the separation of bioactive constituents [16–19]. According to its composition, ATPS can be divided into three types, i.e., two or more polymers, a polymer and a salt, or two surfactants [20–23]. However, almost all phase-forming polymers have high viscosity and narrow polarity range that are not conducive to mass transfer and limit selectivity. In this context, ionic liquids (ILs) are characterized by negligible volatility, chemical stability, comprehensive solubility for organic compounds, and most ILs are environment-friendly [24–28]. These features render IL-APTSs with better performance than other ATPSs for the separation of bioactive constituents. Compared to the traditional ATPS, the IL-ATPS gathers the advantages of both IL and ATPS, such as quick phase separation, high extraction performance, and environmental biocompatibility [12,29,30]. At present, IL-ATPS has not been applied to the separation and purification of ACT (Acteoside), but it has been determined that IL-ATPS can be applied in the extraction and separation of polysaccharides [31], vanillin [32], aloe polysaccharides [33], ginseng saponins [11], etc. For example, He et al. extracted bioactive ginseng saponins using IL-ATPS, which was demonstrated as a simple and effective method for the extraction of ginsenosides with a high extraction efficiency of 99.5% and a partition coefficient of 651. Gao et al. extracted astaxanthin in the [P4448]Br-rich phase using an ILATPS comprising [P4448]Br and K3PO4 with an extraction efficiency of up to 93.08% [34]. These results show that IL-ATPS has potential application prospects for separating bioactive constituents from natural products, and it can be expected to achieve highly selective separation of ACT from Cistanche tubulosa.

In this study, a simple, efficient, and green technique based on-ATPS extraction for the highly selective separation of ACT (Acteoside) from crude extracts of Cistanche tubulosa is explored. Different ILs([C4mim][CF3SO3], [C4mim]Cl, [C4mim]BF4, [C2mim]Br, [C4mim]Br, [C6mim]Br, and [C8mim]Br) and salt types (NaCl, Na2SO4,Na3C6H5O7, (NH4)2SO4, and NaH2PO4) were investigated to determine the optimal IL-ATPS for extracting ACT (Acteoside). Moreover, the extraction conditions were adjusted to achieve highly selective separation of ACT in the IL-rich phase. The effect of different extraction conditions (e.g., extraction temperature, IL concentration, sample solution concentration, salt concentration, pH, and extraction equilibrium time) on the ACT extraction performance is discussed in detail. Furthermore, molecular simulations were conducted on the ACT, [C4mim]BF4, and H2O to reveal the extraction mechanism.

cistanche tubulosa vs deserticola

2. Experimental

2.1. Materials

The stems of Cistanche tubulosa were supplied by Cong Rongtang Biological Technology Co., Ltd. (Xinjiang, China). The standards of ACT (Acteoside) (purity - 98%, Fig. 1a) and echinacoside (purity - 98%, Fig. 1b) were supplied by Sunny Biotech Co., Ltd. (Shanghai, China). 1-Butyl-3-methylimidazolium trifluoro methanesulfonate ([C4mim][CF3SO3]), 1-butyl-3-methylimidazolium chloride ([C4mim]Cl), 1-butyl-3-methylimidazolium tetrafluoroborate ([C4mim]BF4), 1-ethyl-3-methylimidazolium bromide ([C2mim]Br), 1-butyl-3-methylimidazolium bromide ([C4mim]Br), 1-hexyl-3-methylimidazolium bromide ([C6mim]Br), and 1-octyl-3-methylimidazolium bromide ([C8mim]Br) were supplied by Chengjie Chemical Co., Ltd. (Shanghai, China). Analytical grade ammonium sulfate ((NH4)2SO4), sodium dihydrogen phosphate (NaH2PO4), sodium sulfate (Na2SO4), dibasic sodium phosphate (Na2HPO4), dipotassium hydrogen phosphate (K2HPO4), sodium chloride (NaCl), and trisodium citrate anhydrous (Na3C6H5O7) were supplied by Shengao Chemical Reagent Co., Ltd. (Tianjin, China). Methanol, acetic acid, and acetonitrile of chromatographic grade were supplied by Aladdin Chemical Co., Ltd. (Shanghai, China). Ethanol of analytical grade was supplied by Fuyu Fine Chemical Co., Ltd. (Tianjin, China).

Fig. 1. Chemical structures of (a) acteoside (ACT), and (b) enchinacoside.

2.2. Preparation of crude extracts of Cistanche tubulosa

A powdered sample of Cistanche tubulosa (20 mg) was soaked in500 mL of a 50% ethanol solution with ultrasound for 2 h. The residue in the solution was separated by centrifugation at a speed of5000 rpm for 10 min. The supernatant after centrifugation was sequentially filtered using 0.45 and 0.22 lm filters. Then, the filtrate was concentrated and dried at 323 K in a vacuum oven. Finally, the dried product was dissolved in water, and a reddish-brown extract of Cistanche tubulosa was obtained.

2.3. Extraction of ACT (Acteoside) by IL-ATPS

A schematic for the efficient extraction of ACT (Acteoside) by IL-ATPS is shown in Fig. 2. Accordingly, 3.75 mL [C4mim]BF4, 3.5 mL distilled water, 11.11 wt% salt, and 1.0 mL sample solution with the same concentration of ACT and echinacoside were added to a 50 ml centrifuge tube. Another IL-ATPS with the same components except for the sample solution was prepared as a contrast system. The salt of IL-ATPS was completely dissolved by ultrasonication, and the formation of two clear phases was accelerated by centrifugation at 8000 rpm for 8 min. The bottom phase of IL-ATPS was the IL-rich phase, and the top phase was the salt-rich phase. The components of both phases were quantitatively analyzed by high-performance liquid chromatography (HPLC).

The concentration of ACT (Acteoside) and echinacoside in the two phases was determined by HPLC, and the extraction performance was evaluated by the extraction efficiency (E, %), partition coeffificient (K), and selectivity (S). The following Eq. (1) equation was used to calculate K

K=Cb/Ct (1)

where Cb and Ct (mg/mL) is the ACT (Acteoside) concentration in the bottom and top phases, respectively. Eqs. (2) and (3) were used to calculate E and S, respectively.

E=Cb*Vb/Mv*100% (2)

S = K/Kt = Cb*Cet / Ct*Ceb (3)

where mv (mg) is the weight of ACT (Acteoside) in the sample solution, Vb (mL) is the volume in the bottom phase, and Cet and Ceb echinacoside concentrations in the top and bottom phases (mg/mL), respectively.

Fig. 2. Schematic for the efficient extraction of ACT by IL-ATPS

2.4. Selective experiments

ACT (Acteoside) and echinacoside are considered competitors for evaluating the selectivity of IL-ATPS to ACT (Acteoside) due to the similar molecular structure and chemical properties of ACT (Acteoside) and echinacoside. 3.75 mL[C4mim]BF4, 3.5 mL distilled water, 11.11 wt% salt, and 1.0 ml sample solution with the same concentration of ACT and echinacoside were added to a 50 ml centrifuge tube. The salt of IL-ATPSwas completely dissolved by ultrasonication, and the formation of two clear phases was accelerated by centrifugation at8000 rpm for 8 min. The bottom phase of IL-ATPS was the IL-rich phase, and the top phase was the salt-rich phase. The components of both phases were quantitatively analyzed by HPLC.

2.5. HPLC analysis

The concentration of the sample solution in the two phases was analyzed by HPLC (2695, Waters Co., USA) with a C18 column at 330 nm using an ultraviolet detector. The gradient elution method was adopted to separate and detect samples at a column temperature of 303 K. The mobile phases were (a) acetonitrile and (b) acetic acid/water (1:44, v/v), and the flow rate was set to 1 mL/min. The mobile phase and samples were filtered through a 0.22 lm filter before injection. All experiments were performed three times, and the error bar is given in all figures.

2.6. Characterization

ACT, [C4mim]BF4, and the [C4mim]BF4-rich phase were analyzed by Fourier transform infrared spectroscopy (FTIR) using Bruker Vertex 70 (Bruker Optics, Ltd., Germany). The FTIR spectra ofACT, [C4mim]BF4, and the [C4mim]BF4-rich phase were obtained from 4000 to 500 cm 1 with a resolution of 2 cm 1 using potassium bromide as the reference.

2.7. Molecular simulation

To obtain insights into the extraction mechanism of IL-ATPS as well as the interactions modes between ACT and [C4mim]BF4 in ILATPS, first-principle calculations were conducted based on the density functional theory (DFT). Optimizations and single-point energy calculations were performed using the M06–2X functional [35,36], and the basic set 6–31 + G (d) was used to describe C, H, F, B, N, and O atoms [37]. To accurately simulate weak interactions, the DFT– D3 method was used for the adequate description of dispersive interactions [38]. According to previous reports [39], molecular simulations were conducted on the ACT, [C4mim]BF4, and H2O. First, the individual conformations of ACT and [C4mim]BF4 models were optimized, and then the binding conformation of [C4mim]BF4 and ACT was optimized based on their optimal individual configuration.

The binding energies (DE) of ACT (Acteoside) and [C4mim]BF4 and binding energies (DE) of ACT and H2O were evaluated using Eqs. (4) and (5), respectively.

△E = Ecom - (EACT + EIL)
△E = Ecom - (EH2O + EACT)

where EH2O, EACT, EIL, and Ecom are the potential energies of ACT (Acteoside), [C4mim]BF4, H2O, and their complexes, respectively. All the calculations were performed using the Gaussian16 program [40], and all molecules were drawn using the VMD v.1.9.3 software. Meanwhile, an independent gradient model (IGM) was established to identify and quantify the net electron density gradient attenuation in Multiwfn v.3.5 software.

Acteoside in Cistanche tubulosa

3. Results and discussion

3.1. Optimization of IL-ATPS

3.1.1. Screening of ILs

To evaluate the effect of the type of IL on the extraction performance in IL–ATPS, seven kinds of ILs having different cations and anions were selected as phase-forming components for extracting ACT (Acteoside). As shown in Fig. 3 (a), the interaction between ACT and IL anions was investigated by examining the combination of [C4mim]+ as cation with different anions including Br-, Cl-, [CF3SO3] -, and BF4-. The results showed that the IL anions enhanced the ACT (Acteoside) extraction performance in the order BF4- > [CF3SO3] - > Br- > Cl- . The optimal IL anion was BF4 , which can be ascribed to the fact that the corresponding IL can provide more hydrogen bond receptors for the hydroxyl groups of ACT (Acteoside) than other IL anions [21]. This suggests that hydrogen bonding is the main interaction between IL anions and ACT.

Next, the combination of Br with different cations including[C2mim]+, [C4mim]+, [C6mim]+, and [C8mim]+ was investigated to explore the optimal steric configuration between ACT (Acteoside) and the ILscations. The results showed that the ACT (Acteoside) extraction performance increased with decreasing alkyl chain length from [C8mim]+ to[C2mim]+. Since [C2mim]+ did not form the corresponding-ATPS with BF4 , [C4mim]+ was selected as the optimal IL cation

This can be attributed to the high polarity of the short alkyl chain of [C4mim]+, which was beneficial for dissolving ACT (Acteoside) and extracting it in the IL–rich phase. In addition, the imidazole cation of [C4mim]+ has an aromatic p system, which can form p–p interactions with the benzene ring of ACT to achieve the optimal stereo configuration. Therefore, [C4mim]BF4 was chosen as the optimal for the following experiments.

3.1.2. Screening of salts

To evaluate the effect of the salt type on ACT (Acteoside) extraction performance, five kinds of salts (NaH2PO4, (NH4)2SO4, Na2SO4, NaCl, and Na3C6H5O7) were selected to form an IL-ATPSs with [C4mim]BF4, and the results are shown in Fig. 3 (b). The salt can be divided into acid (NaH2PO4 and (NH4)2SO4), neutral (Na2SO4, and NaCl), and basic (Na3C6H5O7) salts. It can be seen from Fig. 3 (b) shows that (NH4)2SO4 affords an IL-ATPS with excellent ACT extraction performance. The result indicates that the difference in extraction efficiency, partition coeffificient, and selectivity is associated with the stability of ACT (Acteoside) in the different extraction microenvironments.

Fig. 3. Screening of ATPSs. (a) Types of ILs, and (b) types of salts

The glycosides of acidic phenols of ACT are stable in weak acidic systems. Since (NH4)2SO4 is an acidic salt, it can provide a weak acidic microenvironment for IL-ATPSs. This is conducive to increasing the extraction efficiency and maintaining the stability of ACT (Acteoside) in the IL-rich phase. In addition, the salting-out ability of different types of salt was different from that of (NH4)2SO4 was stronger than that of NaH2PO4 [24,41]. The (NH4)2SO4 salt competes against ACT (Acteoside) for H2O molecules in the salt-rich phase, thus reducing the solubility of ACT in the salt-rich phase, and the ACT can be effectively extracted in the IL-rich phase. Thus, (NH4)2SO4 was chosen as the optimal salt in the following experiments.

3.2. Optimization of extraction conditions in IL–ATPS

3.2.1. Effect of extraction temperature

According to previous studies [42], extraction temperature has a significant impact on the extraction performance for bioactive constituents in IL-ATPS, low temperature is more beneficial for the formation of IL-ATPS. Fig. 4 (a) presents the effect of [C4mim] BF4-(NH4)2SO4 as IL-ATPS with an extraction temperature in the range of 278–318 K on the extraction performance of ACT (Acteoside). Fig. 4 (a) shows that the extraction efficiency, partition coefficient, and selectivity decreased with increasing the extraction temperature, indicating that a high-temperature condition was unsuitable for ACT extraction in IL–ATPS.

The reason mainly attributed to that the salting-out effect was weakened, and it resulted from the water in the IL-rich phase being transferred to the salt-rich phase in IL-ATPS at high extraction temperatures [42]. In addition, the experiments study showed that high extraction temperature was not conducive to the stable existence of ACT (Acteoside). Thus the optimal extraction temperature was established to be 278 K

3.2.2. Effect of [C4mim]BF4 concentration

Fig. 4 (b) exhibits the effect of [C4mim]BF4 concentration from 20 to 60 wt% on the extraction performance for ACT (Acteoside) in IL-ATPS. As shown in Fig. 4 (b), the ACT extraction performance increased obviously when the [C4mim]BF4 concentration increased from 20 to 50 wt%. However, the extraction performance slightly decreased when the [C4mim]BF4 concentration was increased from 50 to 60 wt%. Thus, a high [C4mim]BF4 concentration was conducive to extracting ACT in the [C4mim]BF4-rich phase, but excessive [C4mim]BF4 concentration above 50 wt% resulted in a decrease in the ACT extraction performance.

This is most likely due to the increase of the solubility of ACT in the [C4mim]BF4-rich phase at high [C4mim]BF4 concentration owing to the high affinity between ACT and [C4mim]BF4. Thus, ACT was easily extracted in the [C4mim]BF4-rich phase by increasing the [C4mim]BF4 concentration. However, above a [C4mim]BF4concentration of 50 wt%, the viscosity of the IL-ATPS increased, resulting in poor mass transfer for extraction. Thus, the optimal[C4mim]BF4 concentration for extracting ACT was 50 wt%.

3.2.3. Effect of sample solution concentration

The sample solution concentration was an important condition for ACT (Acteoside) extraction. Fig. 4 (c) exhibits the effect of the sample solution concentration on the extraction efficiency, partition coefficient, and selectivity, which can reflect the migration ability of ACT between the top and bottom phases. Fig. 4 (c) shows that the extraction efficiency, partition coefficient, and selectivity of ACT (Acteoside) increased with increasing the sample solution concentration from 0.5 to 2.5 mg/mL. However, the ACT extraction performance slightly decreased with a further increase of the sample solution concentration from 2.5 to 3.0 mg/mL. A high sample solution concentration promoted the solubility of ACT (Acteoside) in the [C4mim]BF4-rich phase due to the strong affinity between ACT and [C4mim]BF4.

The decrease in extraction performance can be attributed to the following reasons: On one hand, an excessive increase in the sample solution concentration did not improve the extraction performance of IL-ATPS when the [C4mim]BF4-rich phase was already saturated in the ACT. On the other hand, an increase in the sample solution concentration caused an enhancement of intramolecular hydrogen bonding and aggregation of ACT (Acteoside) molecules, which was not conducive to mass transfer. Thus, the optimal sample solution concentration was determined to be 2.5 mg/mL.

3.2.4. Effect of (NH4)2SO4 concentration

The effect of different (NH4)2SO4 concentrations on the extraction efficiency, partition coefficient, and selectivity are shown in Fig. 4 (d). The experimental result showed that the three parameters increased with increasing (NH4)2SO4 concentration from 2.22 to 11.11 wt%. However, the ACT extraction performance decreased with a further increase of the sample solution concentration from 11.11 to 16.66 wt%.

This can be attributed to the salting-out effect, which is a key feature for extracting ACT (Acteoside) in the [C4mim]BF4-rich phase. The increase in (NH4)2SO4 concentration can increase the salting-out effect, resulting in a decrease in the solubility of ACT in the(NH4)2SO4–rich phase, which favors ACT extraction in the [C4mim]BF4-rich phase. However, an excessive increase in the(NH4)2SO4 concentration (>11.11 wt%) resulted in a decrease in the water content of the [C4mim]BF4-rich phase, which would weaken its affinity for the ACT. This caused a decrease in extraction efficiency, partition coefficient, and selectivity. Therefore, the optimal (NH4)2SO4 concentration was 11.11 wt%.

3.2.5. Effect of pH

The effect of pH on ACT (Acteoside) extraction efficiency, partition coefficient, and selectivity in IL-ATPS were investigated in the 2.0–7.0 pH range. As observed in Fig. 4 (e), the three extraction parameters increased as the pH increased from 2.0 to 6.0. However, the extraction performance decreased at a pH of 7.0.

On one hand, the glycosides of acidic phenols of ACT (Acteoside)would be stable in an acid environment, while it would be dissociated in alkaline solutions [6]. On the other hand, ACT can undergo ionization according to the equilibrium HA $ H+ + A at different pH values [33], being the nonionized form that tends to enter the IL-rich phase favored under acidic conditions. Thus, the extraction experiment should be performed in a moderately acidic environment at the optimal pH of 6.

3.2.6. Effect of extraction time

The effect of extraction time on the ACT (Acteoside) extraction efficiency, partition coefficient, and selectivity were explored in the range of 10–200 min. As can be seen from Fig. 4 (f), the ACT extraction performance increased with the extraction time, and the maximum extraction efficiency (>99%), partition coeffificient (>35), and selectivity (>225) were obtained at 130 min. In addition, ACT (Acteoside) was found to be stable in a weak acidic microenvironment for a long time. Therefore, 130 min was selected as the optimal extraction time.

3.3. Mechanism of ACT extraction with [C4mim]BF4–(NH4)2SO4 ATPS

3.3.1. FTIR analysis

Fig. 5 shows the FTIR spectra of ACT (Acteoside), [C4mim]BF4, and the [C4mim]BF4-rich phase. As shown in the FTIR spectrum of [C4mim] BF4, the peaks at 3162 and 3122 cm 1 can be attributed to the symmetrical stretching vibration of CAH and the asymmetrical stretching vibration of CAH from the imidazole, respectively. The band at 1012 cm 1 can be assigned to the stretching vibration of BF4-.

In the FTIR spectrum of the [C4mim]BF4-rich phase, no new peaks appeared in comparison to the FTIR spectra of ACT (Acteoside) and [C4mim]BF4. However, the characteristic peak of BF4- at 1012 cm- 1 moved to a higher frequency due to the hydrogen bonding between ACT and the F atoms. Moreover, the characteristic peaks of CAH at 3162 and 3122 cm 1 shifted to lower frequencies. This can be attributed to two reasons: (1) the formation of a hydrogen bonding between ACT and the N atom in [C4mim]+, and (2) the formation of p–p stacking between the benzene ring of ACT and the imidazole ring of [C4mim]+. This result was consistent with the molecular simulation results.

Fig. 5. FTIR spectra of ACT, [C4mim]BF4, and the [C4mim]BF4-rich phase.

3.3.2. Molecular simulation

To explore the extraction mechanism, possible interaction modes between ACT (Acteoside) and [C4min]+, BF4- , and H2O were investigated using molecular simulations. Fig. 6 shows the optimal interaction modes and binding conformation of ACT–[C4min]+, ACT–BF4 , and ACT–H2O. As shown in Fig. 6a–h, ACT and [C4mim]+ had shown two optimal binding conformations, i.e., ACT–[C4mim]+ –1 and ACT–[C4mim]+ –2, ACT and BF4 had one optimal binding conformation, ACT–BF4-, and ACT and H2O had the five optimal binding conformations ACT–H2O–1, ACT–H2O–2, ACT–H2O–3, ACT–H2O–4 and ACT–H2O–5.

Furthermore, the binding energy can be invoked to explain the interactions between two molecules. According to the molecular simulation results, the binding energies of ACT–[C4mim]+–1, ACT–[C4mim]+–2, ACT–BF4 , ACT–H2O–1, ACT–H2O–2, ACT–H2O–3, ACT–H2O–4 and ACT–H2O–5 were - 129.71, - 136.53,-130.82, - 60.78, - 53.00, - 76.76, -66.55, and -53.49 kJ/mol, respectively. These values suggest that the binding energy of with [C4mim]BF4 was stronger than that of H2O molecules. Thus, ACT had high affinity for [C4mim]BF4 and easily entered into the[C4mim]BF4–rich phase.

To further investigate the interactions between the different molecules, the interaction modes of ACT–[C4min]+, ACT–BF4-, and ACT–H2O were illustrated using IGM analysis. As shown in Fig. 6a, and b, the interaction between ACT (Acteoside) and [C4mim]+ mainly comprised hydrogen bonding and van der Waals forces. In addition, ACT–[C4mim]+ –1 and ACT–[C4mim]+ –2 displayed similar conformational characteristics, with the benzene ring of ACT and the imidazole ring of [C4mim]+ being parallel to each other and located at a distance of about 4 Å. This indicates the existence of a p  p stacking interaction between ACT (Acteoside) and [C4mim]+ [43]. Fig. 6c reveals the presence of strong hydrogen bonding and van der Waals interactions between ACT and BF4-. Moreover, as shown in Fig. 6d–h, when ACT was dissolved in H2O, ACT, and H2O molecules were mainly bound through hydrogen bonding and van der Waals. Thus, on the basis of the conformational characteristics and binding energy values, it can be concluded that ACT was extracted in the [C4mim]BF4-rich phase through hydrogen bonding, van der Waals interactions, and p - p stacking.

Fig. 6. IGM analysis of the ACT–[C4min]+, ACT–BF4- , and ACT–H2O interactions: (a) ACT–[C4mim]+–1, (b) ACT–[C4mim]+–2, (c) ACT–BF4 , (d) ACT–H2O–1, (e) ACT–H2O–2, (f)ACT–H2O–3, (g) ACT–H2O–4, and (h) ACT–H2O–5.

3.4. Extraction of crude extract

Under optimal extraction conditions, the IL-ATPS was used to investigate the extraction of crude extract. As shown in Table 1, IL-ATPS can effectively extract ACT (Acteoside) from crude extract of Cistanche tubulosa to the IL-rich phase, and IL-ATPS has almost no change in the extraction performance of ACT (Acteoside) from the crude extract compared with the extraction performance of the standard solution. Therefore, this method can effectively extract ACT (Acteoside) from the crude extract of Cistanche tubulosa, which can provide a reference for the extraction of bioactive constituents in the future.

3.5. Separation of ACT from IL-rich phase

Solvent extraction and adsorption methods for the recovery of ACT (Acteoside) and the reuse of IL are explored in this section. First, organic solvents were considered for back extraction, such as n-butanol (NBA), ethyl acetate (EA), isopentenyl (IPA), and chloroform (CR). However, the recovery efficiency of organic extractants was low, as shown in Fig. 7. Moreover, a large number of volatile organic extractants were used for back extraction, which will affect the ‘‘green” feature of the separation process.

The adsorption method was selected for the separation of the ACT (Acteoside) from the IL-rich phase, such as ordered mesoporous carbon (CMK-3), graphene (GR), and single-wall carbon nanotubes (SWCNT). The IL-rich phase after IL-ATPS extraction was diluted 10 times with deionized water. The recovery efficiencies of the above three adsorbents were compared for ACT under the same adsorption conditions as follows: solid-liquid ratio of 1:1 (mg/mL), 303 K, and 12 h.

As shown in Fig. 7, the recovery of SWCNT was better than that of CMK-3 and GR. In addition, the desorption efficiency of ACT (Acteoside) can reach 95.3% using methanol, acetic acid, and ultra-pure water with a volume ratio of 1:1:8 at 318 K. Therefore, SWCNT can be used for the recovery of ACT from the IL-rich phase.

Fig. 7. The effect of type with different extractants and adsorbents for the separation of ACT (Acteoside) from the IL-rich phase.

4. Conclusions

In this study, a simple and efficient method based on IL-ATPS for the extraction of ACT (Acteoside) from the crude extracts of Cistanche tubulosa was successfully developed. The optimal IL-ATPS was determined to be [C4mim]BF4–(NH4)2SO4, and the optimal extraction conditions were an extraction temperature of 278 K, an IL concentration of 50 wt%, a sample solution concentration of 2.5 mg/mL, a salt concentration of 11.11 wt%, a pH of 6.0, and an extraction equilibrium time of 130 min. Under the optimal extraction conditions, the extraction efficiency, partition coefficient, and selectivity of ACT (Acteoside) were found to be 99.78% ± 0.84%, 33.82 ± 1.82, and230.51 ± 8.26, respectively. Therefore, in conclusion, the [C4mim]BF4-(NH4)2SO4 system offers a simple and efficient method for the highly selective separation of ACT from the crude extracts of Cistanche tubulosa. The acidic salt (NH4)2SO4 provides a weak acidic microenvironment for ensuring the stability of ACT (Acteoside) in the [C4mim]BF4-rich phase. Moreover, the high polarity of [C4mim]BF4 having multi-hydrogen bond receptors is conducive to extracting ACT in the [C4mim]BF4-rich phase. Finally, the ACT and [C4mim]BF4 interactions were shown to comprise hydrogen bonding, van der Waals forces, and p–p stacking. This study provides insights for future research on the highly selective separation of phenolic compounds from natural plants.

CRediT authorship contribution statement

Helin Xu: Conceptualization, Methodology, Investigation, Data curation, Writing - original draft, Writing - review & editing. Xueqin Li: Conceptualization, Project administration, Writing - review& editing, Supervision. Yanyan Hao: Data curation, Writing -review & editing. Xiaobin Zhao: Resources, Formal analysis. YunCheng: Resources, Formal analysis. Jinli Zhang: Project administration, Writing - review & editing.

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.

Acknowledgments

This work was supported by the Regional Science Found of the National Natural Science Foundation of China [grant number22068032]; National Natural Science Foundation for Young Scientists of China [grant number 21706166]; Program for Young andMiddle-aged Scientific and Technological Innovation Leaders inBingtuan [grant number 2019CB024]; the Program for Young Innovative Talents of Shihezi University [grant number CXRC201802]and the Major Science and Technology Project of Xinjiang Bingtuan[grant number 2017AA007/01]. We wish to thank the Analysis andTesting Center of Shihezi University for the microscopy and microanalysis of our specimens.

anthocyanin

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