1. Introduction
Cembratriene-diols (CBT-diols) are cembranoid diterpenes with complex molecular structures [
1] that were first extracted from burley tobacco leaves using traditional organic liquid–liquid extraction [
2,
3]. The parent skeleton is a fourteen-membered macrocycle composed of four isoprene units connected end-to-end [
3]. CBT-diols are important aroma precursors that can be degraded to produce aroma components, such as solanone, solanifuran, and ketamine, which are key contributors to the aroma of tobacco [
2]. Notably, CBT-diols have extremely high biological activity and are increasingly used in medicine and healthcare [
4,
5,
6]. Therefore, the investigation of the biological activities of CBT-diols has recently become popular.
Tobacco (
Nicotiana tabacum L.) is an important cash crop belonging to the
Solanaceae family, which is distributed worldwide. Many chemical components are present in tobacco, and 5229 were confirmed [
2]. Tobacco plants have well-developed glandular hairs and other secretory tissues in their epidermis, which produce many adhesive secretions during growth; CBT-diols are mainly present in these glandular hair secretions [
2]. Additionally, recent research found that the content of CBT-diols in tobacco flowers is four to seven times higher than that in fresh tobacco leaves [
2,
7]. To date, traditional liquid–organic solvent extraction methods have been widely used for the effective extraction of CBT-diols from tobacco plants [
2,
8]. However, to minimize environmental pollution, new solvents are required to replace the organic solvents used for such extraction processes.
Natural deep eutectic solvents (DESs), also known as green eutectic solvents, are composed of natural compounds, for example, primary metabolites such as amino acids [
9,
10]. Owing to their unique physicochemical properties, DESs are considered a third liquid phase naturally present in organisms, independent of water and lipids [
11,
12]. Depending on the compounds used in their synthesis, DESs can be divided into five categories: ionic liquid, neutral, neutral acid, neutral base, and amino acid types [
12,
13]. Currently, choline chloride is the most widely used hydrogen-bond acceptors (HBAs) in DES systems and can form DESs with various hydrogen-bond donors (HBDs), such as urea, alcohols, carboxylic acids, and sugars [
13,
14]. The nontoxic, biodegradable, reusable, and green advantages of DESs make them popular for the extraction of active compounds [
15,
16]. For example, flavonoids [
17] and phenolic acids [
18] have been successfully extracted from different plant materials using DESs. Therefore, DESs are expected to replace traditional organic solvents and resolve many problems hindering the extraction of natural compounds.
In particular, microwave-assisted DES extraction can provide a safe, clean, and green extraction technique. Thus, the present study is the first to report on the extraction of CBT-diols from waste tobacco flowers using DESs in combination with microwaves. In particular, different DESs combined with microwave heating were used in this study to extract CBT-diols from tobacco flower waste, and the optimal conditions for the microwave-assisted extraction of CBT-diols from waste tobacco flowers were determined using single-factor experiments and a response surface methodology design. To demonstrate the biological activity of the CBT-diols extracted using DESs, their antimicrobial and antitumor activities were studied.
3. Materials and Methods
3.1. Materials and Reagents
Fresh waste tobacco flowers (N. tabacum L., cultivated variety NC55) (WTFs) were collected from the Xinxing Experimental Station (Zhucheng City, Shandong Province, China; 36.048° N, 119.558° E). The WTFs were fully freeze-dried in a vacuum freeze-dryer (LGJ-10N/A, Beijing Yaxing Yike Technology Development Co., Ltd., Beijing, China) at −50 °C for 48 h. The dried WTFs were crushed into 40-mesh powder and then stored hermetically in a refrigerator at 4 °C before the extraction and quantification of the CBT-diols. Lactic acid, citric acid, glycerol, urea, d-(+)-glucose, tartaric acid, and choline chloride were obtained from the Aladdin Reagent Company (Shanghai, China). Acetonitrile for high-performance liquid chromatography (HPLC) analysis was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). The other reagents and chemicals used in this study were of analytical grade and obtained from China National Medicines Co., Ltd. (Beijing, China).
3.2. Preparation and Characterization of DESs
All DESs used in this study were synthesized using the heating and mixing method described previously [
13,
41]. The components were added to capped flasks according to the molar ratios listed in
Table 4, and a transparent homogeneous DES liquid was obtained by magnetic stirring in a water bath at 80 °C and 100 r/min for 3 h. Then, deionized water was added to the DES system to adjust the water content to 30% (
w/
w), and the samples were denoted DES-1 to DES-7. DESs diluted with water were used to extract CBT-diols from WTFs and to determine their viscosity and density.
3.3. Screening of DESs for the Microwave-Assisted Extraction of CBT-Diols from WTFs
To obtain the best DES system for the microwave-assisted extraction of CBT-diols from WTFs, the extraction capacities of the seven different DESs prepared in this study were evaluated by extracting WTF powder (100 mg) using 1 mL of DESs as the extraction solvent. The extraction processes were performed in triplicate at 40 °C for 20 min at a microwave power of 300 W. The yields of CBT-diols extracted from WTFs were used to compare the extraction efficiencies of the DESs with those of conventional solvents (80% methanol, 80% ethanol, and 80% ethyl acetate) [
13,
42].
3.4. Quantification of CBT-Diols Extracted from WTFs Using HPLC-UV Analysis
Quantitative analyses of the
α-CBT-diols and
β-CBT-diols isolated from WTFs were performed using ultra-HPLC (UPLC; H-CLASS, Waters, MA, USA). The chromatographic column was an Acquity UPLC BEH C18 column (1.7 μm, 50 mm × 2.1 mm). The mobile phase A and B were acetonitrile and pure water, respectively. The isogradient elution procedure was as follows: 0–8 min, 50% A and 50% B. The column temperature, injection volume, flow rate, and detection wavelength were 40 °C, 5 μL, 0.3 mL/min, and 200 nm, respectively. The chromatograms of
α-CBT-diols and
β-CBT-diols are shown in
Figure 6. The calibration curve equation used for
α-CBT-diols and
β-CBT-diols determination were y = 20910x − 21414 (r
2 = 0.9997, n = 8, with a linear range of 10–1000 μg/mL), and y = 15297x − 14940 (r
2 = 0.9996, n = 8, with a linear range of 10–1000 μg/mL), respectively.
3.5. Single-Factor Optimization for the Extraction of CBT-Diols from WTFs
The single-factor experiments were designed and conducted to improve the efficiency of CBT-diols extraction from WTFs using DESs. Microwave power (200, 300, 400, 500, and 600 W), microwave temperature (30, 40, 50, 60, and 70 °C), microwave time (10, 20, 30, 40, and 50 min), and solid/liquid ratio (10, 20, 30, 40, and 50 mg/mL) were selected as the investigation factors. Based on the different parameters, CBT-diols were extracted from WTFs using DESs according to the methods described in
Section 3.3. Single-factor experiments were performed using the extraction yields of CBT-diols as an optimization index.
3.6. Response Surface Methodology Optimization of the Extraction of CBT-Diols from WTFs
Based on the results of the previous single-factor experiments, Box–Behnken design (BBD) was applied to select suitable levels of each factor for response surface methodology (RSM) optimization using the central composite design principle. The four main influencing factors, microwave power, microwave temperature, solid/liquid ratio, and microwave time, were selected as independent variables. The final factor levels and coding values are presented in
Table 5. After microwave extraction, the yield of CBT-diols isolated from the WTFs using DESs was used as the response value. The prediction model for the microwave-assisted extraction and correlation analysis of the responses and independent factors was developed using Design Expert Ver. 8.0 (Stat-Ease Inc., Minneapolis, MN, USA). Three-dimensional (3D) response surface plots were used to reveal the interactions between the various variables visually.
3.7. Recovery of CBT-Diols from DES Extracts Using Macroporous Resins
To recover the CBT-diols from the DES after extraction, six different macroporous resins (HPD-500, S-8, HPD-300, AB-8, D101, and X-5, Tianjin Yunkai Resin Technology Co., Ltd., Tianjin, China) were selected as separation and purification systems (
Table 6). The pretreated macroporous resins (20 g) were added into a 100 mL syringe, and then 10 mL of the DES extract was added. Subsequently, the following elution procedure was used: thorough washing with 100 mL of deionized water (a flow rate of 2 mL/min) and elution with 50 mL of 50% ethyl acetate and 50 mL of 100% ethyl acetate (a flow rate of 1 mL/min). The ethyl acetate elution phases were entirely collected and dried using vacuum rotary evaporation. A crude extract of CBT-diols from the DES system was obtained and used for further analysis of biological activity. The recovery efficiency (%) of CBT-diols was calculated using the following equation:
where
Cea is the concentration of CBT-diols in the ethyl acetate elution phase,
Vea is the total volume of the ethyl acetate elution phase,
Ce is the concentration of CBT-diols in the DES extract, and
Ve is the volume of the DES extract.
3.8. Evaluation of In Vitro Bioactivity of CBT-Diols Extracted from WTFs Using DESs
3.8.1. Evaluation of Antimicrobial Activity
The inhibitory effects of CBT-diols from WTFs by using DESs on
Salmonella (CMCCBC2184B),
Staphylococcus aureus (CMCCBC03068),
Bacillus subtilis (CMCCB63501),
Escherichia coli (CMCCBC00148), and
Pseudomonas aeruginosa (CMCCBC0055B) were evaluated using filter paper diffusion methods, as described by Shang et al. [
42]. These five bacteria were inoculated into Luria–Bertani (LB) agar medium, respectively, and cultured continuously at 37 °C for 12 h. A single bacterial colony was then selected and placed in 5 mL of liquid LB medium. The bacterial solution was incubated with oscillation until the optical density at 600 nm (OD
600) was 0.6, and then it was stored at 4 °C. Under sterile conditions, a total of 100 μL of the bacterial suspension was uniformly coated on the surface of the LB agar medium. Sterile blank drug-sensitive tablets were soaked in CBT-diols dissolved in dimethylsulfoxide (DMSO, 150 μg/mL) for 30 s, dried slightly, and then attached to the surface of the medium. Drug-sensitive tablets containing penicillin–streptomycin solution (1000 U/mL) and DMSO solution were used as positive and solvent controls, respectively. All plates were incubated upside down at 37 °C for 24 h, and each treatment was repeated three times in parallel. Finally, the diameter of the inhibition zone was determined and used as an evaluation index for the antimicrobial activity of the CBT-diols.
3.8.2. Evaluation of Antitumor Activity
The inhibitory effects of the isolated CBT-diols on the human liver cancer HepG2 and SMMC-7721 cell lines (Cell Bank of Chinese Academy of Sciences, Qingdao, China) in vitro were also determined using the colorimetric MTT assay (Shanghai Yuanxin Biotechnology Co., Ltd., Shanghai, China) [
35,
37]. The cell suspension was transferred into a culture bottle filled with complete culture medium, fully shaken, and then placed in a cell incubator (37 °C) containing 5% CO
2 for incubation. DMSO solutions having different concentrations of CBT-diols (80, 40, 20, 10, 5, 2.5, and 1.25 mg/L) were used to detect the inhibition rate of the HepG2 and SMMC-7721 cells at 24, 48, and 72 h. Cancer cell culture media without CBT-diols were used as negative controls.
3.9. Statistical Analysis
Statistical analysis was conducted by analysis of variance (ANOVA) using SPSS 24.0 (Chicago, IL, USA). Means were compared by the Duncan test at a 95% confidence level. The experimental analysis was performed in triplicate, and data are represented as mean ± standard deviation.
4. Conclusions
The findings of this study demonstrate the feasibility of recovering CBT-diols from WTFs using DESs and the potential applications of CBT-diol-rich DES extracts. Among the tested DESs, DES-3, composed of choline chloride and lactic acid (molar ratio of 1:3), showed the highest CBT-diol extraction efficiency compared to the conventional organic solvents (80% methanol, 80% ethanol, and 80% ethyl acetate). The optimal conditions for microwave-assisted DES extraction were determined using RSM to be a microwave power of 425 W, microwave time of 32 min, microwave temperature of 40 °C, and solid/liquid ratio of 20 mg/mL. The yield of CBT-diols was 6.23 ± 0.15 mg/g under these conditions. The recovery of CBT-diols from the DES extraction systems was also investigated, and the macroporous AB-8 resin was selected as the most suitable separation resin. Furthermore, the antibacterial and antitumor activities of CBT-diols were evaluated in vitro. The isolated CBT-diols exhibited inhibitory effects against Salmonella, S. aureus, E. coli, P. aeruginosa, and B. subtilis also showed dose-dependent cytotoxic effects on HepG2 and SMMC-7721 human liver cancer cell lines. These results suggest that CBT-diols are promising antitumor agents and their mechanism of action warrants further investigation.