Evaluation of the Potential of Duckweed as a Human Food, Bioethanol Production Feedstock, and Antileukaemia Drug

Guo, Ling; Liu, Jing; Wang, Qingyan; Yang, Yan; Yang, You; Guo, Qulian; Zhao, Hai; Liu, Wenjun

doi:https://doi.org/10.1155/2023/6065283

Journal of Food Biochemistry

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Abstract Introduction Materials and Methods Results Discussion Conclusion Abbreviations Data Availability Conflicts of Interest Authors’ Contributions Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 6065283 | https://doi.org/10.1155/2023/6065283

Evaluation of the Potential of Duckweed as a Human Food, Bioethanol Production Feedstock, and Antileukaemia Drug

Ling Guo,¹Jing Liu,¹Qingyan Wang,²Yan Yang,¹You Yang,¹Qulian Guo,¹Hai Zhao,³and Wenjun Liu¹

Academic Editor: Rotimi Aluko

Received06 Dec 2022

Revised06 Feb 2023

Accepted17 Mar 2023

Published27 Mar 2023

Abstract

This study evaluated the potential of duckweed as a human food, ethanol feedstock, and anticancer drug. First, the nutritional value of wild duckweed was reported for the first time. Its main composition was similar to that of artificially cultivated duckweed, and thus, wild duckweed can serve as a great human food source. In addition, high-starch duckweed induced by nutrient starvation was fermented into bioethanol. A yield of 0.262 g/g, the highest duckweed-ethanol yield reported thus far, was achieved, indicating that duckweed is an excellent feedstock for ethanol production. Finally, the anticancer effects of duckweed flavonoids (DFs) were assessed for the first time using acute myeloid leukaemia (AML) cells as models in vitro and in vivo. The results revealed that DFs possessed antileukaemia activity and were safe and effective for AML therapy. In conclusion, duckweed was demonstrated to be helpful for humans for food security, energy crisis remediation, and tumour treatment.

1. Introduction

Food security, energy crises, and tumours are the three major challenges that threaten human society worldwide. Studies on novel food alternatives, renewable energy, and anticancer drugs have thus become hotspots in recent years. However, the current research mostly focuses on only one particular field, although investigating a combination of multiple fields may be more promising to solve the abovementioned three problems.

Recently, duckweed, a globally distributed aquatic plant, has received increasing interest due to its intrinsic advantages, such as its rapid growth rate and lack of competition for arable land [1]. First, duckweed has been considered a human food source owing to its high content of protein, beneficial pigments, and sufficient source of essential amino acids recommended by the World Health Organization [2–6]. However, the duckweeds reported in previous studies were artificially cultivated in laboratory medium or semiartificially cultivated in sewage; the nutritional value of wild duckweed has rarely been reported. In our opinion, the scale of artificially cultivated duckweed is far lower than that of wild duckweed worldwide, and wild duckweed is the major source for duckweed biomass production. Thus, the nutritional value of wild duckweed deserves to be systematically studied. In addition, duckweed has been identified as a potential bioenergy crop [7] due to its high starch accumulation capability [8]. Duckweed biomass has been reported as an industrial feedstock for the production of glycerol, 1,3-propanediol [9], succinic acid [10], and especially, bioethanol, an acknowledged alternative to fossil energy [11]. However, the duckweed-ethanol conversion rate is still low, and the fermentation process needs to be further optimized. Finally, duckweed has been used as a folk medicine for a long time in China [12]. It possesses various pharmaceutical activities, such as antibacterial, antifungal, antioxidant, immunomodulatory, and antiadipogenic effects [13]. However, the anticancer effect of duckweed has not been elucidated, and its further investigation is therefore essential.

In this study, the main composition of wild duckweed (mainly Landoltia punctata) was determined, and its nutritional value was systematically evaluated for the first time. In addition, to improve the bioethanol yield, high-starch duckweed biomass induced by nutrient starvation was fermented into ethanol using separate hydrolysis and fermentation. The annual duckweed-to-ethanol yield was estimated according to the annual yield of duckweed biomass. Finally, the anticancer effects of duckweed were identified in vitro and in vivo using acute myeloid leukaemia cells as models, as this disease is a common refractory haematological malignancy in children with ease of relapse.

2. Materials and Methods

2.1. Plant Material and Cultivation

Wild duckweed (Landoltia ≥90%, Spirodela 3–5%, Lemna 2–4%, Wolffia ≤1%, and without Wolffiella) was directly harvested from ponds and paddies across the cities in Sichuan Province. The harvested duckweed materials were rinsed with tap water until the impurities were negligible. A part of the duckweed was used for component determination, and the other parts were cultivated under nutrient deficiency to accumulate starch and flavonoids for subsequent experiments. The cultivation conditions were as follows: the fronds were seeded in tap water in 500 mL plastic beakers (9.0 cm outer diameter × 12.0 cm tall); photoperiod of 16 h/8 h (day/night); light intensity of 130 μM photons/m²/s; temperature of 25°C/20°C (day/night); cultivation time of 10 d; and the sampling time points were at 0, 3, and 10 d. The growth rate (GR) based on dry matter was calculated as follows:where W₀ (g) is the initial dry weight of the sample, W₁ (g) is the final dry weight of the sample (g/m²), s is the cultivation area (m²), and t is cultivation time (d).

2.2. Duckweed Composition

Fresh duckweed and dry duckweed powder were applied to determine the main components of duckweed. First, fresh duckweed samples were dried at 105°C to a constant weight, and moisture was calculated as follows:

The carbon (C%) and nitrogen contents (N%) were determined by an elemental analyser (Elementar Analysensysteme, Vario Macro Cube, C/N mode, TCD, Germany), and the crude protein content was calculated as 6.25 × N%. The duckweed starch content was determined according to the method of Yin et al. [14] with some modification. In brief, 30 mg of dry powder was accurately weighed and transferred to a 10°mL tube. Then, 2.0 mL of distilled water was added. After sonication at 70°C for 30 min, the mixture was centrifuged at 12,000 rpm for 10 min, and the supernatant was discarded. This step was repeated twice. Then, 1.0 mL of distilled water and 1 μL of thermostable α-amylase (Novozymes, China) were added. The mixture was sonicated at 90°C for 45 min. After cooling, 1.0 mL of Na acetate buffer (200 mM, pH 4.8) and 1 μL of glucoamylase (Suhong GAII, Novozymes, China) were added. The mixture was maintained at 60°C for 2 h of hydrolysis and centrifuged for 10 min at 12,000 rpm. Thereafter, the supernatant was filtered and analysed by HPLC (Thermo 2795, Thermo Corp.) with an evaporative light scattering detector (All-Tech ELSD2000, All-tech Corp.). The starch content was determined using the total sugar content (starch content = glucose content × 0.909). The soluble sugar content was measured according to Gao et al. [15]. A dry sample of duckweed (30 mg) was accurately weighed and placed in a 10°mL EP tube. Two millilitres of distilled water was added and the solution was sonicated for 30 min. Then, the mixture was incubated in a water bath at 85°C, and the supernatant was collected by centrifugation. This step was repeated twice, and then distilled water was added to a final volume of 10 mL. The soluble sugar content was determined by sulfuric acid-anthrone colorimetry at 620 nm. Ash and amino acid composition were assayed according to the methods described by Hu et al. [4].

2.3. Duckweed Flavonoid Extraction and Analysis

Duckweed flavonoids (DFs) were extracted according to the methods described by Tsolmon et al. [16] with some modification. In brief, 100 g of duckweed powder was extracted with 2.0 L of 70% (v/v) methanol by three rounds of sonication (30 min each). The mixture was centrifuged at 12,000 rpm. The precipitate was collected and dried in an oven at 60°C for subsequent bioethanol fermentation. The supernatant was combined and concentrated with a rotary evaporator under reduced pressure at 60°C. The concentrate was then lyophilized to a dry powder, 0.1 g of which was accurately weighed and dissolved in 10 mL of 70% methanol. Finally, the solvent was filtered through a PTFE membrane syringe filter (0.22 μm) prior to use. The flavonoid content was analysed by HPLC (AS3000 Auto Sampler, Thermo Electron Corp, USA)-UV (UV6000 DAD Detector, USA) using luteolin-7-O-glucoside as a standard [17]. The mobile phase consisted of methanol (A) and deionized water supplemented with 0.5% (v/v) acetic acid (B). Additional HPLC parameters are as follows: detection wavelength: 326 nm; flow rate: 0.6 mL/min; column: Kromasil 100-5C18 (250 mm × 4.6 mm, 5 μm); column temperature: 35°C; gradient elution procedure: 0–20 min 30–40% A, 20–30 min 40–53% A, 30–40 min 53–90% A, and 40–45 min 90% A.

2.4. Starch Hydrolysis

Duckweed starch was hydrolysed using the two-step enzymatic hydrolysis method described by Perez-Rea et al. [18]. In brief, 19 g of duckweed powder (starch content = 50.8%) was mixed with 110 mL of distilled water in a 250°mL glass Erlenmeyer flask. The mixture was sonicated for 30 min, and 10 μL of liquefaction enzymes (Liquozyme Supra, Novozymes, China) were added. The mixture was incubated at 100°C for 1 h. After adjusting the pH to 4.5, 10 μL of saccharification enzymes (Suhong GAII, Novozymes, China) were added. Then, the mixture was maintained at 60°C for 2 h. Finally, the duckweed mash was autoclaved at 115°C for 20 min. The sterile mash was weighed and stored at room temperature prior to use.

2.5. Ethanol Fermentation

Ethanol fermentation was carried out according to Economou et al. [19]. Saccharomyces cerevisiae (CCTCC M206111) strains (isolated from wine lees in our laboratory) were added to the sterile mash at a 10% v/w ratio and fermented at 30°C for 24 h. Then, 2.5 g of fermentation mash was weighed and diluted with distilled water to a final volume of 25 mL. The suspension was centrifuged at 13,400 × for 10 min, and the supernatant was filtered for ethanol content determination. The ethanol content was determined by gas chromatography (GC, Agilent Technologies 7820A, California, USA) [20].

The ethanol conversion rate (Y_E/G), ethanol fermentation efficiency (%), and ethanol yield based on the duckweed biomass input (Y_E/B) were calculated as follows [21]:where Y_E (g) is the total ethanol yield in the final fermentation mash, M_B (g) is the initial duckweed biomass input, S_D (%) is the duckweed starch content, and 0.511 is the theoretical ethanol conversion rate.

2.6. Cell Lines and Cell Culture

Two human AML cell lines, HL60 and Thp1 (Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology), were cultured at 37°C in 5% CO₂ in RPMI 1,640 medium (Gibco, Beijing, China) containing fetal bovine serum (10%, Biological Industries, Israel), penicillin (100 IU/mL), and streptomycin (100 μg/mL, Gibco, NY, USA).

2.7. Cell Viability Assay

CCK8 kits were used to evaluate cell viability [22]. In brief, 2.0 × 10⁴ cells were seeded in a 96-well culture plate. Then, different concentrations of DFs were added. After 24 h of treatment, cell viability was determined by CCK8 assay according to the manufacturer’s instructions (Beyotime, Shanghai, China).

2.8. Apoptosis Assay

HL60 and Thp1 cells exposed to different concentrations of DFs were collected and incubated in the dark at 37°C in 1 mL of fresh medium containing 1 μg/mL Hoechst 33,258 for 30 min. Subsequently, the cells were washed twice with PBS and monitored by fluorescence microscopy (OLYMPUS IX73 Microscope, Tokyo, Japan).

Apoptotic rates were determined using an apoptosis detection kit (BD Biosciences Pharmingen, San Diego, CA) [23]. First, 2.5 mL of cells (5.0 × 10⁵ cells) were seeded in 6-well culture plates and exposed to DFs (20, 40, and 60 μg/mL for HL60 cells; 40, 60, and 80 μg/mL for Thp1 cells). After 24 h of treatment, the cells were collected and resuspended in 500 μL of 1 × Annexin V binding buffer. Then, 5 μL of Annexin V-FITC and propidium iodide were added in turn. Cells were incubated in the dark at RT for 15 min. Finally, apoptotic cells were quantified by flow cytometry (BD FACSVerse flow cytometer, San Diego, CA).

2.9. Western Blot Analysis

Treated cells were lysed in precooled RIPA buffer containing protease and phosphate inhibitors (Roche, Mannheim, Germany). The protein concentration was quantified by a Bradford dye-binding assay. Then, 30 μg of protein was separated by SDS-PAGE and transferred to a PVDF membrane. Subsequently, the membranes were incubated with the primary antibodies at 4°C overnight. After three washes with PBST, the membranes were incubated with the secondary antibodies at RT for 1 h. Finally, the proteins were visualized by an enhanced chemiluminescence system (Beijing 4A Biotech Co. Ltd., China) [24].

2.10. In Vivo Animal Experiments

All animal experiments were approved by the Ethics Committee of Southwest Medical University (protocol number: 20211019-002). Healthy BALB/c-nu/nu mice (female, 4-5 weeks old) were purchased from Huafukang Biotech. Co. Ltd. HL60 or Thp1 cells (5 × 10⁶) were injected subcutaneously into the right lower limb of each mouse. When the average tumour volume reached approximately 100 mm³, the mice were randomly divided into treatment and control groups with five mice in each group, and the mice were intragastrically injected with 60 mg/kg DFs and vehicle, respectively. Tumour volume was measured every 2 days. The tumour volume was calculated as follows: tumour volume (mm³) = (length) × (width)² × 0.5. At the end of treatment, the tumours were dissected, imaged, and weighed.

2.11. Statistical Analysis

Three independent repeats were performed in all experiments in this study. SPPS software (version 21.0) and GraphPad Prism (version 8.0) were applied for statistical tests. One-way ANOVA and unpaired t tests were used to analyse the statistical significance of differences among the different groups. was considered significant.

3. Results

3.1. Main Composition

As presented in Table 1, the dry weight of wild duckweed was approximately 8% of the fresh weight. Based on the dry weight, the contents of the other components were determined. First, the carbon content was 38.77%, and the nitrogen and crude protein contents were 3.97% and 24.80%, respectively. Second, the starch and soluble sugar contents reached 8.87% and 1.44%, respectively. Moreover, the ash content was 8.97%, indicating abundant mineral contents. Finally, to further evaluate the nutritional quality of wild duckweed, the amino acid composition of the total protein was determined. As shown in Table 1, 17 amino acids were found in duckweed, and the total amino acid (TAA) content was 7.5% of the dry weight. Among these amino acids, essential amino acids (EAAs) accounted for 41% of the TAAs, while nonessential amino acids (NEAAs) accounted for 59%, which were more abundant than the EAAs. The abovementioned results indicated that duckweed possessed potential as a food feedstock.

3.2. Starch and Flavonoid Accumulation

To promote starch and flavonoid accumulation, duckweed was cultivated in tap water (nutrient starvation) for 10 days. The results are shown in Figure 1. First and foremost, the biomass increased continuously from the original 15.04 ± 0.62 to 72.53 ± 2.78 g/m² on the 10th day, achieving a mean growth rate of 5.75 ± 0.29 g/m²/d. In addition, starch accumulation was tested during the cultivation process. As shown in Figures 1(c) and 1(d), the starch content significantly increased from 8.97% to 36.35% DW in 3 days. The highest starch content reached 51.45% DW on the 10th day, which was 5.74 times higher than that on the first day. Finally, the flavonoids were extracted and quantified. As illustrated in Figures 1(e) and 1(f), the flavonoid content exhibited an increasing trend. The initial flavonoid content was 1.81 ± 0.22% DW, which then gradually increased to 2.33 ± 0.03% DW on the 3rd day. Thereafter, the flavonoid content changed insignificantly to 2.53 ± 0.12% DW on the 10th day. The flavonoid extract was concentrated and stored at −20°C. The residues after flavonoid removal were then used for bioethanol fermentation.

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3.3. Bioethanol Fermentation

To determine whether high-starch duckweed can serve as a feedstock for industrial production, the quality of duckweed biomass should be evaluated. In this study, we converted duckweed biomass into ethanol using separate hydrolysis and fermentation (SHF). After 24 h of SHF, the mean ethanol yield reached 4.98 ± 0.06 g in each Erlenmeyer flask, achieving an ethanol productivity of 0.208 ± 0.002 g/h. As shown in Table 2, the ethanol conversion rate (Y_E/G) reached 0.469 g/g glucose, and 91.83% fermentation efficiency was achieved. In addition, the ethanol yield (Y_E/B) under SHF reached 0.262 g/g biomass; based on the biomass yield (26.11 t/ha/y), a potential volume of duckweed-bioethanol of 8670 L/ha/y can be produced (Table 3).

3.4. The Antileukaemic Effect of Duckweed Flavonoids In Vitro

3.4.1. Cell Viability

To confirm the antileukaemic effect of duckweed flavonoids (DFs), the cytotoxicity of the DFs was first measured. Two AML cell lines (HL60 and Thp1) were exposed to different concentrations of DFs (10, 20, 40, 60, 80, and 100 μg/mL). After 24 h of treatment, the viability of both cell lines showed a sharp decrease with increasing DF concentration, indicating that DFs inhibited the growth of AML cells in a dose-dependent manner (Figure 2(a)). Furthermore, the DFs gave IC₅₀ values of 31.35 ± 4.45 μg/mL against HL60 cells and 62.93 ± 7.73 μg/mL against Thp1 cells (Figure 2(b)). Based on these IC₅₀ values, 0, 20, 40, and 60 μg/mL DFs were chosen to treat HL60 cells, while 0, 40, 60, and 80 μg/mL DFs were chosen to treat Thp1 cells in the subsequent experiments.

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3.4.2. Apoptosis

After the cell viability assay, apoptosis was detected. First, apoptosis was morphologically monitored using an inverted fluorescence microscope. The results displayed clear apoptotic cells of both lineages after DF treatment (Figures 2(c) and 2(d)). The number of apoptotic cells increased as the DF concentration increased, which indicated that DFs can induce apoptosis in AML cells in a dose-dependent manner. Then, apoptosis was quantified by flow cytometry. The changes in the apoptotic rates in both cell lines were in good agreement with the morphological results (Figures 2(c) and 2(d)). The apoptotic rate significantly increased in both types of treated cells. The highest apoptotic rates of 76.31 ± 3.79% and 38.08 ± 16.95% were observed in HL60 cells in the 60 μg/mL treatment group and in Thp1 cells in the 80 μg/mL treatment group, respectively. Finally, DF-induced apoptosis was further demonstrated at the protein expression level. The Western blot results showed clear increases in cleaved PARP and cleaved caspase 3 expression levels after DF treatment (Figure 2(e)), which confirmed that DFs can indeed induce apoptosis in HL60 and Thp1 cells in vitro.

3.5. The Antileukaemic Effect of Duckweed Flavonoids In Vivo

To further evaluate the antileukaemic effect of the DFs in vivo, AML cell-xenografted nude mice (5 × 10⁶ HL60 or Thp1 cells/mouse) were intragastrically administered 60 mg/kg DFs per day for 12 days. As shown in Figures 3(a) and 3(b), DF administration significantly inhibited the growth of the tumours from both AML cell lines. At the end of treatment, the wet tumour weights in the DF treatment groups were 1.57 ± 1.06 g (HL60-xenograft tumours) and 2.65 ± 0.88 (Thp1-xenograft tumours). Compared to the control groups (4.27 ± 1.35 g, HL60-xenograft tumours and 5.13 ± 0.72 g, Thp1-xenograft tumours), the inhibition rates reached 63.33% and 48.37%, respectively. These results indicated that 60 mg/kg DFs can efficiently suppress the growth and reduce both the volume and weight (Figures 3(a)–3(c)) of tumours of these two AML cell lines. Regarding the inhibitory effects, HL60 cells were more sensitive to DFs than Thp1 cells, which were consistent with the results of the in vitro experiment. Finally, haematoxylin and eosin (H&E) staining of tumour tissues reconfirmed that DFs can efficiently induce AML cell death (Figure 3(d)). In summary, these results revealed that DFs possessed powerful antileukaemia activity in vitro and in vivo.

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4. Discussion

4.1. The Potential of Duckweed as a Novel Food Feedstock

Reportedly, nearly 690 million people were suffering from hunger in 2019, accounting for 8.9% of the world’s population [25]. It is estimated that the world’s population will reach 9 billion by mid-century, which will cause more severe food insecurity. Due to the limited amount of arable land and bottlenecks in traditional crop cultivation technology worldwide, novel aquaculture crops have been proposed as important additions to the global food system [29]. Hu et al. [4] and Appenroth et al. [30] determined the nutritional value of Wolffia. Similarly, Pagliuso et al. [5] found that duckweed can serve as a promising food source worldwide. Furthermore, Appenroth et al. [2] reported that the nutritional component distribution of duckweed was close to the WHO recommendations. However, all of the duckweeds in these studies were cultivated under artificial conditions, and the nutritional value of natural duckweed is still undetermined. In this study, we first reported the main composition of natural duckweed. As the biggest concern for food nutrition, the protein and TAA contents of natural duckweed were 24.80% and 7.5% DW, respectively, which was highly consistent with the results reported by Pagliuso et al. [5]. Importantly, duckweed contained an abundance of the EAAs recommended by the WHO (Table 2). This result indicated that natural duckweed was also a promising source of plant protein. Interestingly, people in some countries or regions, such as Thailand, Myanmar, Laos, and Southwest China, have long consumed duckweed as food [13]. Finally, the safety of duckweed as a food must be focused on. A previous study revealed that duckweed did not have any detectable adverse effects on human cells [31]. Zeinstra et al. [32] reported that no notable adverse reactions occurred after duckweed intake. Kaplan et al. [33] similarly concluded that duckweed was safe to eat. Taken together, these data show that natural duckweed is inexpensive, easily obtained, and capable of being a human food feedstock.

4.2. The Potential of Duckweed as a Novel Feedstock for Bioethanol Fermentation

In addition to being a human food feedstock, duckweed has been considered a bioenergy crop [7]. As listed in Table S1, duckweed biomass can be efficiently converted into bioethanol, and the duckweed-ethanol conversion rate is positively correlated with the starch content in duckweed. Thus, duckweed was cultivated under nutrient starvation to induce starch accumulation. The starch content and biomass yield were then evaluated. As shown in Figure 1(c), the starch content increased to 51.45% DW after 10 d of cultivation, which is similar to that in some grains [1]. Therefore, the duckweed starch content was high enough for bioethanol fermentation. Notably, 72.53 g/m² high-starch duckweed biomass can be harvested every 10 days (Figure 1(a)), thus leading to an annual yield of 26.11 t/ha/y, which was significantly higher than that of maize and wheat (Table 3). Finally, duckweed biomass was fermented into ethanol using SHF. The ethanol conversion rate (Y_E/G) reached 0.469 g/g glucose with 91.83% fermentation efficiency, revealing that in duckweed, starch can be efficiently converted into ethanol (Table 2). The efficiency of duckweed fermentation into ethanol (91.83%) was comparable to that of maize to ethanol and wheat to ethanol [28]. In addition, the ethanol yield (Y_E/B) reached 0.262 g/g, which, to our knowledge, is the highest ethanol yield based on duckweed fronds reported thus far (Table S1). Based on the biomass yield (26.11 t/ha/y), a potential duckweed-bioethanol volume of 8670 L/ha/y can be achieved, which is far higher than that of wheat-bioethanol (1,552 L/ha/y) and maize-bioethanol (2,585 L/ha/y) (Table 3). Consequently, high-starch duckweed can be used as a profitable feedstock for large-scale bioethanol production.

4.3. The Potential of Duckweed as an Antileukaemia Drug

Another important feature of duckweeds that has attracted scientific attention worldwide is their pharmaceutical effects. Duckweed has long been used as a folk medicine for the treatment of asthma, diabetes, rhinitis, and so forth in China and even in some European nations [12]. However, the anticancer effect of duckweed flavonoids has rarely been reported. Although Pagliuso et al. [34] speculated that duckweeds had anticancer effects based on their main flavonoids (apigenin, luteolin, and their derivatives), the real anticancer effects, especially antileukaemic effects, of duckweed flavonoids have not been systematically investigated. Therefore, the antileukaemic effects of duckweed were evaluated in vitro and in vivo for the first time. Acute leukaemia (AL) is the most common cancer among children and accounts for one-third of all childhood cancers [35]. Acute myeloid leukaemia (AML) accounts for only 15∼20% of the cases but more than 30% of the deaths from AL [36]. In particular, the survival of refractory childhood AML patients is poor (<22%) [37]. Thus, it is essential to find more promising drugs for AML therapy. In this study, HL60 and Thp1 cells were chosen for further investigation. As shown in Figure 2, DFs showed a notable inhibitory effect on the growth of both types of cells. Based on the IC₅₀ values, HL60 cells were more sensitive to DFs than Thp1. Thus, we can preliminarily conclude that DFs possess an antileukaemic effect. As shown in Figures 2(c)–2(e), DF induced apoptosis in both cell lines in a dose-dependent manner, indicating that DF effectively suppressed AML cell growth through apoptosis induction in vitro. To further confirm this conclusion, in vivo experiments were performed. The results indicated that DFs at 60 mg/kg inhibited tumour growth in xenograft mice. The DF inhibitory rates exceeded 45% for tumours originating from both cell lines and over 60% for those of the HL60 lineage. These results were in complete agreement with those of the in vitro experiments. Importantly, the body weights of the mice in the treatment group and control group exhibited no apparent difference, indicating that DFs can effectively inhibit AML in vivo without influencing body weight (Figure S2). Furthermore, DFs showed insignificant cytotoxicity on human or monkey normal cells (Figure S1). Collectively, the in vitro and in vivo experiments suggested that the use of DFs is a safe and effective strategy for AML therapy.

5. Conclusion

In this study, the nutritional value of natural duckweed was systematically evaluated for the first time. Considering the high yield of duckweed in the natural environment worldwide, it may be a great candidate for the global human food supply. Furthermore, the potential to produce bioethanol from duckweed was assessed. High-starch duckweed biomass was fermented into bioethanol, and the yield reached 0.262 g/g biomass, which was the highest ethanol yield using duckweed fronds reported thus far. Based on the duckweed biomass yield (26.11 t/ha/y), a potential duckweed-bioethanol yield of 8670 L/ha/y can be achieved, which is 4.6 and 2.4 times higher than that of wheat-bioethanol and maize-bioethanol, respectively. Duckweed, therefore, seems to be an excellent energy crop for bioethanol production. Finally, the antileukaemic effects of duckweed flavonoids were evaluated for the first time. DFs significantly inhibited AML cells in vitro and in vivo, and the outcome supported DFs as effective agents for AML therapy. In conclusion, duckweed is a promising crop that could be helpful for humans to meet the challenges posed by food security, energy crises, and tumours. Certainly, the anticancer effect of DFs and the relevant molecular mechanism need to be investigated further.

Abbreviations

DF:	Duckweed flavonoid
AML:	Acute myeloid leukaemia
GR:	Growth rate
SHF:	Separate hydrolysis and fermentation
TAA:	Total amino acid
EAA:	Essential amino acid
NEAA:	Nonessential amino acid
DW:	Dry weight
FW:	Fresh weight
AL:	Acute leukaemia
RT:	Room temperature.

Data Availability

The data used to support the findings of this study are included within the article and the supplementary information file(s).

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Authors’ Contributions

Ling Guo, Jing Liu, and Qingyan Wang contributed equally to this work. Ling Guo conceptualized the study, performed the methodology, provided funding acquisition, performed supervision, performed investigation, and wrote the original draft. Jing Liu performed investigation and wrote the original draft. Qingyan Wang conceptualized the study, performed the methodology, and performed supervision. Yan Yang performed investigation and contributed to visualization. You Yang performed formal analysis and performed data curation. Qulian Guo performed supervision, performed the methodology, and provided resources. Hai Zhao conceptualized the study, performed the methodology, provided resources, and provided funding acquisition. Wenjun Liu conceptualized the study, performed the methodology, provided funding acquisition, and performed supervision. All authors wrote, reviewed, and edited the manuscript.

Acknowledgments

This work was funded by the Natural Science Foundation of Sichuan Province (2022NSFSC0723), the Opening Project of the State Key Laboratory of Non-Food Biomass and Enzyme Technology (2022-36-Z06-02), the Medical Science and Technology Project of Health Commission of Sichuan Province (no. 21PJ091), the Science and Technology Strategic Cooperation Project between Luzhou Municipal People’s Government and Southwest Medical University (2021LZXNYD-J22), the Special Project of Science and Technology Research of Sichuan Administration of Traditional Chinese Medicine (2020JC0135), the Applied Basic Research Project of Southwest Medical University (2021ZKQN083, 2020ZRQNB029), the Doctoral Research Initiation Fund of the Affiliated Hospital of Southwest Medical University, Sichuan Science and Technology Program (2022YFS0622), the Biological Resources Program, Chinese Academy of Sciences (KFJ-BRP-008), the Innovation Academy for Seed Design, CAS, and the National Aquatic Biological Resource Center (NABRC).

Supplementary Materials

Table S1: the data of different technologies and ethanol yields from different duckweed varieties in the previous studies and this study. Figure S1: the cytotoxicity of duckweed on human or monkey normal cells. Figure S2: the data of mouse body weight after DFs treatment. The results indicated that 60 mg/kg DFs had no significant effect on mouse body weight. (Supplementary Materials)

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Copyright © 2023 Ling Guo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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