Protective effect of the methanol extract of edible insects against oxidative damages in C2C12 myoblasts and myotubes

Abstract

This study investigated the protective effect of edible insect methanol extracts (EIMEs) against oxidative stress induced by H₂O₂ in C2C12 cells. Total phenolic and total flavonoid contents of EIMEs were 7.43–47.15 mg gallic acid equivalents/g of residue and 2.01–31.00 mg catechin equivalents/g of residue, respectively. Among edible insects, Oxya chinensis showed the highest antioxidant activity, followed by Allomyrina dichotoma. EIMEs (100 µg/mL) significantly prevented H₂O₂-induced cytotoxicity in both myoblasts and myotubes. H₂O₂-induced oxidative stress causes reactive oxygen species (ROS) production, glutathione depletion, and lipid peroxidation, all of which were markedly ameliorated by EIMEs in myoblasts in our study. In addition, A. dichotoma prevented the generation of ROS and release of creatine kinase (CK) and lactate dehydrogenase (LDH) in myotubes. EIMEs increased the diameter of myotubes and mRNA and protein expression levels of myosin-heavy chains. Our study provides insights into the application of edible insects to regulate muscle differentiation and alleviate oxidative damage to muscles.

Introduction

Oxidative damage in human is caused by an imbalance between protection of the antioxidant system and the generation of reactive oxygen species (ROS). ROS is a major initiating factor in various age-related diseases (Birben et al., 2012). Elevated ROS production increases oxidative damage and induces cellular dysfunction and cell death (Kang et al., 2015). In addition, it causes proteolysis and leads to skeletal muscle atrophy (Leitner et al., 2017). The skeletal muscle comprises approximately 40 % of the body weight in humans. Myosatellite cells proliferate and differentiate into myoblasts, which fuse with each other and pre-existing myofibers to form myotubes and myofibers (Frontera and Ochala, 2015). Myosin- heavy chain (MyHC), lactate dehydrogenase (LDH), and creatine kinase (CK) are used as myogenesis markers during muscle differentiation (Barjot et al., 1998). Dietary protein consumption is crucial for skeletal muscle mass maintenance and health. Proteins from animal-based sources including fish, eggs, and meat are major sources of dietary protein. However, production of sufficient amounts of these animal-based proteins to meet global food demands is difficult. Thus, more sustainable alternatives to these high-quality protein sources have attracted interest.

Edible insects have recently attracted global attention for their nutritional, environmental, and economic benefits. Edible insects are a highly nutritious source of protein, fat, minerals, fiber, and vitamins (Payne et al., 2016). Orkusz (2021) indicated that Gryllus bimaculatus and Bombyx mori have similar thiamine content to that of meat. Their report also showed that insects are characterized by a higher content of vitamin C, riboflavin, and tocopherol than meat, regardless of the species and form of development. Edible insects can reduce the risk of oxidation-related diseases because of their high levels of functional compounds (Nowakowski et al., 2022). Previous studies have reported that some edible insects alleviate inflammation and inhibit the metabolic syndrome (Santos, et al., 2010; Zielinska et al., 2020). Consumption of the cricket Brachytrupes orientalis decreases intracellular ROS levels and lipid peroxidation via the upregulation of Nrf2 expression (Dutta et al., 2017). A recent study documented the approval of mealworm powder as a protein supplement for the treatment of sarcopenia in the elderly and the sick (Kim et al., 2016). As the regulation of oxidative stress is essential for alleviating sarcopenia, edible insects possessing abundant nutrition are attractive materials with fewer side effects for the management of muscle-related diseases (Meng and Yu, 2010). Therefore, in the present study, we attempted to gain a better understanding of the therapeutic potential of edible insects in C2C12 skeletal muscle cells following the induction of oxidative stress by hydrogen peroxide.

Materials and Methods

Preparation of methanol extracts from edible insects  The dried edible insects including silkworms (Bombyx mori), rice grasshoppers (Oxya chinensis sinuosa), mealworms (Tenebrio molitor), white-spotted flower chafers (Protaetia brevitarsis), rrhinoceros beetles (Allomyrina dichotoma), two-spotted crickets (Gryllus bimaculatus) and white stiff silkworms (Bombycis corpus) were obtained from a local market (Busan, Republic of Korea). Whole edible insects (approximately 7 g) were extracted with 200 mL of methanol for 24 h using a shaker. The extracts were filtered through Whatman No. 2 filter paper (Whatman International, Kent, UK) and evaporated. The dried residues were dissolved in dimethyl sulfoxide (DMSO).

Determination of total polyphenolic content (TPC), total flavonoid content (TFC), and antioxidant activities  TPC and TFC were assayed using the Folin-Ciocalteu phenol reagent and aluminum chloride colorimetric method, respectively (Noh et al., 2018). The extracts were mixed with 2 % Na₂CO₃ solution and 50 % (v/v) Folin-Ciocalteu reagent solution. After incubation for 5 min, the absorbance was measured at 750 nm on a spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA). The TPC was calculated using a gallic acid standard curve. The extracts were mixed with water and 5 % NaNO₂. After incubation for 5 min, 150 µL of 10 % AlCl₃•6 H₂O and 500 µL of 1 M NaOH were added. The absorbance was measured at 510 nm on a spectrophotometer. The TFC was calculated using a catechin standard curve. ABTS and DPPH radical scavenging activities and reducing power were measured using the method described by Yang et al. (2019). 1 mL of ABTS radical solution was mixed with 20 µL of extracts. After 60 min, the absorbance was read at 734 nm. 1 mL of DPPH radical solution was mixed with 20 µL of extracts. After 30 min, the absorbance was read at 520 nm. 250 µL of the extracts was mixed with a 250 µL of 0.2 M phosphate buffer and 250 µL of 1 % potassium ferricyanide. The mixture was incubated at 50 °C for 20 min and mixed with 250 µL of 10 % trichloroacetic acid. 500 µL of supernatant was mixed with 500 µL of water and 100 µL of 0.1 % ferric chloride. The absorbance was read at 700 nm. The ABTS and DPPH radical scavenging activity and reducing power were expressed as trolox equivalent.

Cell culture  C2C12 cells were purchased from ATCC (Rockville, MD, USA) and grown in DMEM supplemented with 10 % FBS, 100 units/mL penicillin, and 50 µg/mL streptomycin in a humidified incubator with 5 % CO₂ at 37 °C. C2C12 cells were seeded onto 96-well plates at a density of 6.0 × 10⁴ cells/mL or 6-well plates at a density of 1.0 × 10⁵ cells/mL. After 24 h, the cells were pre-treated with edible insect methanol extracts (EIMEs) (100 µg/mL) for 2 h and then co-treated with H₂O₂ (600 µM) for 24 h. The protective effects of the extracts were evaluated using the MTT assay. The cells were exposed to MTT reagent (0.5 mg/mL) and maintained at 37 °C for 2 h. The formazan product was dissolved in DMSO after discarding the culture medium. The absorbance at 550 nm was measured using a spectrophotometer (BioTek Instruments Inc.).

Measurement of intracellular, glutathione (GSH), and malondialdehyde (MDA) levels  Intracellular ROS production was determined by fluorescence intensity using the DCF-DA assay. The fluorescence intensity was measured using a fluorescence spectrophotometer (Perkin-Elmer, Norwalk, CT, USA). MDA and GSH levels were measured using the TBA reactive substance method and DTNB-GSSG reductase recycling assay, respectively (Lee et al., 2019; Choe et al., 2021).

Measurement of LDH and CK levels and muscle diameters  The levels of LDH and CK, released from the cytosol of damaged cells into the supernatant, were assayed using LDH and CK assay kits according to the manufacturer's instructions (Abcam, 102526 and 155901). The LDH and CK release (%) was estimated by dividing the amount of LDH and CK in the medium by the total amount of LDH and CK in the medium and cell lysate. The diameters of the myotubes were measured from 10 random fields using Image J software 1.50i (NIH, Bethesda, MD, USA).

Western blot analysis  C2C12 myoblasts were cultured in a 100 mm dish and differentiation was induced using differentiation medium with or without EIMEs (100 µg/mL) for 6 d. Total protein was extracted from the cells using the PRO-PREP™ protein extraction solution (iNtRON Biotechnology, Inc., Seongnam, Korea). Membranes were incubated with primary (1:500 dilution for MyHC and 1:1 000 dilution for β-actin) and secondary (1:1000 dilution for anti-mouse) antibodies (Santa Cruz, CA, USA). The bands were visualized using X-ray film.

Quantitative real-time polymerase chain reaction (qPCR)  To investigate the mRNA expression of MyHC, the synthesized cDNA was analyzed using qPCR (Applied Biosystems, Carlsbad, CA, USA). MyHC (Mm01332489_m1) and GAPDH (Mm99999915_g1) transcripts were quantified using gene-specific primers.

Statistical analysis  Data were analyzed using Duncan's multiple comparison test and Tukey's post-hoc test using SAS (version 8.1; SAS Institute, Cary, NC, USA) and GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA). p < 0.05, p < 0.01, and p < 0.001 were considered to be statistically significant.

Results and Discussion

Total polyphenolic content, total flavonoid content, and antioxidant capacity of edible insects  Seven edible insects including silkworms (B. mori), rice grasshoppers (O. chinensis sinuosa), mealworms (T. molitor), white-spotted flower chafers (P. brevitarsis), rrhinoceros beetles (A. dichotoma), two-spotted crickets (G. bimaculatus), and white stiff silkworms (B. corpus) have been accepted as regular food items in Korea. Edible insects have attracted attention for their abundant nutrition and health benefit. In recent years, bioactive compounds discovered from edible insects have induced bioactivities including anticancer, anti-diabetes, antiinflammation, and antioxidant (Nowakowski et al., 2022). However, information on the comparative efficacy of edible insects in the protection of skeletal muscle cells is limited. To examine the bioactivity of EIMEs, total phenolic and total flavonoid contents and antioxidant activities were measured (Table 1). The total polyphenol content of the extracts was 7.43–47.15 mg gallic acid equivalent/g residue; total flavonoid content of the extracts was 2.01–31.00 mg catechin equivalent/g residue; ABTS, DPPH radical scavenging capacities, and reducing power were 5.34–31.85, 9.81–34.73, and 9.64–40.91 mg trolox equivalent/g residue, respectively. Among all extracts, the methanol extract of O. chinensis showed the highest antioxidant activity followed by that of A. dichotoma. Similarly, a recent study showed that the TPC of A. dichotoma, T. molitor, Rhynchophorus ferrugineus, and Zhophobas morio was in the range of 10.9–38.3 mg gallic acid equivalent/g dry matter (Botella-Martinez et al., 2021). Chakravorty et al. (2016) informed that the TFC values were 4.97 and 6.15 mg/g in Oecophylla smaragdina and Odontotermes sp., respectively. Zielińska et al. (2017) showed the antioxidative activities of some edible insects using free radical-scavenging capacity, ion chelating capacity, and reducing power assays. Our data indicated high positive correlations (r = 0.911–0.996, P < 0.01) between antioxidant contents and antioxidant activities (data not shown). Hence, our findings suggest that edible insects that have a high concentration of antioxidant compounds may provide antioxidant capacities.

Table 1 Total polyphenolic content, total flavonoid content, and antioxidant capacity in edible insects.

Samples	Total polyphenolic contentA	Total flavonoid contentB	ABTSC	DPPHD	Reducing powerE
Bombyx mori	15.45 ± 0.27d	3.42 ± 0.00d	11.55 ± 0.53d	15.65 ± 1.69b	19.67 ± 0.37b
Oxya chinensis	47.15 ± 0.65a	31.00 ± 0.63a	31.85 ± 0.26a	34.73 ± 0.22a	40.91 ± 0.08a
Tenebrio molitor	17.36 ± 0.18c	1.86 ± 0.13e	12.25 ± 0.21c	9.30 ± 1.62e	9.64 ± 0.48f
Protaetia brevitarsis	14.67 ± 0.18e	2.01 ± 0.24e	12.14 ± 0.12c	11.41 ± 0.70cd	12.77 ± 0.19d
Allomyrina dichotoma	20.05 ± 0.31b	4.33 ± 0.20c	14.98 ± 0.04b	15.21 ± 0.97b	14.23 ± 0.23c
Gryllus bimaculatus	12.81 ± 0.73f	5.48 ± 0.23b	9.61 ± 0.16e	12.17 ± 0.53c	14.32 ± 0.42c
Bombycis corpus	7.43 ± 0.10g	4.18 ± 0.17c	5.34 ± 0.19f	9.81 ± 0.28de	11.96 ± 0.25e

^A  Total polyphenolic content is expressed as gallic acid equivalent mg/g residue.

^B  Total flavonoid content is expressed as catechin equivalent to mg/g residue.

^C  ABTS radical scavenging activity is expressed as Trolox equivalent mg/g residue, and

^D  DPPH radical scavenging activity is expressed as Trolox equivalent mg/g residue.

^E  Reducing power is expressed as Trolox equivalent mg/g residue. Data are presented as mean ± standard deviation (n = 3). Different letters in the same column indicate significant differences according to Duncan's test (p < 0.05).

Protective effect of edible insects against H₂O₂-induced cytotoxicity in C2C12 myoblasts and myotubes  The cytotoxicity of C2C12 cells in the presence of edible insects was investigated. Treatment with 100 µg/mL EIMEs for 24 h did not affect the cytotoxicity of C2C12 myoblasts and myotubes (Fig. 1A and 1C). Treatment with 600 µM H₂O₂ significantly decreased cell viability. However, treatment with EIMEs significantly enhanced H₂O₂-induced cell viability (Fig. 1B and 1D). Remarkably, we found that O. chinensis treatment showed the highest protective effect against hydrogen peroxide treatment. Previous studies have reported that edible insects exhibit various biological activities. G. bimaculatus protects against gut-derived inflammatory responses and liver damage (Hwang et al., 2019). A recent study confirmed that edible insects regulate pancreatic beta cell function in the diabetic state via the modulation of apoptosis (Park et al., 2020). Moreover, tetrahydroquinolines from edible insects have been shown to exert protective effects against LPS-induced inflammation (Park et al., 2020). These various effects may be due to the antioxidant capacity of edible insects. Hence, our results suggest that O. chinensis extracts exert cytoprotective effects on C2C12 myoblasts and myotubes because of its high antioxidant activity, thus protecting against H₂O₂-induced oxidative stress.

Fig. 1.

Effects of edible insects methanol extract on (A and C) cell viability and (B and D) protective effect against H₂O₂-induced C2C12 myoblast and myotubes. Each value was expressed as the mean ± SE (n = 3). ^###p < 0.001 versus non-treated cells. _*p < 0.05, ^**p < 0.01, and ^***p < 0.001 versus H₂O₂ treated cells. CON, control; BM, Bombyx mori; OC, Oxya chinensis; TM, Tenebrio molitor; PB, Protaetia brevitarsis; AD, Allomyrina dichotoma; GB, Gryllus bimaculatus; BC, Bombycis corpus.

Protective effect of edible insects against H₂O₂-induced cellular oxidative damage in C2C12 myoblasts  Acceleration of ROS generation and lipid peroxidation, and depletion of GSH are associated with H₂O₂-induced cytotoxicity (Lee et al., 2021). H₂O₂ induces aberrant ROS accumulation in skeletal muscle cells, resulting in muscle impairment (Bosutti and Degens, 2015). Elevated MDA levels have been used as index of oxidative stress. Prathapan et al. (2012) suggested that low cellular GSH levels might be due to either decreased GSH biosynthesis or increased utilization of GSH during stress. To examine whether the methanol extract of edible insects contributed to the protection of C2C12 myoblasts against H₂O₂-mediated oxidative stress, we measured intracellular ROS levels (Fig. 2A and 2B). H₂O₂ exposure significantly increased the ROS generation. However, EIMEs treatment group showed a significant decrease in ROS generation. We found that O. chinensis, P. brevitarsis, A. dichotoma, and G. bimaculatus strongly reduced ROS level by 20.1 %, 21.9 %, 22.8 %, and 22.6%, respectively, at a concentration of 100 µg/mL, compared with the H₂O₂ treatment group. Treatment with H₂O₂ induced significantly reduced GSH levels (Fig. 2C). However, all EIMEs completely prevented H₂O₂-induced decreases in GSH levels. O. chinensis and A. dichotoma markedly prevented the lipid oxidation caused by H₂O₂ (Fig. 2D). These results clearly indicate that edible insects alleviate intracellular ROS production, GSH depletion, and lipid peroxidation, which support the maintenance of the normal redox status of myoblasts.

Fig. 2.

Effects of edible insects methanol extracts on (A and B) ROS generation, (C) GSH depletion, and (D) MDA production against H₂O₂-induced C2C12 myoblast. Each value was expressed as the mean ± SE (n = 3). ^###p < 0.001 versus non-treated cells. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 versus H₂O₂ treated cells. CON, control; BM, Bombyx mori; OC, Oxya chinensis; TM, Tenebrio molitor; PB, Protaetia brevitarsis; AD, Allomyrina dichotoma; GB, Gryllus bimaculatus; BC, Bombycis corpus.

Protective effects of edible insects against H₂O₂-induced cellular oxidative damage in C2C12 myotubes  Skeletal muscle cells can produce ROS during differentiation and muscular activity. Previous studies have reported that ROS generation decreases the antioxidant defense system in muscle fibers, resulting in the accumulation of oxidatively modified nucleic acid molecules, lipids, and proteins (Kavazis et al., 2009; Powers et al., 2011). LDH is a known marker of cellular injury (Stein and Wade, 2005). Likewise, CK is considered a differentiation marker and a good substitute for evaluating myotube functions (Mittal et al., 2010). Under pathological conditions, injured membranes increase the release of LDH and CK from the cells (Li et al., 2014). We confirmed that edible insects regulate myotube membrane integrity to protect cells against oxidative stress. Treatment with H₂O₂ significantly increased ROS, LDH, and CK release compared to that in control cells (Fig. 3). However, A. dichotoma significantly prevented the production of ROS and release of LDH and CK. A recent study showed that treatment with cinnamaldehyde increased intracellular LDH and CK levels, which means maintaining cell membrane integrity and preserving the functional and bioenergetic status of myotubes (Kaur et al., 2019). These results indicate that A. dichotoma can alleviate increased oxidative damages in myotube membranes.

Fig. 3.

Effects of edible insects methanol extracts on (A and B) ROS generation, (C) released LDH, and (D) released CK against H₂O₂-induced C2C12 myotubes. Each value was expressed as the mean ± SE (n = 3). ^#p < 0.01 and ^###p < 0.001 versus non-treated cells. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 versus H₂O₂ treated cells. CON, control; BM, Bombyx mori; OC, Oxya chinensis; TM, Tenebrio molitor; PB, Protaetia brevitarsis; AD, Allomyrina dichotoma; GB, Gryllus bimaculatus; BC, Bombycis corpus.

Effect of edible insects on differentiation in C2C12 myotube  Myogenic differentiation of C2C12 cells is a process characterized by the expression of muscle-specific genes in myocytes and the fusion of myocytes into multinucleated myotubes (Langen et al., 2002). Skeletal muscle hypertrophy and atrophy contribute to the regulation of the diameter of pre-existing muscular fibers (Rommel et al., 2001). Recent studies have demonstrated applications of natural products that have the potential to improve myogenesis and muscle regeneration. Lee et al. (2018) reported that black ginseng increased the diameter and thickness of larger multinucleated myotubes. In addition, MyHC, myogenin, and MyoD expression levels were higher in the black ginseng group than in the control group in the same study. To confirm the effects of EIMEs on the differentiation of myotubes, we assessed the diameter of myotubes and the expression of MyHC mRNA and protein as differentiation markers using qPCR and western blotting, respectively. We used the 6-day time-point for the measurement of myotube diameter (Fig. 4A). Treatment of B. corpus significantly increased the diameter by 29.2 % compared to the control group (Fig. 4B). In addition, treatment with B. corpus, P. brevitarsis, and O. chinensis increased MyHC mRNA expression by 28.7 %, 19.7 %, and 42.1 %, respectively (Fig. 4C). Similarly, B. corpus showed the highest MyHC protein expression (Fig. 4D). These results indicate that B. corpus can help stimulate muscle differentiation.

Fig. 4.

Effects of edible insects methanol extracts on differentiation of myotubes; (A) phenotypic change, (B) myotube diameter, and (C and D) MyHC mRNA and protein expression levels. Scale bar indicates 100 µm. Each value was expressed as the mean ± SE (n = 3). ^#p < 0.01, ^##p < 0.05, and ^###p < 0.001 versus non-treated cells. CON, control; BM, Bombyx mori; OC, Oxya chinensis; TM, Tenebrio molitor; PB, Protaetia brevitarsis; AD, Allomyrina dichotoma; GB, Gryllus bimaculatus; BC, Bombycis corpus.

In conclusion, O. chinensis extracts show high antioxidant capacity, which may contribute to their protective activities in C2C12 myoblasts and myotubes. O. chinensis extracts strongly protected cells against H₂O₂-induced damage in C2C12 cells via modulation of intracellular ROS generation, lipid peroxidation, and GSH depletion. In addition, they alleviated the release of LDH and CK in myotubes. Based on these results, we found that treatment using EIMEs can alleviate the damage to the myotube membrane by H₂O₂. The diameter and MyHC expression levels, which are markers of muscle differentiation, increased in edible insects. Thus, this study demonstrates the protective activity of O. chinensis extracts in myoblasts and myotubes against oxidative damage. Further studies are required to explain in-depth diagnostic parameters for treatment of edible insects in diverse skeletal muscle pathophysiological conditions.

Conflict of interest  There are no conflicts of interest to declare.

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