Skin irritation and potential antioxidant, anti-collagenase, and anti-elastase activities of edible insect extracts

Insect extracts

Thai edible insects (Fig. 1) were extracted and yield of each extract is shown in Fig. 2. Hexane extracts of most insects, except for P. succincta, provided the highest yield, followed by ethanolic extracts, and aqueous extracts, respectively. The reason might be due to a high amount of fat content of insects. Since these fat components are hydrophobic, they could be extracted well using nonpolar solvent, e.g. hexane. Semi-polar solvent like ethanol could also be used to extract hydrophobic compounds but with less extraction efficacy⁵. Several previous studies reported that fat was abundant in biomass of insects, ranging from 4.2 to 77.2%, which was accounted for about 26.8% on average dried insects^6,7.

Among several insect extracts, Euconocephalus sp. yielded the significantly highest extract content when extracted by hexane (29.3 ± 2.3% w/w) (p < 0.05), followed by A. domesticus (23.5 ± 4.0% w/w), O. fuscidentalis (19.6 ± 1.6% w/w), B. mori (17.1 ± 1.2% w/w), P. succincta (13.8 ± 1.2% w/w), and L. indicus (11.5 ± 1.7% w/w), respectively. Similarly, Euconocephalus sp. yielded the significantly highest extract content when extracted by ethanol (25.2 ± 0.7% w/w) (p < 0.05), but followed by P. succincta (21.2 ± 1.6% w/w), A. domesticus (17.8 ± 2.0% w/w), B. mori (11.5 ± 1.3% w/w), L. indicus (9.0 ± 0.5% w/w), and O. fuscidentalis (2.5 ± 0.7% w/w), respectively. The reason might be due to high fat content in the hexane and ethanolic extracts of Euconocephalus sp. The previous studies reported that fat content of some grasshopper species belonging to Acrididae family was in range of 4.2 to 22.2% of body weight⁸, whereas fat content of A. domesticus was around 10% of body weight⁹ and P. succincta contained a low amount of fat (~ 1.5% of the body weight)⁷.

In contrast, yields of aqueous extracts of all insects, which were extracted by digestion, were the lowest among various solvents. Owing to duration of extraction and numbers of extraction cycles.

Protein content of insect extracts

The protein content of each insect extract is shown in Fig. 3. The present study demonstrated that aqueous extracts of most insects contained higher protein content than ethanolic extracts. Additionally, protein was not detected in hexane extracts of most insects. The reason was due to the less extraction efficiency of ethanol to extract protein comparing to DI water since DI water is more hydrophilic. Additionally, some protein might be denatured or precipitated during the extraction process¹⁰. Similarly, hexane which is non-polar organic solvent could not extract protein well because of its lipophilicity that was incompatible with protein. Apart from protein component, the compositions of hexane extract were nonpolar compounds, e.g. flavonoids, lipids, saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids¹¹. These results corresponded well to a previous study noted that high protein content (68.8 ± 0.4% w/w) was detected in aqueous extract of Protaetia brevitarsis larvae¹².

The present study revealed that the protein content of the insects extracted by maceration and digestion ranged from 4.7 ± 2.6 to 87.3 ± 0.5% w/w which were related to a previous study reported that dry basis protein content of the insects ranged from 15 to 81%⁷. Interestingly, the aqueous, ethanolic, and hexane extracts of P. succincta had the significantly highest protein content among several insects, which were 87.3 ± 0.5, 53.8 ± 2.5, and 37.2 ± 7.4% w/w, respectively (p < 0.05). These findings were in good agreement with a previous study that reported the protein content of defatted locust extract, investigated using the Kjeldahl method, was 82.3% of dry weight, although the estimated protein content of a sample may be varied depending on the technique applied¹³. Kjeldahl is a method that involves digesting food with a strong acid, which results in the release of nitrogen, which is then measured using a titration approach¹⁴. Although the Kjeldahl technique is regarded as the worldwide standard and therefore simple to compare findings with other laboratories, it does not measure real protein and can result in overestimation of protein owing to the use of the standard nitrogen correction factor¹⁴. On the other hand, the BCA technique, which is used in the present study, is based on two chemical reactions, including the biuret reaction, which reduces cupric ions (Cu²⁺) to cuprous ions (Cu¹⁺) via peptide bonds and the chelation of one Cu¹⁺ molecule with two BCA molecules to form a bright purple complex which is spectrophotometrically detected¹⁵. Besides, BCA is simpler, ease of use, takes less time for the experiment, requires fewer instruments, high sensitivity, and tolerance of interfering species, e.g. common surfactants^15,16,17. The findings revealed that the protein contents analyzed by BCA in the current investigation were equivalent to those found in the prior study utilizing the Kjeldahl method.

Therefore, protein might be a major component in most insect extracts and was efficiently extracted by water, an environmentally friendly method. However, several previous studies demonstrated that protein content of insects could be affected by different environmental factors, including origin, stage of life, and feeding¹⁸.

Antioxidant activities of insect extracts

Antioxidant activities of Thai edible insect extracts were determined by 4 different methods, including ABTS, DPPH, FRAP, and FTC assay since multiple reactions and mechanisms are reported which involve antioxidant process. Both ABTS and DPPH assays indicate the abilities of test compound to scavenge free radicals and are expressed as TEAC value and DPPH^· inhibition percentage, respectively¹⁹. While FRAP assay represents antioxidant potential of test sample through the reduction of ferric iron (Fe³⁺) to ferrous iron (Fe²⁺), which is expressed as EC₁ value. Additionally, FTC assay is the most studied biologically relevant free radical chain reaction that indicates a protective effect on lipid peroxidation of test compound²⁰. Therefore, these methods were used to confirm the antioxidant activities of insect extracts.

The antioxidant activity of each insect extract is shown in Fig. 4. The hydrophilicity of extracted solvent tended to affect the antioxidant activities since both aqueous and ethanolic extracts possessed dominant antioxidant activities in ABTS, DPPH, and FRAP assay, whereas hexane extracts possessed dominant inhibitory activity against lipid peroxidation in FTC assay. The likely explanation might be due to the compatibility of test compounds with test systems²¹. Interestingly, the aqueous extracts, which were obtained from digestion for only 3 h, tended to possess higher antioxidant activities than the ethanolic extracts. The likely explanation might be due to higher hydrophilic property of aqueous (ε = 78.4) comparing to ethanol (ε = 24.5)²². Additionally, higher temperature used in digestion process could be another factor leading to higher extraction efficiency of aqueous. Therefore, the aqueous extracts of most insects possessed the highest scavenging activities on ABTS^·+ and DPPH^·, as well as ferric reducing abilities comparing to other solvents. However, B. mori and L. indicus ethanolic extracts possessed the dramatically highest ferric reducing abilities. This could be explained by various bioactive compounds which have been previous detected in B. mori and L. indicus, e.g. alkaloids, phenolic, and flavonoid compounds, which could be extracted well by ethanol^21,23.

Among various insect extracts, A. domesticus and P. succincta aqueous extracts were predominant as radical scavenger and reductant with the significantly highest TEAC values of 8.8 ± 0.1 and 8.7 ± 0.1 μg Trolox/mg sample, DPPH^· scavenging activities of 19.5 ± 3.8% and 16.1 ± 1.8%, and EC₁ values of 12.1 ± 0.7 and 12.9 ± 0.3 µM FeSO₄/ mg sample, respectively (p < 0.05). These findings were in good accordance with a previous study reported that Gryllodes sigillatus, which is one of cricket species, possessed the highest ABTS^·+ and DPPH^· scavenging activities among several insects²⁴. Additionally, these results corresponded well to a previous study reported that the aqueous extracts of some grasshopper species and A. domesticus possessed the potent ferric reducing abilities with EC₁ values of 2.1 ± 0.2 and 1.8 ± 0.1 mmol Fe²⁺/100 g sample²⁵. Thus, the mechanisms of aqueous insect extracts on oxidation inhibition were various, including radical scavenging activities and ferric reducing abilities.

Apart from A. domesticus and P. succincta aqueous extracts, the significantly highest ferric reducing abilities were also detected in ethanolic extract of B. mori and L. indicus, with EC₁ values of 16.0 ± 0.4 and 15.1 ± 1.4 µM FeSO₄/mg sample, respectively (p < 0.05). The likely explanation might be due to several antioxidant compounds, e.g. quercetin-3,4,-O-diglucoside, phenolics, flavonoids, and riboflavin (vitamin B2), which have been detected in B. mori and L. indicus²⁶. Since B. mori was normally fed with mulberry leaves, quercetin-3,4,-O-glucoside, a derivative of quercetin with two beta-D-glucosyl residues attached at positions 3′ and 4′ which is rich in mulberry leaves, was also detected in B. mori²⁷. According to a potent ferric reducing ability of quercetin-3,4,-O-glucoside²⁸, B. mori ethanolic extract also exhibited strong ferric reducing ability.

On the other hand, the results of lipid peroxidation inhibitory activities showed a different trend in comparison to the other methods. The hexane extract of Euconocephalus sp. and L. indicus had the significantly highest lipid peroxidation inhibitory activities with lipid peroxidation inhibition of 62.5 ± 2.6% and 61.6 ± 2.0%, respectively (p < 0.05). The reason would be lipid peroxidation test system was more compatible with hydrophobic test compounds, which were extracted well using non-polar solvent, e.g. hexane²⁹.

The correlations between the protein content of aqueous insect extracts and their antioxidant activities from various tests, including ABTS, DPPH, FRAP, FTC assays, are shown in Fig. 5. A strong positive correlation was detected in ABTS^·+ scavenging activities with an R² of 0.8013. Additionally, these graphs showed moderate positive correlations between the protein content of aqueous insect extracts and antioxidant activities in DPPH and FRAP assay with R² of 0.7489 and 0.7961, respectively. Hence, protein from aqueous insect extracts was a major antioxidant compound that possessed radical scavenging activities and ferric reducing abilities. A previous study suggested that amino acids were found to be efficient antioxidants due to their chelating properties³⁰. The amino acids had an ability to convert hydroperoxides into imines and sulfur-containing amino acids and could reduce hydroperoxides into the respective inactive hydroxylic derivatives³⁰.

In contrast, there was no relationship between the protein content and inhibitory activities against lipid peroxidation (R² = 0.0924). The explanation might be due to the incompatibility of protein with the lipid peroxidation inhibition test system since most of protein was hydrophilic and soluble well in polar solvent. Therefore, hexane insect extracts might contain some other bioactive compounds, which owning lipid peroxidation inhibitory activity, e.g. flavonoid, lipids, saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids¹¹.

Anti-aging activities of insect extracts

Collagen and elastin are predominant extracellular matrix components presented in dermal layer of human skin³¹. Collagen fibers, which are produced by fibroblasts, are responsible for tensile strength and toughness of skin. These fibers can be degraded by matrix metalloproteinase-1 (MMP-1), also known as collagenase, and resulting in skin aging³². Additionally, elastin fibers comprising about 5% of the dermis layer provide elasticity and resilience of skin. The cleavage of elastin fibers by elastase leads to sagging and wrinkling skin^19,32. Therefore, the bioactive compounds with anti-collagenase and anti-elastase properties could delay skin aging process.

The anti-aging activity of each insect extract is shown in Fig. 6. Among various solvents, aqueous extracts of most insects possessed the highest anti-collagenase activities, followed by ethanolic extracts, and hexane extracts, respectively. Interestingly, the aqueous extract of A. domesticus, B. mori, and P. succincta possessed the significantly highest anti-collagenase activities with the collagenase inhibition of 60.8 ± 2.1%, 54.4 ± 3.9%, and 53.5 ± 1.9%, respectively (p < 0.05). These findings supported a previous study reported that the extract of Gryllus bimaculatus De Geer, which is one of cricket species in Gryllidae family, had a protective effect against wrinkle formation via collagen degradation inhibition³³.

Besides, the significantly highest elastase inhibitory activities were found in the aqueous extract of A. domesticus (17.0 ± 0.1%), P. succincta (16.9 ± 2.7%), and Euconocephalus sp. (12.2 ± 0.9%), as well as hexane extracts of B. mori (22.1 ± 2.5%) and L. indicus (17.1 ± 2.8%) (p < 0.05). These results were in good accordance with a previous study reported that A. domesticus possessed strong pancreatic elastase inhibitory activity³⁴.

Irritation properties of insect extracts

The irritation properties of Thai edible insect extracts were investigated using HET-CAM assay which had been verified for reliability. In the present study, no irritation sign on CAM was induced by a negative control (0.9% w/v NaCl solution) and vehicle controls (DI water and 0.05% w/v DMSO).

In contrast, severe irritation signs on the CAM were detected in a positive control (1% w/v SLS) with IS score of 10.0 ± 0.5 (Table 1). All signs of irritation, including hemorrhage, coagulation, and vascular lysis were detected on the CAM exposed to 1% w/v SLS within 5 min and more pronounced after 60 min as shown in Fig. 7.

All insect extracts in the present study were safe since they induced no irritation on CAM, except for P. succincta. Ethanolic and hexane extracts of P. succincta induced moderate irritation with IS of 6.2 ± 0.5 and 7.4 ± 0.4, respectively. All irritation signs were detected on the CAM after exposed to P. succincta hexane extract, whereas only vascular lysis and hemorrhage were detected on the CAM after exposed to the P. succincta ethanolic extract as shown in Fig. 8. The results were related well to a previous study reported that P. succincta could trigger hypersensitivity reaction and P. succincta was identified as a cross-reacting allergen with crustaceans³⁵. Therefore, the hexane and ethanolic extracts of P. succincta might be noted as a skin irritant and had a precaution for using topically on human skin.

Since HET-CAM assay was usually employed as an alternative irritation test for human tissue, including eye and/or skin irritation³⁶. Extracts from B. mori, O. fuscidentalis, Euconocephalus sp., A. domesticus, and L. indicus were suggested as safe for using topically on human skin.

Skin irritation effects of insect extracts on human volunteers

The irritation effects of Thai edible insect extracts on human skin are shown in Fig. 9. Since the ethanolic and hexane extracts of P. succincta induced irritation signs in HET-CAM test, they were excluded from a clinical study in humans. Among various insects, B. mori and L. indicus extracts induced skin irritation in humans. The aqueous extracts of B. mori and L. indicus induced itching in 2 among 30 individual volunteers. Additionally, hexane extract of L. indicus induced mild erythema, whereas ethanolic extract of B. mori showed severe signs of skin irritation, including papules and itching. However, there were no signs of edema, vesicles, bullae, or weeping in any volunteer. These results related well with a previous study reported that tropomyosin, which was a dimeric coiled-coil protein for muscle regulation, might contribute significantly to B. mori allergy. Furthermore, B. mori has been reported to be one of a strong IgE cross-reactivity with crustacean³⁷. Additionally, a previous study also reported that L. indicus appeared to be the insect that was most likely to cause an allergic reaction³⁸. Nonetheless, there have not been identified allergen of L. indicus in previous studies. Thus, the extracts of B. mori and L. indicus, which caused irritation in human volunteers, should avoid for further topical applications.

In conclusion, there was a significant difference among different insect species (Wilks’ lambda = 0.000, F (45,128.3) = 350.975, p = 0.000) and extraction method (Wilks’ lambda = 0.000, F (18,56) = 1164.921, p = 0.000). Aqueous extracts of A. domesticus exhibited the significantly highest antioxidant, anti-collagenase, and anti-elastase activities without irritation in both HET-CAM and clinical studies. As a result, A. domesticus was proposed for further usage as a cosmeceutical active ingredient for anti-wrinkle skin care. However, it was advised that the chemicals responsible for these biological activities should be further identified. Although utilizing the entire body of the insect would facilitate scale-up production in future applications, further independently investigation for each part of the insect, such as the head, thorax, and abdomen of A. domesticus would be suggested. The current study’s findings would lead to the selection of an appealing insect family for further intensive comparative research.

Source: Ecology - nature.com