2.1. Chemical Composition of Aqueous Plant Extracts
In our previous research, we determined the compositions of garlic (
Allium sativum L.) and clove (
Syzygium aromaticum L. Merr. & Perry) aqueous extracts [
10]. The main components of the clove extract were eugenol and eugenol acetate, amounting to 82.39% and 4.56% of the total composition, respectively. The garlic extract was dominated by 5-HMF (33.24%), followed by furan-2-carboxaldehyde (7.09%), acetaldehyde (5.17%), and 3-methylbutanal (4.27%) [
10]. The other aqueous extracts used in the present study were sage (
Salvia officinalis L.) and turmeric (
Curcuma longa L.). The volatile components of the plant extracts were analysed and identified by gas chromatography–mass spectrometry (GC-MS) combined with Kovats retention index. This is a commonly used method for the characterization of volatile compounds in various plant extracts [
14]. The chemical compositions of the tested extracts are presented in
Table 1 and
Table 2. The chromatograms show great variation in the chemical profiles of the plant extracts tested. The most important chemical compounds are listed int the tables, but the synergistic effects of other compounds should not be excluded.
Salvia spp. belong to the family of Lamiaceae and are associated with potent antimicrobial activity, primarily due to their chemical constituents [
15,
16]. According the Drugs and Lactation Database LactMed
® [
17], sage leaves contain mainly tannins (salviatannin), essential oils (including α-thujone,
β-thujone, 1,8 cineole, and camphor), flavones, phenolic acids, phenylpropanoid glycosides, triterpenoids, and diterpenes. The tested sage extract was dominated by camphor (31%), α-thujone (24%), and eucalyptol (18%) (
Table 1,
Figure 1).
The antimicrobial activity of these compounds is well known [
18,
19]. Sage essential oil is active against the mycelial growth of the strawberry pathogens
Colletotrichum acutatum [
20],
Alternaria alternata,
Botrytis cinerea, and
Fusarium oxysporum [
16]. It is worth noting the presence in the profile of 2-metoksy-4-vinylphenol (11%). This compound often causes severe skin burns and eye damage [
21].
Curcuma longa (turmeric) belongs to the family Zingiberaceae. Turmeric essential oil acts as a natural food preservative and alternative to chemical fungicides. It has been shown to have a significant antifungal effect against
Penicillium expansum,
Fusarium solani,
Rhizopus stolonifera, and
Alternaria alternata [
22]. In our study, the turmeric extract was composed mainly of 1-hydroxy-2-propanone (acetol) (21%), ar-turmerone (19%), and ethanol (14%) (
Table 2,
Figure 2).
These compounds have known potential antimicrobial activity [
23,
24]. However, it is worth noting the significant share (17%) of unidentified compounds in the profile. The effects of these compounds on living organisms are unknown.
2.2. Cytotoxicity of Plant Aqueous Extracts
Sf-9 (
Spodoptera frugiperda) and HeLa cells were exposed to the extracts for 24 h. Non-cytotoxic concentrations (IC
0) and IC
50 were determined for all extracts and both cell lines (except for the garlic extract, for which only the IC
20 value was determined due to its weak cytotoxicity towards HeLa cells). Against the Sf-9 insect cell line, the extracts with the highest cytotoxicity were garlic (IC
50 41.6 mg/mL), followed by turmeric and sage, and the weakest cytotoxicity was shown by clove (
Table 3). Against the model HeLa cell line, sage extract showed the strongest cytotoxicity (IC
50 49.6 mg/mL), followed by clove, turmeric, and finally garlic. Overall, the garlic and turmeric extracts showed stronger cytotoxicity against Sf-9 insect cells than against HeLa cells. λ-Cyhalothrin always showed the highest cytotoxicity, which was more than 200 times stronger than the cytotoxicity of the garlic extract against Sf-9 cells.
S. frugiperda insects are common pests of plants belonging to the Solanaceae Juss. family. Sf-9 cells are the most established insect cells. They are used in agricultural research and toxicity studies. They are particularly valuable for assessing the ecotoxicity of environmental pollutants, studying the control of plant pests and crop protection strategies (especially in mass production systems), evaluating the cytotoxicity of insecticides, and supporting the development of new biopesticides against crop pests [
25]. In vitro studies using Sf-9 cells allow for rapid toxicity screening of biopreparations and their components.
The cytotoxicity and mechanism of cytotoxic action of garlic extracts have been investigated for many human cell lines, including tongue squamous carcinoma (SCC-15), normal skin fibroblasts (BJ), epithelial papilloma (KB), leukemic (U-937, Jurkat, E6-1, K-562, TIB-152), cervix adenocarcinoma (HeLa), colorectal cancer (DLD-1), lung squamous carcinoma (SK-MES-1), colon adenocarcinoma (Caco-2), hepatic carcinoma (Hep-G2), prostate cancer (PC-3), breast cancer(MCF-7), and normal human keratinocytes (HaCaT) [
26,
27,
28,
29,
30,
31,
32]. The main mechanisms of the cytotoxic action of garlic extracts are the induction of reactive oxygen species (ROS) generation, inhibition of proliferation, and cell death by necrosis or apoptosis [
27,
28,
29,
32]. In our study, we used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to evaluate cytotoxicity. The MTT assay is widely used to measure cell metabolic activity, which is an indirect indicator of cell viability. The high cytotoxicity of the garlic extract in our study may be due to the content of acetaldehyde. Although it did not account for a large proportion of the extract’s composition (about 5.2%), acetaldehyde can exhibit strong mitophagy-related cytotoxicity, which involves the removal of damaged mitochondria through lysosomal degradation. The results of the MTT assay may also reflect impaired and reduced mitochondrial function after exposure to this compound [
33]. This may be linked to a decrease in mitochondrial membrane potential (MMP), ROS production, and the ATP level in the cell [
33,
34]. Tigu et al. [
32] obtained IC
50 values ranging from about 8.8 to 63 mg/mL, depending on the cell line. In our study, the IC
50 value for the Sf-9 cells was within this range, at 41.6 mg/mL. As in the study by Tigu et al., normal cells were much more sensitive to garlic extract than cancerous cells.
The cytotoxic potential of clove extracts has likewise been tested on a number of human cell lines, including colon adenocarcinoma (HCT 116, HT-29), breast adenocarcinoma (MCF-7, AU 565), HeLa, Hep-G2, and lung alveolar carcinoma (A-549) [
35,
36,
37,
38,
39,
40,
41]. The main mechanism of cytotoxic action of clove extract is the induction of apoptosis. The main component responsible for cytotoxic effect is eugenol [
42]. In our study, eugenol and eugenol acetate accounted for more than 86% of all components in the clove extracts. Various extracts from different parts of clove (e.g., leaves, stems, fruits, buds, rhizomes) display cytotoxic effects on cell lines by inhibiting proliferation, inducing ROS, and causing cell cycle arrest [
43].
The cytotoxicity of curcumin may be related to its uptake into the cell. As a lipophilic molecule, curcumin interacts with the cell membrane and is then transported into the cell [
44]. The more curcumin enters the cell, the greater its cytotoxicity. This mechanism has been demonstrated for cell lines including mouse T lymphoblast EL4, human MCF-7, and murine fibroblasts NIH3T3 [
44]. Ar-turmerone accounted for almost 19% of the components in the studied turmeric extract. This compound has been shown to contribute to the uptake of curcumin into the cell in vitro [
45]. Many other studies have reported that curcumin inhibits the growth of various cells, including mouse skin melanoma—B16-F1, human colon adenocarcinoma—COLO 205, HCT 116, and Caco-2, human hepatic stellate—LX-2, Hep-G2, and HeLa, in assays based on tetrazolium salts (MTT, MTS, WST-1) [
46,
47,
48,
49]. In HeLa cells treated with curcumin, apoptotic changes and cell cycle arrest (in G0/G1 and S phases) were observed in proportion to its concentration [
49]. Ar-turmerone may be responsible for this cytotoxic effect and contribute to the induction of apoptosis in cells [
50]. Curcumin-based secondary metabolites have well-documented potent cytotoxic activities [
51]. Curcumin-based linear diarylheptanoids 35a and 35b have been shown to display antiproliferative activity on human cancer cells (MCF-7 and Hep-G2). Furthermore, 35b increases GADD45B (growth arrest and DNA-damage-inducible, beta) expression, leading to inhibition of MCF-7 proliferation and death. The same study confirmed the importance of the conjugated group for antiproliferative activity [
51]. Some linear diarylheptanoids, due to their chemical structure (at least two substituents on aryl rings), demonstrate higher cytotoxic and anticancer properties against cancer cell lines than curcumin [
52]. Sage extract may be cytotoxic to Hep-G2 cells, causing morphological changes in cells and inhibiting proliferation, while decreasing the intracellular ATP level, lactate dehydrogenase (LDH) leakage, and apoptosis [
53]. Sage extract can also induce apoptosis in human lymphoma and leukaemia cells [
54] and show cytotoxicity against other human-derived cell lines, including HeLa and MCF-7 [
55]. One of the main components of the tested sage extract was eucalyptol, which can display cytotoxicity in cells by inducing apoptosis [
56]. Other components included camphor and α-thujone. All these compounds (camphor, eucalyptol, α-thujone) can be cytotoxic to colon carcinoma cells (MRC-5, HT-29, and HCT 116) [
57].
To the authors’ best knowledge, the studied aqueous extracts have not previously been considered as bioinsecticides against Sf-9 cells. Lopes et al. [
58] investigated seven different plant extracts (in water, ethanol, or dichloromethane as solvents) against Sf-9 cells, observing that the less polar samples were more cytotoxic. Dichloromethane extract caused a loss of cell viability that exceeded the effect of the commercial insecticide chlorpyrifos. In our study, λ-cyhalothrin always showed more cytotoxicity than the tested extracts. λ-Cyhalothrin belongs to the class of Type II pyrethroid insecticides. This compound has insecticidal properties and is used to control a wide range of insect pests and diseases. It is one of the most powerful pyrethroid insecticides in the world. Due to its acute toxicity, the insecticide is extremely toxic in mice and induces hepatic and renal toxicity in rats. In cells in vitro it induces micronuclei, nucleoplasmic bridges, nuclear buds, necrosis, and apoptosis, which is positively related with carcinogenicity [
59]. The main mechanisms of cytotoxicity of λ-cyhalothrin are hepatotoxicity, neurotoxicity, nephrotoxicity, and reproductive toxicity, as well as induction of oxidative stress leading to mitochondrial, lipid, DNA, and protein damage in cells [
60]. The overall mechanism of toxic action of λ-cyhalothrin is due to its chemical structure. However, this mechanism is not fully understood and is the subject of systematic research [
60].
2.3. Cytotoxicity of Microbial Metabolites
Sf-9 and HeLa cells were exposed to the microbial metabolites for 48 h.
L. plantarum KB2 LAB 03 metabolites were more cytotoxic to Sf-9 cells at physiological pH than at neutral pH (
Table 4).
L. plantarum KB2 LAB 03 metabolites showed greater cytotoxicity after culturing in MRS than after culturing in AM at physiological pH. The cytotoxicity of
L. plantarum KB2 LAB 03 fermentation by-products against the HeLa cell line showed less variation. However, the metabolites cultured in MRS also showed greater cytotoxicity than those cultured in AM. Similarly, greater cytotoxicity was observed for yeast metabolites towards Sf-9 cells at physiological pH. The highest cytotoxicity was observed for metabolites cultured on sYPG medium and the lowest after culturing in sYP. In the case of the metabolites cultured on sYPG, no cytotoxicity was observed at pH 6.2, but an enhanced pro-proliferative effect was noted. Yeast cultured in sYP at both pH options had the same effect on HeLa cells (
Table 4). The yeast metabolites cultured in sYPG also showed the strongest cytotoxicity towards HeLa cells.
In our previous studies, we identified the main metabolites of
L. plantarum KB2 LAB 03 after cultivation in MRS medium. The metabolites were mostly lactic, acetic, and isobutyric acids. The main fermentation by-products of the strain cultured in AM (acid whey-based) medium were lactic, acetic, and propionic acids, as well as ethanol [
5]. We also studied the main metabolites of
M. pulcherrima TK1 after cultivation in YPD medium. The main metabolites were ethanol and glycerol, as well as lactic, succinic, and acetic acids. After fermentation in sYP (acid whey-based) medium, the by-products were lactic acid, ethanol, glycerol, acetic, and succinic acids [
8]. Organic acids such as propionic acid can also suppress the viability of HeLa cells, by inducing ROS generation and dysfunction of the mitochondrial membrane, leading to autophagy [
61]. Elsewhere, we showed that pure lactic, acetic, and propionic acids inhibit the growth of Caco-2 cells [
62]. We screened the cytotoxicity of metabolites of 39 LAB strains against Caco-2 cells (at neutralised pH). The metabolite concentrations ranged from 10 to 200 mg/mL. Cytotoxicity depended on the concentration of metabolites: the higher their concentration, the higher the cytotoxicity.
In the current research, we found LAB metabolite concentrations ranging from 0.001 to 200 mg/mL. Their cytotoxic effect may be related to the acidic pH of some LAB metabolites. In the case of Sf-9 cells, cytotoxicity increased at lower sample pH. Such a relationship was not observed for HeLa cells cultivated with LAB fermentation by-products and not at all for yeast metabolites. This may indicate that a different mechanism is responsible for their cytotoxicity. It has been suggested that the protein nature of secreted metabolites may be responsible for their cytotoxicity [
63]. According to the literature, several cell components (cytoplasmic fractions, fermentation by-products including organic acids, cell wall components, exopolysaccharides (EPS), peptidoglycan, conjugated linoleic acids, and S-layer proteins) can have strong cytotoxic effects on various cell lines [
64,
65,
66]. Bacteriocins such as plantaricin A produced by
L. plantarum and enzymes such as arginine deiminase produced by some strains of LAB can also inhibit the growth of some cells in vitro [
66]. Cell wall components of yeast, β-glucan and chitin, can have the same effect [
67,
68]. Generally, the cytotoxicity of microbial fermentation by-products depends on the strain being tested, the cell line, and the culture medium. The main mechanisms of their cytotoxic activity are cell cycle arrest, apoptosis, necrosis, increased ROS production, and decreased intracellular ATP and MMP [
60,
62,
69].
2.4. Genotoxicity of Plant Aqueous Extracts
After screening for cytotoxicity, extracts with concentrations close to or below the IC
50 values were selected for further testing for genotoxicity in the comet assay (
Table 5 and
Table 6). Due to their different cytotoxic activity against the two cell lines, different concentrations were used. Concentrations higher than the IC
50 values induced severe, unmeasurable DNA damage (apoptotic cells), so were not tested. Due to the wide variety of concentrations tested in the cytotoxicity assays, they could not be standardized in genotoxicity tests.
The rate of DNA damage in the negative controls was 2.35% ± 5.74% (for HeLa cells) and 4.54% ± 0.48% (for Sf-9 cells). At the tested concentration of 1.3 mg/mL, λ-cyhalothrin was 4-fold more cytotoxic against Sf-9 cells than HeLa cells. It was noticed that λ-cyhalothrin always displayed the strongest genotoxicity, which was also greater against insect Sf-9 cells than human HeLa cells. The genotoxicity of λ-cyhalothrin against Sf-9 cells was 42.32% ± 3.59% at a concentration of 0.63 mg/mL and 42.62% ± 3.31% at a concentration of 1.3 mg/mL. Against HeLa cells, its genotoxicity was 10.46% ± 2.56% at 1.3 mg/mL and 35.20% ± 4.00% for concentrations of 1.3 and 2.5 mg/mL, respectively.
Sf-9 cells were more sensitive to garlic and turmeric extracts than HeLa cells at similar concentrations, and genotoxicity was not proportional to concentration. Garlic extract showed the highest genotoxicity against Sf-9 cells (close to 50%), and clove extract showed the highest genotoxicity against HeLa cells (up to 44.55%) at a concentration of 50 mg/mL (
Figure 3).
The variability of the raw material and extracted components is a significant factor that limits studies on the toxicity of extracts from different plants. Aqueous and alcoholic garlic extracts have been shown to induce genotoxicity in peripheral blood lymphocytes, as evaluated in the comet assay, and cause chromosome aberrations in human leucocytes [
70,
71]. The main component of our garlic extract was 5-HMF, which can demonstrate genotoxicity at higher concentrations [
72]. 5-HMF can induce DNA damage in Hep-G2 cells, suggesting a weak genotoxic effect, probably due to its rapid cell repair capacity [
73]. This weak genotoxic effect may explain why in our study HeLa cells were practically resistant to the activity of garlic extract. According to the literature, eugenol—the main component of clove extract—can induce moderate to severe genotoxic effects in hamster lung fibroblasts V79, in a dose-dependent way [
74]. In our study, eugenol may have been responsible for the medium genotoxicity of clove extract against the tested cells. Beltzig et al. [
75] investigated the genotoxicity of ethanolic solvent and micellar curcumin against several primary and cancerous cell lines. They showed that curcumin had similar genotoxic effects against the cancerous cell lines, but also observed high efficiency of DNA repair after its removal. Crude extract of
Curcuma longa L. demonstrates genotoxic activity against genomic DNA in the human tumour cell lines: prostate DU-145 and colon HT-29 [
76]. Curcumin at high concentrations displayed a significant increase in MN frequency in the micronucleus (MN) test [
77]. The main components of sage extract, camphor, eucalyptol, and α-thujone induced evident DNA breaks in human colon HT-29 and HCT 116 cells, foetal lung fibroblasts MRC-5, and Vero (kidney monkey) cells [
57]. These compounds may be responsible for the genotoxicity of sage extract observed in our study.
2.5. Genotoxicity of Microbial Metabolites
After screening for cytotoxicity, extract concentrations close to or below the IC
50 value were selected for further testing (
Table 7,
Table 8,
Table 9 and
Table 10,
Figure 4). Due to their different cytotoxic activity against the two cell lines, the tested concentrations were also different. Concentrations higher than the IC
50 values induced very strong unmeasurable DNA damage (including apoptotic cells), so they were not used. In general, the genotoxicity of the bacterial metabolites was not dependent on the pH or concentration (
Table 7). The genotoxicity of the metabolites against Sf-9 cells was not dependent on their concentration and remained at similar levels. Metabolites of
L. plantarum in AM at physiological pH showed the highest genotoxicity (54.52 ± 4.45%) at a concentration of 50 mg/mL compared to the other bacterial samples (
p ≤ 0.05). Yeast metabolites showed generally higher genotoxicity than LAB metabolites. Independently of pH, yeast metabolites in sYPG showed the strongest genotoxicity (
Table 8), which was correlated with cytotoxicity. On YPG, greater genotoxicity was observed at physiological (acidic) pH.
The bacterial and yeast metabolites showed no or weak genotoxicity against HeLa cells, compared to the Sf-9 line (
Table 9 and
Table 10). Genotoxicity was weakly or uncorrelated with metabolite concentration. Metabolites of
L. plantarum KB2 LAB 03 in MRS showed the highest but weak genotoxicity regardless of pH, and in AM these metabolites showed the lowest genotoxicity. In the case of HeLa cells, yeast metabolites showed greater genotoxicity than LAB metabolites. Yeast metabolites in sYPG medium showed the weakest genotoxicity at the tested concentrations.
To the best knowledge of the authors, the genotoxicity of by-products of fermentation by LAB and yeast on cell lines has not previously been studied. Since one of the mechanisms for the toxic effects of the tested microbial fermentation by-products may be increased intracellular ROS production, they can induce oxidative damage to DNA, leading to apoptosis [
65,
78]. Ethanol can also be genotoxic, as was shown in a comet assay using peripheral blood lymphocytes as well as human primary gastric and colon mucosa [
79].