Chemical characterization of SRE
The spectrophotometry analysis shows that there are about 83.5 g of saponins per 100 g of extract. This amount is higher than the results obtained by Bhargava et al.31 (20 g/100 g to 40 g/100 g of extract), Segura et al.8 (60% saponins found in quinoa husk extract), and Castillo-Ruiz et al.32 how found an amount of 36% w/w of total saponins after aqueous extraction and alkaline treatment, which confirms the richness of our extract in saponin. On the other hand, Muir et al.33 found an amount greater than our finding (85–90% w/w saponins) after alcohol extraction followed by acid hydrolysis of quinoa brain. The ethanol is considered the best solvent for the extraction of saponins because of their high solubilization properties34. Overall, the variation of the amount of saponins is due to several parameters, among them the extraction method, the used solvent, and the initial saponin content of the raw material35.
FTIR analysis
The infrared spectrum of the saponin extract (Table 1; Fig. 1a) disclosed the presence of broad and strong -OH signaling hydroxyl groups (3290 cm− 1) in the side chain of the saponin oligosaccharide. The band at 2945 cm− 1 attributable to C-H is a trait of alkane compounds36. Whereas, the bonds at 2883 cm− 1, 1718 cm− 1, and 1620 cm− 1 are attributable to the stretching of CH2, C=O, and C=C respectively37,38,39. Therefore, the absorption between 1150 cm− 1 and 1350 cm− 1 indicates the presence of C–O–C group. This spectrum was compared with the spectrum of Quillaija saponin standard (Fig. 1b). According to the obtained spectrum and spectrophotometry analysis (Sect. 3.1), the detected OH, C=O, C–H and C=C bands confirm the richness of the extract on saponin, which is in line with the claims of Mishra & Thakur40.
IR spectra of SRE (a) and saponin Quillaija standard (b).
Effect of SRE on T. castaneum by contact action
Effective dose estimates of SRE
The saponin rich extract of quinoa showed a toxicity effect on T. castaneum adults. Table 2 presents the data of Effective Dose Estimates (ED) (in mg/mL) under their respective 95% confidence intervals (95% Limits) with the mortality percentages of 10% (ED10), 50% (ED50), 90% (ED90), and 99% (ED99). As shown, the concentration of 13.10 mg/mL SRE was necessary to attain the mortality of 10% of the population, and the median lethal concentration (ED50) was 43.26 mg/mL after one day of exposure. Furthermore, the mortality of 99% of the population was achieved with 378.52 mg/mL SRE. The noticeable effects displayed by quinoa bran extract can be related to its main triterpene components, which is supported by previous research studies that have shown that saponins possess insecticidal properties by topical application against different storage insects3,24,41.
Observation of the epidermis and surface structure of T. castaneum adults treated with SRE by Scanning Electron Microscopy (SEM)
SEM was used to investigate the effects of SRE treatment on the epidermis and surface of the T. castaneum adults (Fig. 2). In comparison to the control treatment (distilled water) (Fig. 2, a1), the epidermis waxy layer of insects treated with SRE was ablated (Fig. 2, b1), resulting in the loss of certain pits and the formation of holes in the epidermis of the insects. Hence, SRE induces a dysfunction of the epidermis as the waxy layer protects the skin from drying out, senses the environment, and gives mechanical support and mobility to the insect42. The Fig. 2, b2, presents T. castaneum adult abdomen treated with aqueous solution containing SRE. The depicts showed a damage in the abdomen compared to the control (Fig. 2, a2). This alteration of the epidermis of insects was previously remarqued by researcher. Cui et al.13 proved that tea saponins can penetrate in E. obliqua via its cuticle because of their elevated viscosity and low interfacial tension, resulting in higher bioavailability of saponins than other natural compounds. Consequently, higher disruption of the insect waxy layer. Wakil et al. suggested that the main activity of diatomaceous earth on storage insects is abrasion of the insect cuticle and sorption of the wax layer43. May because of the polar characteristic of saponin molecules, the waxy covering of the insect body is damaged, allowing the active component to permeate the surface of the body and reach the target location, resulting in a significant contact toxicity44.
Scanning electron microscopy images of the surfaces of T. castaneum adults. Control treatment (a) and SRE treatment (b) (1 and 2, corresponding to thorax layer 3000x and abdomen 250x, respectively).
Effect of quinoa saponin rich extract on the diet and nutritional indexes of T. castaneum adults
Nutritional indices
The nutritional study was carried out to assess the relative palatability and growth response of T. castaneum to the flour disks amended with different concentrations of SRE (Table 3). The results showed a negative correlation between the growth rate (RGR) of insects and the concentration of the extract, with a significant decrease noted in the insects under the diet with SRE concentrations of 25 mg/g and beyond. A negative value of RGR was registered from the concentration of 50 mg/g to 100 mg/g and at the level of the starved group, which means that the insects have lost weight. It is noted that the insects under the 100 mg/g SRE diet lost more weight (-0.32 ± 0.05 mg/d), followed by the insects under the 75 mg/g and 50 mg/g SRE diets, and finally the starved group with a RGR equal to -0.19 ± 0.01 mg/d.
Likewise, the RCR of the studied insects decreased with increasing SRE concentrations, and from concentrations above 75 mg/g, the insects no longer consumed the disks. In previous studies, Lima et al.24 noted the growth-inhibiting and antinutritional activities of crud extracts containing saponins from G. americana against T. castaneum with loss of insects’ weight from the concentration of 100 mg of extract. However, at low extract concentrations, the insects fed but did not convert it to biomass, which confirms the positive correlation between the insect weight and the food consumption rate23,24.
Food deterrence index (FDI)
The Fig. 3a presents the results of the FDI in proportion to SRE doses. Overall, SRE extract demonstrated to be a feeding deterrent agent. The doses of 5 mg/g and 10 mg/g induced low food deterrence levels of about 44.32% and 44.64%, respectively. Under these doses, the insects were able to feed from amended disks. The value of FDI for the insects under the diet with 25 mg/g SRE was increased drastically up to 84.20%, and the insects in the box of the treated disks with 50 mg/g, 75 mg/g, and 100 mg/g were increased up to 92.69%, 93.60%, and 96.82%, respectively, with a non-significant difference. These findings are in accordance with the results obtained at the level of food consumption rate. In the same context, previous research studies have found that pest insects fed an artificial diet containing saponins consume less food due to digestive disruption3,24. The inclusion of total saponins from alfalfa in the diet of Colorado potato beetle larvae lowered their dietary intake, growth rate, and survival45. Nielsen et al. investigated the function of saponins in Barbarea vulgaris resistance to the flea beetle (Phyllotreta nemorumi L.) (Coleoptera: Chrysomelidae). They examined the feeding-deterrent action of isolated saponins from B. vulgaris and found that hederagenin cellobioside has a significant feeding deterrent and defensive chemical against the flea beetles46.
Food Deterrence Index (FDI%) in proportion to SRE doses (a), Variation of T. castaneum survive under the diet with different doses of SRE for 60 days (b). Different letters indicate significant differences (p < 0.05) among treatments by Tukey’s test.
Effect of SRE on insect survival
The number of surviving insects was recorded over 60 days to assess the long-term effect of saponins. Figure 3b shows a positive correlation between the insect mortality rate and the dose of saponin extract. A mortality rate of 20% of the population was observed at the concentration of 100 mg/g after 10 days, whereas this mortality rate was achieved for 25 and 50 mg/g after 30 and 20 days, respectively. For 75 mg/g and starved insects neededn17 days to attain this 20% mortality. On the other hand, the population under a 0, 5, and 10 mg/g saponin diet was found to need 60 days to lose about 20% of insects. However, the population under the diet treated with 100 mg/g of saponins was the first to achieve 100% mortality after 60 days of incubation, earlier than the starved one. This result shows that the mortality of T. castaneum is attributable to the toxicity of the product and not just the starvation action. These findings are consistent with the result of Lima et al.24 previously found that the mortality of T. castaneum was up to 73% at 250 mg of extract rich in saponins from Genipa americana per g of wheat flour after 28 days of consuming the diet. In comparison with 100 mg/g in our case, a mortality rate of 73% was reached also after around 28 days. Shahriari et al.47 also reported the mortality effect of tea saponins on the larvae of Ephestia kuehniella Zeller. Moreover, Szczepanik et al.45 investigated the effect of saponins from several Medicago species on the larvae of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae). Their results showed that the extracted saponins reduced the growth rate of the insect and affected the survival of the larvae. On the other hand, McCartney et al. showed that quinoa bran extract was insecticidal only against non-quinoa feeders (Pseudaletia impuncta) and had no observable negative effects on survival or developmental time for quinoa feeders (Feltia subterranea and Trichoplusia ni), suggesting that the abundances and/or types of saponins produced by explored quinoa genotypes are insufficient to protect these adapted species48.
Effect of SRE on enzymatic activities of T. Castaneum
Phenoloxidase activity
Phenoloxidase (PO) in insects has a key role in the immune defense system, and some studies have shown its role in sclerotization and brown pigmentation of the T. castaneum cuticle49,50. The effect of SRE on PO activity of T. castaneum was determined using L-DOPA as a substrate. For the antifeeding test, results showed a significant decrease in PO activity by introducing SRE into the diet of insects. A low value of PO activity was noted in the insects under a diet of 50 mg/g of SRE (0.015 U/mg protein) (Fig. 4a). This aligns with Mirhaghparast et al. findings, where saponin was found to suppress the immune system by decreasing the PO activity51. The same significant reduction in PO activity was revealed in starved insects. The research of Ebrahimi and Ajamhassani52 reported that the PO activity was considerably lowered in the larvae of Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) because of starvation. Therefore, the low concentrations of SRE have reduced the level of phenoloxidase activity by acting as an inhibitor, while the decrease of PO activity from the concentration 75 mg/g can be explained by the starvation effect based on the results of nutritional indices. The study of Zibaee & Bandani showed that the plant extract can interfere with the PO activity of Eurygaster integriceps (Heteroptera: Scutellaridae), and a significant decrease in PO activity was noted with the increase of extract concentrations53. For the topical test, Fig. 4b shows a slight increase of PO activity in insects treated with 5 and 25 mg/mL. In contrast, a non-significant difference was observed between the control and the extract at 50, 75, and 100 mg/mL. This results can be explained by the capacity of saponins, at low concentrations, to induce the activation of PO, reflecting as a defense strategy of the insect against saponin extract as a stressful agent54. On the other hand, at concentrations above 50 mg/mL, the saponins act as inhibitors of the phenoloxidase due to their antioxidant capacity50, which confirm that the defense system of T. castaneum from all kinds of inhibitors and activators largely reliant on PO activity55,56. Otherwise, the variation of the effect of SRE concentrations between the nutritional and topical tests demonstrated the advantageous impact of the diet on the insect immune defense system52; At the level of nutritional test the low concentrations can inhibited the PO activity compared with the topical test the inhibition start at concentrations above 50 mg/mL.
Phenoloxidase (PO) activity in the adult of T. castaneum exposed to SRE by a flour disk diet amended with different concentrations of SRE for antifeeding test (a) and treatment with SRE aqueous solution for the topic test (b). The enzyme values are expressed in U/ mg protein as mean (± SD) values and analyzed by one way analysis of variance (ANOVA). Different letters indicate significant differences (p < 0.05) among treatments by Tukey’s test. S: Starved group.
Catalase activity
The effect of SRE on the catalase activity of T. castaneum is shown in Fig. 5. Overall, there was a significant increase of catalase activity in insects under SRE diet (Fig. 5a) and in insects exposed to SRE at the level of topical assay (Fig. 5b) when compared to the control (P ≤ 0.001). Additionally, a positive correlation was observed between the catalase activity and the SRE doses in the nutritional study. A similar activity increase in antioxidant enzymes, including the catalase activity of E. kuehniella (Zeller) (Lepidoptera: Pyralidae), under diets containing different concentrations of saponins isolated from tea, was reported by Shahriari et al.47. Also, Manjula et al.28 reported a significant rise in catalase activity in Spodoptera litura (Lepidoptera: Noctuidae) after its exposure to the leaf extract of M. esculenta. For the starvation effect, starved insects demonstrated higher catalase activity. Likewise, Peter et al.57 registered a significant increase in catalase activity in the starved group of juvenile loaches (Paramisgurnus dabryanus) (Cypriniformes: Cobitidae) comparing with the feed group. Following starvation stress, insects produce more reactive oxygen species (ROS), which activate their antioxidant system. Antioxidant enzymes contribute to eliminating the harmful free radicals generated because of the toxicant exposure28,58. Herein, the catalase primarily involves the decomposition of H2O2, resulting in the synthesis of water and molecular oxygen. The increase in catalase activity protects insects from oxidative damage and stress58. Conversely, the decrease in catalase activity can lead to high levels of superoxide anion radicals, which can inhibit or slow down the catalase enzyme function26.
Catalase activity in the adult of T. castaneum exposed to SRE by a flour disk diet amended with different concentration of SRE for antifeeding test (a), and treatment with SRE aqueous solution for the topic test (b). The enzyme values are expressed in µmol H2O2 / mg protein as mean (± SD) values and analyzed by one way analysis of variance (ANOVA). Different letters indicate significant differences (p < 0.001) among treatments by Tukey’s test. S: Starved group.
Glutathione S-transferase (GST) activity
Detoxification enzyme bioactivity, including GST activity, has a crucial role in an insect’s ability to survive and grow under stressful environments, including pharmacologically active compounds59. Figure 6 illustrates the SRE effects on GST activity of T. castaneum adults. For the nutritional study (Fig. 6a), the highest GST activity in insects was observed under diet with 5 mg/g of SRE, while lowest GST activity was registered at 100 mg/g of SRE. However, results disclosed a non-significant difference in the GST activity of insects under the diet with other concentrations, i.e., 10 to 75 mg/g of SRE when compared to the control (P ≤ 0.05). For the topic test (Fig. 6b), an upregulation of GST activity was noted with the rise of SRE concentrations from a mean of 0.82 mU/mg protein in the control to 2.10 mU/mg protein in 100 mg/mL.
GST activity in the adult of T. castaneum under a flour disk diet amended with different concentrations of SRE for antifeeding test (a), and exposure to SRE aqueous solution for the topic test (b). The enzyme values are expressed in mU/mg protein as mean (± SD) values and analyzed by one way analysis of variance (ANOVA). Different letters indicate significant differences (p < 0.05) among treatments by Tukey’s test. S: Starved group.
These findings are consistent with Shahriari et al.39, where tea saponins were found to induce a high GST activity in E. kuehniella. Likewise, Dhivya et al.58 reported that the levels of GST enzymes in S. litura raised after 2 h of exposure to the hexane extract of Prosopis juliflora. Also, Li et al.60 observed a similar pattern in T. castaneum treated with various doses of paeonol extract from Moutan cortex. In the same vein, Manjula et al.28 revealed a significant increase in GST activity in S. litura (P ≤ 0.001) after 4 h of exposure to leaf extract of M. esculenta. It was also disclosed that GST activity decreased gradually after 12 and 24 h of leaf extract exposure, compared to the control (P < 0.05). This drop might be attributed to elevated ROS levels, which have a dramatic effect on cells and can induce cellular damage, inhibiting GST enzymes. In line with above, Czerniewicz et al.61 demonstrated that GST enzyme bioactivity was significantly boosted after a low dose exposure of aphids into clove oil, while this bioactivity was reduced at higher doses. In this study, GST activity was significantly simulated by low concentrations of SRE i.e., 5 mg/g. However, at concentrations exceeding 75 mg/g of SRE, GST activity decreased in insects, indicating that GST activity is an SRE concentration dependent parameter as it is influenced by the concentration of the toxic compound applied. This pattern is positively correlated with observed oxidative damage of insect tissues at higher concentrations. Noteworthy to mention that GSTs play critical roles in the evolution of insecticide resistance and antioxidant defense62. Hence, the increase of GST activity is positively correlated with insect resistance to insecticides, which means that the reduction of this enzyme activity will hinder insect resistance evolution60,63,64.
Amylase activity
The α-amylase variation in T. castaneum exposed to a disk diet amended with different concentrations of SRE is illustrated in Fig. 7. the results showed a significant reduction in α-amylase activity as contrast to the control. This saponin effects on α-amylase as a digestive enzyme have been documented in several studies. Maazoun et al.65 demonstrated that saponins isolated from A. americana leaf extract may have role in the inhibition of Sitophilus oryzae (Coleoptera: Curculionidae) digestive enzymes. Shahriari et al.47 reported the suppression of digestive enzymes of E. kuehniella under a saponin tea diet. Furthermore, Li et al.66 noted a significant inhibition of pea aphids (A. pisum) α-amylase after treatment with isolated triterpenoid saponins from Clematis lasiandra. While, Ye et al.67 reported a suppression of α-amylase in aphids after treatment with triterpenoid saponins from Oxytropis hirta Bunge. Other triterpenoids also showed the inhibition effect of α-amylase, e.g., ecdysteroids Makisterone A and 22-hydroxyhopane in T. castaneum68,69 and 22-hydroxyhopane from Adiantum latifolium in Oryctes rhinoceros Linn70.
Amylase activity in the T. castaneum adult under a flour disk diet amended with different concentrations of SRE for the antifeeding test. The enzyme values are expressed in mU/mg protein as mean (± SD) values and analyzed by one way analysis of variance (ANOVA). Different letters indicate significant differences (p < 0.05) among treatments by Tukey’s test. S: Starved group.
Saponins’ toxicity might be attributed to their capacity to promote membrane fluidity65. Saponins can diminish cell viability by enhancing membrane permeability, causing organelle injury in midgut tissues. Consequently, this can cause digestive problems, nutritional deficiencies, and even death65,67. Moreover, the inhibition of the activity of digestive enzymes such as amylase, carbohydrases, proteases, exopeptidases, and TAG-lipase by saponins can be a sign of organelle damage in the midgut tissues51,66,67. These findings sparked interest in studying the midgut as a target to illuminate the mechanism of action for the most active saponin against insects66. Saponins are thought to facilitate food transit in the insect stomach while affecting digestion by interfering with the secretion of digestive enzymes10. Rane et al.71 identified three α-amylase inhibitors (αAIS) specific against coleopteran storage pests i.e., Amaranthus hypochondriacus (AhAI), Alternanthera sessilis (AsAI), and Chenopodium quinoa (CqAI). The highest inhibition potency on T. castaneum α-amylase was observed for AhAI, followed by AsAI and CqAI. The selectivity of these inhibitors against coleopteran α-amylases highlights their potential for controlling storage grain pests. α-amylase is an essential digestive enzyme required for the growth and development of insects, which can catalyze the hydrolysis of carbohydrates, and αAIS target α-amylases and interfere with carbohydrate digestion in insects, resulting in their starvation and death4.
In this study, a significant reduction was observed in α-amylase activity due to the starvation effect. This reduction aligns with the nutritional indices results, indicating that at concentrations of 50 mg/g and above, the insects ceased consuming the disks, which may explain the high mortality rate of insects subjected to diet with higher concentrations of SRE (50, 75, and 100 mg/g flour). Similarly, previous studies have focused on deciphering the underlying causes of insects’ deaths. The results revealed the presence of a strong relationship between starvation and digestive enzyme activities. Abad et al. observed a reduction of α-amylase activity in the Colorado potato beetle (L. decemlineata) (Coleoptera: Chrysomelidae), which was due to starvation72. In the same line, Peter et al. found that in starved group of juvenile loaches (P. dabryanus), the digestive enzyme activities were lower compared to those in the feed group57.
