Characterization of phenolic acids, flavonoids, and caffeine using HPLC
Based on HPLC analysis, fourteen phenolic acids and five flavonoids in addition to caffeine (alkaloid) have been identified in the SCGs methanolic extract (Table 1). Cinnamic (979.38 µg/g), rosmarinic (163.10 µg/g), and gallic (32.51 µg/g) acids were the predominant phenolic acids, followed by sinapic (10.10 µg/g), chlorogenic (8.74 µg/g), salicylic (7.61 µg/g), and caffeic acids (6.41 µg/g) (Table 1). Many researchers have investigated the polyphenolic constituents of coffee waste; however, the yield and type of bioactive compounds extracted from SCGs depend on many factors like the coffee species, instant coffee production technique and efficiency, storage conditions, and the extraction method, which is based on polar solvents. For example, increased the intensity of coffee roasting from medium to dark reduced phenolic acids like chlorogenic due to isomerization, gallic, p-coumaric, ferulic acids, increased ellagic acid without affecting caffeic acid and ruten contents9. Andrade et al.18 have extracted phenolic compounds from SCGs and coffee husks using different techniques like ultrasonic, Soxhlet, and SFE based on ethanol, ethyl acetate, and CO2 solvents. They identified chlorogenic acid as abundant among others in all extracts. Meanwhile, gallic acid, p-hydroxybenzoic, protocatechuic, vanillic, and tannic were also detected in lower concentrations. In contrast, Okur et al.19 identified both chlorogenic and caffeic acids as minor components, which agrees with our findings. The focus on chlorogenic acid is due to its potential health benefits described in previous studies, including free radical scavenging capacity, anti-inflammatory, antidiabetic, and anticancer effects20.
Table 1 Contents of phenolic acids, flavonoids, and caffeine determined in SCGs isopropanol extract
Compound
|
Phenolic acids
(µg/g of the SCGs extract)a
|
Compound
|
Flavonoids
(µg/g of the SCGs extract)
|
Gallic acid
|
32.51±2.08
|
Catechin
|
14.55±1.47
|
Protocatechuic acid
|
2.07±0.74
|
Epicatechin
|
10.08±2.37
|
p-Hydroxybenzoic acid
|
4.37±0.88
|
Naringin
|
86.94±3.15
|
Gentisic acid
|
0.26±0.05
|
Apigenin-7-glucoside
|
1534.22±7.74
|
Chlorogenic acid
|
8.74±1.05
|
Chrysin
|
1.01±0.14
|
Caffeic acid
|
6.41±0.74
|
-
|
-
|
Syringic acid
|
3.41±0.41
|
-
|
-
|
Vanillic acid
|
2.08±0.56
|
-
|
-
|
Sinapic acid
|
10.1±1.24
|
-
|
-
|
Rosmarinic acid
|
0.53±0.22
|
-
|
-
|
Ferulic acid
|
0.37±0.14
|
-
|
-
|
Salicylic acid
|
7.61±1.05
|
-
|
-
|
p-coumaric acid
|
0.16±0.11
|
-
|
-
|
Cinnamic acid
|
979.38±4.78
|
-
|
-
|
Rosmarinic acid
|
163.1±3.74
|
-
|
-
|
Compound
|
Alkaloid (µg/g of the SCGs extract)
|
Caffiene
|
1322.2±5.71
|
a Values represent averages ± standard deviations for triplicate experiments. |
Cinnamic acid, which is predominant in the present study in agreement with Vamanu et al.21, has generally been reported to be phenolic constituents in SCG in previous research but in much lower concentrations with respect to the extraction technique22. As explained above, the variations might be due to many reasons, including solvent, solvent/sample ratio, extraction time, extraction method, and temperature. To our knowledge, rosmarinic acid, an ester of caffeic acid reported in Coffea arabica L.23, is newly identified in the present study and has never been mentioned before in SCGs. Compared to ethanol and methanol, water was reported as the most efficient solvent for the extraction of gallic acid; therefore, it was not always detected in SCGs24. However, gallic acid was identified in the current study with a median concentration, maybe due to the detection and quantification sensitivity of the analysis22. On the other hand, coumaric (0.16 µg/g), gentisic (0.26 µg/g), and ferulic (0.37 µg/g) acids were the lowest compared to the identified constituents, which are in agreement with Angeloni et al.25 (Table 1).
Apigenin-7-glucoside was the main flavonoid identified in SCGs methanolic extract, followed by naringin, catechin, epicatechin, and chrysin (Table 1). Up to our knowledge, only four flavonoids have been identified in SCGs using ethanol and microwave extraction technique; epicatechin, catechin, rutin, and quercetin; however, nothing was reported about apigenin-7-glucoside or naringin9. Generally, both apigenin-7-glucoside and naringin were present in green coffee beans, with an increase in their concentrations upon roasting using either microwave or oven techniques26. It is well-known that flavonoids serve many beneficial health functions and elicit protective effects, including anti-inflammatory, antioxidant, antiviral, and anti-carcinogenic9.
Caffeine is one of the essential bioactive components found in SCGs in a remarkable concentration (Table 1), consistent with Kovalcik et al.7. It is a methylxanthine alkaloid, which showed deleterious effects on the nervous system, on the sensitization of DNA to damage, on the delayed entry of cells into mitosis, and on other aspects of cell division on the development of organisms, on fertility, and on chromatin structure, to mention but a few27.
Oviposition deterrent activities of SCGs extract against female moth Spodoptera littoralis
Daily inspection showed that eggs laid by females in jars treated with the maximum SCGs extract (100%) were rarely observed (0.67± S.D 0.58 batch/jar). Although the mean number of eggs is minimal, females have been putting their eggs on the inner wall of jars, not on the treated leaves, and on the opposite, females in non-treated jars have laid their eggs entirely on the leaves. Meanwhile, the mean number of egg batches laid in each treatment ±S.D were 2.33±0.56, 7.33±1.15, and 8.67±2.57 in each treatment 50, 25, and control, respectively (Fig. 1). The present investigation results agree with Borges et al.28, showed oviposition deterrent and larvicidal activities of the phenolic extracts of T. avellanedae against 3rd instar larvae Aedes aegypti. In the same context, Kovanci29 revealed the feeding and oviposition deterrent activities of microencapsulated cardamom oleoresin and eucalyptol against Cydia pomonella, while Basukriadi and Wilkins30 studied the effect of yam bean seed extract and coumarin, which partially deterred the moth Plutella xylostella.
Insecticidal effect of SCGs extract on the percentage mortality of Spodoptera littoralis
Results showed that SCGs extract (100%) caused mortality with 76% for larvae after four days of treatment. While with 50% extract concentration, the mortality reached 54.5%, and the lowest extract concentration, 25%, recorded 27.5% of mortality compared to control; 7.4% mortality in larvae (Fig. 2). The main remark from our observations during this test was that there was no consumption for treated leaves compared with control dishes where the larvae fed and consumed many parts of leaves. This observation reveals that SCGs extract can act as a repellent or antifeedant agent against larvae of Spodoptera littoralis, which died without feeding on treated leaves.
These results are agreed with Pavela31, who showed antifeedant and larvicidal effects for 12 simple phenols and nine phenolic acids against Spodoptera littoralis (Boisd.). Along the same lines, Malpighia emarginata DC. bagasse acetone extract, which contains many phenolic compounds like gallic acid, epigallocatechin gallate, catechin, p-coumaric acid, salicylic acid, and quercetin, have prolonged the pre-pupal stage and increased the mortality of caterpillars of fall armyworm Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)32.
Survey and population density of insects of P. vulgaris in the field treated with SCGs extract
Data presented in (Fig. 3) showed the mean number of the main insects found in the field, where aphid, Aphis craccevora specimens were 4.5, 10.7, 13.5, and 17.3 at plants treated with 100, 50, 25% of SCGs extract and control, respectively. Jassed, Empoasca fabii recorded 0.75, 4, 9, and 12.7 with treatments 100, 50, 25% of SCGs extract and control, respectively. In the case of the whitefly, Bemisia tabaci, the data obtained were 2.5, 5.7, 10.7, and 15.5 with concentrations of SCGs extract 100, 50, 25%, and control (water). The number of cutworms, Agrotis ipsilon larvae, has been collected during the earlier 35 days of plant age because those larvae after third larval instar sheltered in soil and challenging to be on the foliage part of plants. At the same time, it was accessible to collect larvae of cotton leafworms from the field during all of the season. Both of these two pests are responsible for damage to the foliage of this crop.
Data have been recorded with The mean number of larvae caught from each plot according to concentration for spodoptera was 1.7, 5, 6.7, and 8.0 with 100, 50, 25% of SCGs extract and control, respectively. In earlier plant age, larvae of Agrotis ipsilon were collected from plants according to different treatments was mean number 2, 5, 8.5, and 11.8 specimens/plant with concentrations 100, 50, 25% of SCGs extract and control, respectively. These results show that a high concentration of 100% SCGs extract gave a highly significant difference compared with other lower concentrations of 50, 25%, and much higher than the control.
The decrease in the population number of Spodoptera and Agrotis larvae collected from the field has been supported by the results obtained twice a week. The mean number of leaves damaged by those insects was very low in plots treated by high concentrations of 100% and 50% of SCGs extract compared with the lower concentration 25% and control (Fig. 4). These findings reinforce the assumption; the SCGs extract acts as an antifeeding and repellent agent. The above results are in harmony with many previous works; for example, Pavela31, who showed the antifeedant effect of phenolic on Spodoptera littoralis (Boisd.), and Rahayu et al.33 studied the antifeedant activity of leaf phenolic extract of two cultivars, Carica papaya L. on Spodoptera -litura F. Larvae.
Evaluation Of Molecular Docking
Evaluation for the potential interaction between the predominates of the SCGs methanolic extract, and insecticide AChE enzyme (1QON) was conducted through a molecular docking study. The intermolecular interactions between the ligand and the target receptor were evaluated. The ideal pose was validated by aligning the X-ray bioactive conformer with the best-fitted pose of the same compound for the enzyme. The perfect pose of each molecule was selected according to the energy score, and validation is considered satisfactory when the RMSD is smaller than 2.0 Å regarding the crystallographic pose of a respective ligand34.
The binding free energies (∆G) for the extract predominates docked at AChE are shown in Fig. 5, revealing the best poses obtained in the molecular docking analyses. The larger the peaks, the lower the ∆G and consequently the more significant the interaction between the receptor and the ligands with insecticidal ability. Apigenin 7-glucoside displayed the best binding affinity compared to other ligands or the control (Lannate; -5.49 kcal/mol), with high docking scores (−9.16 kcal/mol), followed by catechin and epicatechin; -8.58 and -8.21 kcal/mol, respectively. Rosmarinic and chlorogenic acids showed median scores with -7.69 and -6.99 kcal/mol, while gallic acid was the least (-3.61 kcal/mol) (Fig. 5). The above results follow Tundis et al.35 and Zengin et al.36, where flavonoids, according to the former, especially apigenin 7-glucoside, showed a potential anti-cholinesterase effect, while the latter proved an enzyme inhibitory effect for the total methanolic extracts of both SCGs and coffee silver skin.
Figure 6a–d shows the interaction of apigenin 7-glucoside, rosmarinic acid, caffeine, and lannate (control) with the AChE receptor. These compounds represent the highest binding affinity from different phenolics, flavonoids, and alkaloids identified in SCGs extract. The higher binding affinity of apigenin 7-glucoside is attributed to the crucial conventional hydrogen bonds formed with GLU237 and ASP375, C-H interaction with GLY481 and HIS480, and finally, Pi-Pi interaction with TYR71, TYR370, and TYR374 (Fig. 6a). The number of Pi-sigma interactions (Pi-alkyl), which primarily involves charge transfer, helps bind rosmarinic acid and caffeine firmly with the receptor residues LEU479, TYR370 TYR374, and TRP83. Again, similar Pi-Pi interactions with TYR370, TRP83, and PHE371 are shown for both, but caffeine has a unique conventional hydrogen bonding with TRP472 and carbon-hydrogen bonding with ASP482 and GLY481, while rosmarinic acid showed unparalleled hydrogen bonds with SER238 and HIS480. Types of bonds and their distances were the main reasons for the differences in binding affinity and free energy between each ligand and the control (Lannate), indicating an insecticidal ability of the tested molecules. The unique Pi-Pi interactions, C-H bonding, and distances of the bonds between ligands and enzyme moieties could make remarkable differences in binding affinity. For example, distances of conventional H-bonding between Apigenin 7-glucoside and AChE moieties are between 1.81-2.04Å, shorter than the same between control (Lannate) and receptor (2.04-2.24 Å). The same trend could be noted for the remaining bonds, as shown in Fig. 7.
According to the literature, the molecular docking method used here identified a conformation that allows the ligand to bind to the residues of the 1QON active sites, around the α-helix between amino acid residues TYR370–TYR374 and around the β-sheet between amino acid residues VAL478–HIS480. For the ligand, it is possible to see hydrogen bonds in common with residues TYR370 and HIS480. There was also a hydrophobic interaction with residues TYR71, TRP83, TYR370, PHE371, and LEU47937. The interactions obtained after molecular docking of the compounds with the amino acid residues TRP83, TYR370, PHE371, and HIS480 of AChE are similar to those reported in the literature38.
Generally, the main task of the insecticidal agents like the potential compounds examined during the current study is to irreversibly inhibit the production of the AChE enzyme, which is responsible for acetylcholine’s hydrolysis, consequently terminating acetylcholine the nerve impulse. The previous concept represents the initial mechanism for an extract or compound to be considered an insecticide in the larval phase39. Therefore, molecular docking represents an essential tool to observe interactions formed inside the active site of the AChE and the mechanism of elucidation of biological action of the potential ligands applied.