Antioxidant and Anticoagulant Activities of Red Palm Weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae) Extracts

Article History


INTRODUCTION
Oxidative reaction has a crucial function in human health.An antioxidant is a chemical that can inhibit the oxidation of other molecules, hence preventing harmful changes.Free radicals are commonly found in the body and are produced by certain physiological processes to combat pathogenic germs.Various disorders such as neurological diseases, cardiovascular diseases, autoimmune skin diseases, chronic renal failure, and cancer result from oxidative stress activation and the lack of intracellular antioxidant defenses (Zhu et al., 2013;Roy et al., 2015;Hasaballah et al., 2019).Antioxidants can diminish unstable and reactive free radicals, protecting cells from their harmful effects (Souri et al., 2008).
Conversely, any vascular injury will result in quick bleeding.The bleeding is quickly halted by the blood coagulation mechanism, which is then followed by the repair of the hemostatic system.Accurate regulation of blood coagulation is crucial for the survival of all animals.Failure to stop bleeding promptly can provide a life-threatening risk.Unwanted clot development, such as in cases like stroke or DIC, can also lead to death.To address various bleeding disorders, a procoagulant or anticoagulant molecule is required.Anticoagulants decrease blood coagulation to avoid deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke (Hossain et al., 2018).Arthropods, especially insects, are valuable and untapped sources of bioactive compounds for modern medicine.Insects make up over 50.0% of total biodiversity and more than 80.0% of all animal diversity, inhabiting various ecological systems (Chernysh et al., 2002;Roy et al., 2015;Hassan et al., 2018;Hasaballah et al., 2019;Mekhlif 2021).
The current study focused on examining the antioxidant and anticoagulant properties of different extracts obtained from red palm weevil larvae (Rhynchophorus ferrugineus) to contribute to the understanding of potential insect-derived materials.

Rhynchophorus ferrugineus Tested:
The last instar larvae of Rhynchophorus ferrugineus, a type of palm weevil, were initially obtained from the Central Laboratory for Date Palm Research and Development in Giza.They were subsequently bred for multiple generations in a controlled environment within the insectary-rearing laboratory at the Zoology Department, Faculty of Science, Al-Azhar University (Cairo).The larvae were maintained under specific conditions of temperature (25-27°C), relative humidity (60-70%), and photoperiods (12-12 hours lightdark rhythm) in a wooden cage measuring 200×120×300 cm (Shahina et al. 2009).

Evaluation of Antioxidant Activity by DPPH Radical Scavenging Method:
The free radical scavenging activity of various leaf plant extracts was assessed using 1,1-diphenyl-2-picryl hydrazyl (DPPH).A 0.1 mM DPPH solution in ethanol was produced.A 1 ml solution was combined with 3 ml of various ethanol extracts at concentrations ranging from 3.9 to 1000 μg/ml.Only the extracts that are soluble in ethanol were employed, and different quantities were generated using a dilution procedure.The mixture was briskly agitated and then left at room temperature for 30 minutes.The absorbance was measured at 517 nm using a UV-VIS Milton Roy spectrophotometer.The experiment was conducted using ascorbic acid as the reference standard chemical and was repeated three times (Shekhar and Anju, 2014;González-Palma et al., 2016).
The IC50 value of the sample, representing the concentration needed to inhibit 50% of the DPPH free radical, was determined using a Log dosage inhibition curve.Decreased absorbance of the reaction mixture suggested increased free radical activity.The DPPH scavenging effect percentage was determined using the formula: Percent inhibition = [(A0 − A1) / A0] × 100, Where A0 represents the absorbance of the control, and A1 represents the absorbance in the presence of the test or standard sample.

Evaluation of the Anticoagulant Activity of Tested Extracts:
The anticoagulant effects of the studied extracts were evaluated utilizing both activated partial thromboplastin time (APTT) and prothrombin time (PT).
The APTT assay was conducted according to the procedure outlined by Seedevi et al. (2017).The APTT was measured using the commercial kit Plasmatrol H-II, Liquicellin-E, and BioMed.The citrated blood plasma was combined with 10 μl of tested extracts at concentrations of 25, 50 and 75 μg/ml and incubated at 37°C for 10 minutes in a glass vial in this assay.100 μl of APTT reagent was added to the mixture, which was then incubated for 3 minutes at 37°C.Following this, 100 μl of pre-heated 0.02 mol/l CaCl2 solution was added to the mixture, and the clotting time in seconds was recorded in comparison to Heparin sodium salt.
The PT potential of the studied extracts was determined using the method outlined by Seedevi et al. (2015).The prothrombin time was evaluated using the commercial kit plasmatrol H-II from BioMed.Preheat the reagent to 37°C for 5-10 minutes before using it in the test procedure.90 μl of citrated normal human plasma was combined with 10 μl of extracts at concentrations of 25, 50, and 75 μg/ml.The combination was then incubated for 10 minutes at 37°C.Following incubation, 200 μl of PT reagent was combined with the mixture, and the clotting time in seconds was measured and compared to Heparin sodium salt used as a standard.

Statistical Analysis:
The data was encoded and inputted with the statistical software SPSS V.22.Data underwent testing to ensure they met the assumptions of parametric tests.Continuous variables were assessed for normality using the Shapiro-Wilk and Kolmogorov-Smirnov tests.The probability and percentile data were normalized for normal distribution using the Arcsine Square Root transformation.The data were given as the mean and standard deviation.Data were displayed graphically, when feasible, with R Studio version 2022.02.4.

Anticoagulant Activity of Tested Extracts:
The anticoagulant properties of Rh. ferrugineus larvae extracts were evaluated using the usual coagulation tests, Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT), with heparin sodium salt as a control.The typical range for the PT commercial kit is 10 to 12 seconds, while for the APTT kit is 28 to 35 sec.
The results indicated that Rh. ferrugineus has anticoagulant properties.Larval extracts were less powerful than heparin and required higher concentrations to achieve the same level of anticoagulation in both APTT and PT testing.

DISCUSSION
The present study proved the capability of Rhynchophorus ferrugineus larval extracts as antioxidant agents.The hexane, n-butanol, dichloromethane, and diethyl ether were extracted from Rh. ferrugineus larvae recorded antioxidant activity with values of 142.67, 122.24, 231.77 and 74.95 µg/ml as compared with 3.16 µg/ml for the standard ascorbic acid, respectively.Free radicals such as hydroxyl groups, peroxyl radicals, and single oxygen are detrimental because they can damage cell components, leading to various illnesses (Bozin et al., 2008).Antioxidants can reduce chronic diseases such as cancer and cardiovascular diseases by neutralizing free radicals.Synthetic antioxidants have been utilized to decrease biological toxicity by causing various harmful effects (Hasaballah et al., 2019).Therefore, there is a high desire to substitute synthetic antioxidants with natural oxidizing agents.Original substances like ground-up bodies have been commonly utilized in medical sectors and are generally deemed safe because they include numerous chemicals with antioxidant and anticancer properties (Suh et al., 2010).The findings support the results of Orhan et al. (2007), who utilized various extracts of Lycopodium clavatum and its alkaloid fraction, showing insignificant antiradical effects on DPPH.Hasaballah et al. (2019) also utilized methanol crude extract from maggots of Musca domestica, Lucilia sericata, and Chrysomya albiceps.
On the other hand, the obtained results exhibited the anticoagulant activity of Rh. ferrugineus (larvae) tested extracts using APTT assay showed 33.8, 44.3 and 50.9 sec.at 25, 50 and 75 µg/ml of hexane extract, respectively, while Rh.ferrugineus n-butanol extract recorded 46.9, 57.2 and 65.5 sec.at the same concentrations.The results align with those reported by Xu et al. (2016) for the crude extract of Holotrichia diomphalia larvae and by Arasukumar et al. (2019), who studied the anticoagulant activity of chitosan extracted from Thenus unimaculatus using APTT and PT assays with heparin sodium as the standard.The results showed that the anticoagulant activity of extracted chitosan was recorded at 49.7 and 20.9 s and at 50 μg/ml by APTT and PT assays, compared with 61.2 and 31.4 s. recorded by heparin, respectively and Hamdi et al. (2020), who reported that anticoagulant activity of the different types of blue crab chitosan was recorded at 31.2 s., 14.8 and 14.4 s. using APTT, Quick time (QT) and thrombin time (TT) in vitro assays.

Conclusion
The study examined the antioxidant and anticoagulant properties of several extracts from the larvae of the red palm weevil, Rhynchophorus ferrugineus, using hexane, n-butanol, dichloromethane, and diethyl ether extracts.The current investigation demonstrated that all Rh.ferrugineus larval extracts tested have antioxidant properties.The anticoagulant properties of Rh. ferrugineus tested extracts were less powerful than heparin and required higher concentrations to achieve the same level of anticoagulation in both APTT and PT experiments.Further research is required to clarify the bioactivity of Rh. ferrugineus larval extracts.Declarations: Ethical Approval: Ethical Approval is not applicable.

Competing interests:
The authors declare no conflict of interest.Funding: No funding was received.

Fig. 4 :
Fig. 4: Step slope chart represents the anticoagulant activity of Rhynchophorus ferrugineus extracts using APTT and PT assays at different concentrations.

Table 4 :
Anticoagulant activity of Rhynchophorus ferrugineus larval crude extracts and heparin based on APTT and PT Values (seconds).