Promising Antioxidant and Insecticidal Properties of Chemically Characterized Hydroethanol Extract from Withania adpressa Coss. ex Batt

Abstract

Withania adpressa Coss. ex Batt is a wild plant belonging to the family Solanaceae, native to the Mediterranean area. The present work was undertaken to study the chemical composition, antioxidant and insecticidal properties of Withania adpressa Coss. ex Batt (W. adpressa) extract. The plant extract was obtained by use of maceration with hydroethanol, and its chemical composition was characterized by use of HPLC. Evaluation of antioxidant potency was achieved by use of DPPH, TAC and FRAP bioassays. Insecticidal activity was tested against Callosobruchus maculatus (C. maculatus) by investigating its mortality, longevity, fecundity and emergence after being immersed with hydroethanol extract of W. adpressa. HPLC analysis revealed that the studied extract is rich in apigenin, luteolin, rutin hydrate, gallic acid, p-coumaric acid, acid gallate, and ferulic acid. The crude extract of W. adpressa recorded excellent antioxidant potencies with calculated values as follows: IC-50 of 49.01 ± 0.65 µg/mL (DPPH), EC₅₀ of 119.61 ± 1.81 µg/mL (FRAP), and 483.47 ± 5.19 µg EAA/mg (TAC). Regarding insecticidal activity, it was revealed that the mortality of C. maculatus after being treated with hydroethanol extract reached 94.21 ± 2.05%. In addition, hydroethanol extract effectively controlled the longevity, fecundity, and the emergence of C. maculatus. The outcome of the present work suggests that W. adpressa derivatives have promising antioxidant and insecticidal properties, and consequently, they may be used as natural insecticides and antioxidants.

1. Introduction

Medicinal herbs have been used to control a variety of diseases and wounds since ancient times around the world [1,2]. They are a part of several countries’ socio-cultural legacies, as demonstrated by ethnobotanical data in their pharmacopeia [3]. Phytochemicals extracted from plants have been a source of several commercial medications, and they are continuously studied for these purposes [4]. Medicinal and aromatic plants are still widely used by many people around the world, particularly in low- and middle-income nations where people face difficulties in accessing modern medications [5]. With the growing use of medicinal plants, some concerns arise, such as the fact that only a few plant species have been systematically investigated for possible medical usage [6]. It is appealing to discover novel therapeutic agents to help society fight illness, acting as potential alternative sources for sustainable medication, which can improve treatment outcomes [7].

Processes of oxidation are critical for the survival of organisms [7]. Reactive oxygen species, particularly peroxides, superoxides, hydrogen peroxide, hydroxyl radicals, singlet oxygen, nitric oxide, and alpha-oxygen radicals play a crucial role in oxidative stress, resulting in the development of many diseases [8]. The antioxidative defense system balances the creation of free radicals in healthy people; however, oxidative stress occurs when the balance favors the overproduction of free radicals due to low antioxidant levels. ROS are capable of causing DNA mutations or harming certain cells or tissues, resulting in cell senescence and death [9,10]. Antioxidants are thought to be potential defense agents against free radicals in the human body. As a consequence, there is an increasing need for substances with antioxidants that may be given to humans and other animal species as components of food or as specific pharmaceutics [11].

Callosobruchus maculatus Fab. (Order: Coleoptera; Family: Chrysomelidae) is a widespread insect pest that feeds on the crops of leguminous plants worldwide. The presence of this insect in inventories of leguminous plants might result in a large loss because of its capacity for rapid reproduction in storage facilities. Previous reports indicate that C. maculatus is capable of wreaking havoc on the organoleptic qualities of goods and has the potential to affect more than fifty percent of stored leguminous in a short time [12,13].

Insecticides are widely used to control insect pests. However, insect resistance to contemporary insecticides remains a significant concern relating to chemically synthesized insecticides. Furthermore, these substances may generate a risk to consumers and may have long-term negative consequences [12]. The plant kingdom provides an opportunity for innovation in the form of a potential resource for naturally occurring agents of pest management. Natural products from aromatic plants possess insecticidal properties. As a result of their insecticidal potencies, extensive research has been conducted on the efficacy of insect-controlling plant-derived compounds against pests that attack stored grains [14].

Withania adpressa Coss. ex Batt (Solanaceae) is a wild species growing in Mediterranean areas and has traditionally been used as a cytotoxic, immunomodulatory, analgesic, healing, and anticholinesterase substance. This species is rich in phenols, particularly glycowithanolides and withanolides, along with volatile compounds [14,15,16,17,18,19,20,21]. The current study was designed to investigate the phytochemical composition, antioxidant, and insecticidal properties W. adpressa extract.

2. Materials and Methods

2.1. Plant Material

W. adpressa was harvested in March from Maghrebian areas before being identified by a botanist (A2/WdBF21). Before undergoing the extraction process, leaves of the W. adpressa plant were allowed to air-dry in the shade for seven days.

2.2. Plant Extraction

W. adpressa leaves were cleaned and ground to a fine powder by use of an electric grinder. Forty grams of powder was extracted by use of maceration with hydroethanol (3/1) for 24 h. Next, a filter paper was used to remove the remaining solids from the mixture before being concentrated at 40 °C by use of a rotary evaporator. The cured extract was meticulously saved at 4 °C until further use.

2.3. Chromatographic Analysis

Hydroethanol extract of W. adpressa was chromatographically analyzed by the use of Shimadzu Prominence high-performance liquid chromatography with a UV-visible detector, a quaternary pump type LC-A/20, and a manual injector type Ry-odine. The separation of compounds was performed by the use of C18/Wakosil II column (length = 150 mm; diameter = 4.6 mm, 5 m, 100 A). The flow rate was one milliliter/minute in a ternary gradient mode consisting of ACN-MeOH/H₂O acidified to 0.2% orthophosphate. The injected volume for analysis was 20 µL, and the chromatographic column was conditioned with the elution (distilled water 0.2% H₃PO₄ (V/V)/methanol/acetonitrile 96/2/2.0 (V/V/V) for 15 min). By infusing 0.l µL of 80/20 (V/V) methanol/water into the HPLC system, an initial empty gradient chromatographic run was performed beforehand.

2.4. Antioxidant Activity

2.4.1. DPPH Test

This test was performed by mixing 980 μL of 2,2-diphenyl-1-picrylhydrazyl (60 μM) with 20 μL of the sample at various concentrations (up to 500 µg/mL). The mixture was incubated at room temperature for 20 min, while a blank composed of 980 μL of 2,2-diphenyl-1-picrylhydrazyl and 20 μL of methanol served as a negative reference. Quercetin and butylated hydroxytoluene (BHT) were used as positive controls [22,23]. Antioxidant potency was assessed by determining the percentage of free radical inhibition according to the formula:

I(%) = [(Ac − At)/Ac] × 100

(1)

where I is the inhibition, Ac is the absorbance of the control, and At is the absorbance of the sample.

2.4.2. Ferric Reducing Power Test

The ferric reducing antioxidant power (FRAP) of the ethanol extract from W. adpressa was determined according to the documented protocol by Bourhia and co-authors [23]. A solution consisting of 25.00 mL of sodium acetate buffer (0.30 M; pH = 3.60), 2.50 mL of tripyridyl-S-triazine (10.00 mM), 2.5 mL of FeCl₃ (20 mM), and 3 mL of distilled water was prepared to conduct this experiment. Next, 30 mL of the ethanol extract was added to 970.00 μL of the mixture before being incubated at 37 °C/30 min. By use of a calibration curve obtained by various concentrations of FeSO₄, results were expressed in mM Fe (II)/g extract.

2.4.3. Total Antioxidant Capacity

Total antioxidant capacity (TAC) was also used to test antioxidant potency as described elsewhere [24]. One milliliter of various concentrations of the ethanol extract (up to 1000 µg/mL) was mixed with one milliliter of a reagent composed of H₂SO₄ (0.60 M), Na₂PO₄ (28 mM) and ammonium molybdate (4 mM). The solution was sealed and incubated for 90 min at 95 °C. The absorbance was well measured at 695 nm after cooling. The negative control consisted of methanol mixed reagents, whereas the positive control consisted of quercetin (1 mg/mL) and BHT (1 mg/mL). The samples and controls were both incubated at the same temperature. The results were represented as mg EAA/g (milligrams of ascorbic acid equivalent per gram of extract).

2.5. Insecticidal Activity

2.5.1. Breeding of Insects

In the current work, C. maculatus was used to test the insecticidal effect of W. adpressa extract. C. maculatus was reared under standard laboratory conditions, humidity (65 ± 5%), temperature (25 ± 1 °C) and a photoperiod of 10 h: 14 h (light/dark). A pellet diet of Cicer arietinum was used for rearing C. maculatus.

2.5.2. Evaluation of Crude Extract on C. maculatus

Five pairs of C. maculatus with ages of up to 24 h were placed in glass Petri dishes containing 25 g of Cicer arietinum before being treated with an extract spray at different doses (0.25, 0.5, 0.75, 1 and 1.25 g per 25 g of chickpea seeds). Control groups of C. maculatus were maintained with untreated chickpea seeds under similar conditions to treatments. The effectiveness of the extract against C. maculatus was evaluated by studying the effect on longevity, fecundity, fertility, and viability of insects.

Adult Longevity

Adult longevity of treatments was determined by counting the insect individuals that died from the start of the experiment onwards.

Fecundity of Females

All eggs deposited in the grains (hatched and unhatched) were counted by use of a binocular magnifying glass after 13 days from the start of experiment.

Egg Hatching Rate

After counting the number of laid eggs, the egg hatch rate was calculated by use of the following formula:

Hatching rate = (number of eggs hatched/number of eggs laid) × 100

Egg Viability Rate (Emergence)

Adults, which started emerging at around the fifth week after the start of experiment, were regularly counted and removed from boxes. The viability rate of eggs was calculated by the following formula:

Viability rate = (number of emerging adults/number of laying eggs) × 100

2.6. Statistical Analysis

In the current work, GraphPad-Prism software version 7 was used to process the results, which were expressed as means of triplicate experiments ± SD (standard deviation). Shapiro–Wilk’s test was used to determine whether or not the distributions were normal, while the Levene test was used to determine homogeneity of variances (95%). Analysis of variance (ANOVA) was used, and for making multiple comparisons, Tukey’s HSD test was employed as a post hoc comparison test. When the p-value was less than 0.05, differences were judged to be significant.

3. Results and Discussion

3.1. Identification of Phytochemical Compositions

Phytochemical analysis by HPLC/MS revealed that the extract of W. adpressa is rich in phenolic compounds, wherein eight compounds were identified, such as apigenin, luteolin, rutin hydrate, gallic acid, p-coumarin, acid gallate and ferulic acid (

Table 1. Phytochemical identification by HPLC for total polyphenols extracted from leaves of W. adpressa.

Retention Time	Identification Compound	Phenolic Class	Content in µg/mg
4.78	Galic acid	Phenolic acid	47.84 ± 0.87 b
5.92	p-Coumaric acid	Phenolic acid	28.64 ± 0.73 c
11.03	Cafeic acid	Phenolic acid	41.17 ± 0.49 b
17.24	Methyl gallate	Phenolic acid	54.37 ± 0.61 b
17.81	Luteolin	Flavonoids	86.13 ± 0.58 a
26.35	Ferulic acid	Phenolic acid	24.73 ± 0.74 d
28.43	Apigenin	Flavonoids	34.51 ± 0.68 c
49.17	Rutin hydrat	Flavonoids	18.48 ± 0.80 d

Row values with the same letters are not significantly different (n = 3, ANOVA, Tukey’s HSD, p-value less than 0.05 considered to be significant).

; Figure 1 and Figure 2). Luteolin and methyl gallate were the predominant compounds in the extract of W. adpressa, representing 86.13 ± 0.58 µg/mg and 54.37 ± 0.61 µg/mg, respectively (Table 1). These results are in agreement with previous works reporting that the genus Withania possesses phenols including steroidal lactones, tannin, alkaloids, flavonoids [25,26,27,28]. More than twelve alkaloids, forty withanolides, and many sitoindosides were isolated from the leaves, fruits, and roots of Withania [25]. Crude extracts from the genus Withania are predicted to possess several compounds, such as 2-(4-hydroxy-3,5 dimethoxyphenyl)-3-oxetanamine, N-4-(3-furoylamine)-1-butanol, withanolides, apocarotenoids, carotenoid, tetra-acetylated apocarotenoid, glucosides, hydroxywithanolide F, withanolide A, withacoagulin, withanoside IV, physagulin D, 27-hydroxywithanone, withanoside V, withaferin A, withastramonolide, withanone, withanolide B, isopelletierine, anaferine, withaferins, and withanolide D [28].

3.2. Antioxidant Activity

Antioxidant potential was carried out by assessing the ability to scavenge radicals in the presence of W. adpressa extract. By means of the use of the DPPH method, W. adpressa extract exhibited potent antioxidant power in a dose-dependent manner wherein DPPH free radical inhibition increased with an increasing concentration of the plant extract (Figure 3A). Notably, the IC₅₀ value of W. adpressa extract was determined to be 49.01 ± 0.65 µg/mL, which is important when compared to the control values, 23.31 ± 0.85 µg/mL (BHT) and 19.09 ± 0.46 µg/mL (quercetin) (Figure 3B).

The studied extract exhibited interesting total antioxidant capacity, wherein doses of 100 and 500 µg/mL showed antioxidant capacities of 106.23 ± 5.35 µg EAA/mg and 483.47 ± 5.19 µg EAA/mg, respectively (Figure 3C). The reducing power of the extract was assessed by use of the FRAP method, and the results were expressed in half-maximal effective concentration (EC₅₀). The findings show that the 50% effective concentration of the plant extract was determined to be 119.61 ± 1.81 µg/mL (Figure 3D). This antioxidant power observed in the extract may be mostly attributable to the abundance of polyphenols in the studied plant extract, especially flavonoids, and also according to the chemical structures of the bioactive molecules [29]. The reducing power of the W. adpressa extract is presumably owing to the abundance of a hydroxyl group in phenolic compounds, which has the potential to act as an electron donor in order to scavenge free radicals [29]. Studies conducted by Van Acker [30] on the chelation of iron ions by flavonoid compounds clearly indicated the essential sites for chelation of metal ions, which were the 3′-hydroxy and 4′-hydroxy groups of the B ring, the 3-hydroxy and 4-oxo groups of the C ring and the 4-oxo and 5-hydroxy groups in position 3 [29,31]. As a consequence, quercetin, which already has all of these substituents, is an exceptionally powerful metal complexing agent [31,32]. The autoxidation process is usually dependent on many factors, such as temperature, pH, the presence of complexing agents, and the amount of metal ions and polyphenols [32,33]. Some previous works have also shown that the reducing capability of chemicals can be a good sign of their potential antioxidant effects [29].

3.3. Insecticidal Activity

The results show that crude extract from W. adpressa effectively controlled the adult C. maculatus longevity, resulting in a significant decrease with increasing doses. The highest concertation of extract exhibited strong mortality with calculated values of 94.21 ± 2.05% (Figure 4A). From this figure, it can be seen that extract of W. adpressa leaves exhibited a serious lethal effect on C. maculatus in a dose-dependent manner. Concerning the effect of extract on female fertility, the results show that this natural product decreased the average number of eggs laid by females in a dose-dependent manner (Figure 4B). The average fecundity recorded for the control group was 155.10 ± 6.48 eggs/female, while for insects treated with the lowest dose (1%) of extract, the average fecundity decreased to 57.25 ± 3.30 eggs/female. With increasing concentration, up to 5%, a serious decrease in the average fecundity of females was noted with a calculated value of 38.01 ± 2.16 eggs/female.

Regarding the effect of hydroethanol extract on egg fertility, the results show that the average rate of hatched eggs significantly decreased with increasing doses of hydroethanol extract. An egg hatchability average of 85.04 ± 1.41% was recorded for C. maculatus used as a control, while the egg hatchability average for C. maculatus treated with hydroethanol extract (1%) deceased to 41.10 ± 0.84% (Figure 5A). At the highest dose (5%), hydroethanol extract decreased the hatching rate of insects by 74.86 ± 0.79%. W. adpressa hydroethanol extract effectively controlled the viability of eggs at various doses used for testing (Figure 5B). The viability of eggs for the treated insects with the highest dose (5%) decreased to 13.50 ± 1.29%, while the viability of eggs for untreated insects (control) was determined to be 63.75 ± 1.83% (Figure 5B).

Many plant species exhibit insecticidal effects on insects and inhibit their reproduction [34]. Previous studies have shown that chemicals in plants adversely affect the fecundity of female insects, resulting in disruptions in insect behavior and oviposition. Fertility was reduced from 59.39% to 8.05% in the presence of leaf powder of Phaseolus vulgaris [35]. Polyphenolic compounds identified in the crude hydroethanol extract of W. adpressa (Figure 1) could be responsible for the recorded activities against C. maculatus. The results presented here are in accordance with those reported by Regnault-Roger and co-authors [36], who showed that hydroethanol extracts rich in phenols compounds possessed important insecticidal effects [36]. Polyphenols are involved in several physiological and ecological activities that confer plant defense against external threats [37], and reduce pests through repellent and anti-nutritional effects [36]. In addition, flavonoids have adverse effects on insects, resulting in oviposition and egg-laying disturbance [38]. Many studies have evaluated the insecticidal effect of several aromatic plants according to Bouchikhi Tani (2011). Hydroethanol extracts from Rosmarinus officinalis and Origanum glandulosum significantly reduced the longevity of insect adults in the recorded literature [39].

Secondary phytochemicals from medicinal and aromatic plants can affect many aspects of insect development and physiology by influencing their life cycles [40]. Notably, natural extracts with flavonoids are known for their larvicidal activity against certain insect pests [41,42]. The mechanism of action of phytochemicals at the molecular and cellular levels remains elusive; however, some secondary metabolites, particularly flavonoids, were found to inhibit acetylcholine esterase activity, resulting in a larvicidal effect, as documented in previous reports [43].

4. Conclusions

The usage of natural products derived from plants as antioxidants and insecticidal agents can have many advantages over current chemically synthetic products. The outcome of the present study showed that the crude hydroethanol extract from W. adpressa is rich in phenolic compounds, particularly apigenin, luteolin, rutin hydrate, gallic acid, p-coumarin, acid gallate and ferulic acid, which exhibited excellent antioxidant and insecticidal properties against the pest Callosobruchus maculatus. Further detailed studies on W. adpressa hydroethanol extract are warranted, particularly on the mechanism of action of the plant extract, assays on isolated compounds along with toxicity tests on non-target organisms.