Insecticidal Activity and Free Radical Scavenging Properties of Isolated Phytoconstituents from the Saudi Plant Nuxia oppositifolia (Hochst.)

Chromatographic purification of the alcoholic extract from the aerial parts of the Saudi plant Nuxia oppositifolia (Hochst.), Benth., resulted in five isolated phenolic compounds. Two flavones, hispidulin (1) and jaceosidin (2), and the phenylethanoid glycosides, verbascoside (3), isoverbascoside (4), and conandroside (5), were identified and their chemical structures were determined by spectroscopic analyses. The insecticidal activity of compounds 1 and 2, in addition to 11 compounds isolated in a previous research (6–16), was evaluated against the Yellow Fever mosquito, Aedes aegypti. Four compounds displayed adulticidal activity with LD50 values of 2–2.3 μg/mosquito. Free radical scavenging properties of the plant extracts and compounds (1–5) were evaluated by measuring the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate radical cation (ABTS•+) scavenging activity. All compounds exhibited notable activity, compared with the positive control, l-Ascorbic acid. This study suggests that N. oppositifolia could be a promising source of secondary metabolites, some with lethal adulticidal effect against Ae. aegypti.


Introduction
The bite of an infected Aedes aegypti L. mosquito can spread several dangerous diseases, such as Dengue, Zika, and Chikungunya fever. These diseases are worldwide public health problems, with particularly severe consequences in developing countries. Controlling the mosquito vector is the best method for disease prevention, and the need for new mosquitocidal agents due to resistance and regulatory loss is a serious issue [1].
Plants and their derivatives have been used for millennia in insect control because of beneficial toxic activity or repellent properties. Phytochemicals responsible for such activity are primarily secondary metabolites synthesized by plants for defense against herbivorous insects [2]. A few phytochemical and biological studies have examined the Nuxia (family: Buddlejaceae) for beneficial compounds and found possible candidate molecules. Secondary metabolites were isolated from the leaves and aerial parts of N. oppositifolia, N. floribunda, and N. sphaerocephala. [3][4][5][6]. Anti-diabetic activity of N. floribunda [7], and anti-inflammatory, antimalarial, and cytotoxic activity of N. verticillata were also previously evaluated [8,9].
Nuxia is represented in Saudi Arabia by two species, N. oppositifolia Benth and N. congesta Fresen. Recently, we isolated three new labdane-type diterpene acids and 11 triterpenes, including a triterpene derivative, and the common phytosterols, β-sitosterol and stigmasterol from the n-hexane and dichloromethane extracts of N. oppositifolia. The compounds demonstrated high in vitro cytotoxic activities against human cancer (cervical, lung, breast) cell lines [5]. Two of the isolated compounds, 3-oxolupenal and katononic acid, showed significant affinity at the binding sites of α-amylase and α-glucosidase and could be useful for treatment of diabetes mellitus type II [10].

Free Radical Scavenging Activity
Phenolic compounds, including the flavonoids, are bioactive secondary metabolites known for a wide range of health benefits [13,20]. They have considerable antioxidant activity, as demonstrated in several in vitro and in vivo assay systems [13,21,22]. Multiple electron-donating phenolic hydroxyl groups in the flavonoid and phenylethanoid glycosides and resonance stabilization of the radicals explain this high radical scavenging activity [23,24]. The vast literature confirming antioxidant activity of phenolic compounds encouraged us to test the radical scavenging potential of N. oppositifolia isolates, as shown in Table 2. All samples displayed the capacity to reduce the DPPH and (ABTS •+ ) radicals with a dose-dependent inhibition. At 1000 µg/mL concentration, the activity of tested samples ranged from 75.7 ± 0.4 to 79.8 ± 1.2% and from 73.7 ± 0.3 to 77.1 ± 0.1% in the DPPH and ABTS assays, respectively. At the same concentration, the L-Ascorbic acid positive control had a scavenging activity of 90.7 ± 1.4 and 88.7 ± 2.1% in the DPPH and ABTS assays, respectively. Jaceosidin (2) had the lowest IC 50 (28.0 ± 0.8 µg/mL) in the ABTS assay, compared to ascorbic acid displaying IC 50 of 6.0 ± 0.85 µg/mL. Conandroside (5), with a structure similar to verbascoside but with a xylose instead of rhamnose moiety attached to the glucose, had the highest activity with a significantly (p < 0.005) lower IC 50 of 27.3 ± 1.1 µg/mL than ascorbic acid (IC 50 of 5.0 ± 0.15 µg/mL). Conandroside has limited abundance in nature and has been isolated from the families, Gesneriaceae, Lamiaceae, and Polypremaceae [25]. To the best of our knowledge, this study is the first report of the antioxidant potential of conandroside.

Apparatus and Chemicals
Mass determination used a Jeol JMS-700 high-resolution mass spectrophotometer, with electron impact mode ionization at 70 ev. IR spectra were recorded on JASCO 320-A spectrometer. The 1 H-and 13 C-NMR spectra were recorded on an Ultra Shield Plus 500 MHz (Bruker, Billerica, MA, USA) spectrometer with a TMS internal standard.

Adulticidal Activity
Screening and toxicities of isolated compounds were evaluated in assays using cohorts of 3-5 day old adult Ae. aegypti females, following previously described procedures from a large natural product screening program [26]. Briefly, compounds were dissolved to a concentration of 100 µg/µL in DMSO with vigorous vortexing or gentle heating if needed. A 10 µg/µL solution for screening was created by diluting the 100 µg/µL stock in acetone. Orlando 1952 strain mosquitoes were cold anesthetized and sorted into groups of 10 females per TK35 cup (Solo Co, USA) and maintained at 4 • C until dosing. Initial screening of compounds at 5 µg/mosq was conducted by application of a 0.5 µL droplet to the ventral aspect of each mosquito using a repeating pipettor and a 25-µL Series 7100 gas tight syringe with a blunt tip (Hamilton Syringe Co.) to avoid damaging the mosquito. Cohorts of 10 mosquitoes were dosed with a specific compound in each assay. After dosing, cups were covered with a screen mesh and secured with a rubber band. Cotton balls saturated with 10% sucrose were provided for each cup and then cups were maintained at 22 • C in an insectary. Mortality was recorded 24 h after application. Permethrin and acetone were used as positive and negative controls, respectively. Initial screening assays were conducted three times. Subsequent dose response assays (three separate assays) were conducted for the four samples that produced screening mortality above 80% in initial screening. Dose response assays used the initial 100 µg/µL stock of each compound to produce a series of concentrations in acetone. These dilutions were applied in the same manner as above to produce a range of mortality values to calculate LD 50 . Prism 8.4.3 was used to analyze dose response mortality data in best-fit sigmoidal plots with the minimum and maximum constrained to 0 and 100%, respectively [27].
3.5. Radical Scavenging Activity 3.5.1. DPPH (2, 2-diphenyl-1-picrylhydrazyl) Scavenging Activity The free radical scavenging activity of compounds 1-5 was determined based on the scavenging activity of stable DPPH, as described by Mothana et al. (2019) [28]. Seven concentrations of each sample (25, 50, 100, 500, 1000 µg/mL) were prepared by mixing with 0.125 mL of 0.2 mM methanol solution of DPPH. The negative control was one mL of methanol. An ascorbic acid positive control was prepared at the same concentrations as the test samples (Table 2). Absorbance was measured at λ = 517 nm after 30 min of incubation in the dark. DPPH percent inhibition of antioxidant effect was calculated using the formula: % of anti-radicle activity = [(Abs control − Abs sample )/Abs control ] × 100 Assays were run in triplicate and means and standard errors were calculated.

ABTS •+ Radical Cation Scavenging Activity
Antioxidative activity of compounds 1-5 was also resolved utilizing the 2,2 -azinobis (3-ethylbenzothiazoline-6-sulfonate radical cation (ABTS •+ ) method, as reported by Alqahtani et al., 2019 [29], with minor modification. In brief, ABTS and potassium persulfate were prepared in deionized water to a 7 and 2.45 millimolar final concentration. To a 50-µg/mL ABTS concentration, various concentration of each extract was pipetted (1:1) and the absorbance reading (λ 734 nm) was taken after 1 h of reaction initiation using UV-vis spectrophotometer. The capacity of each extract to exert antioxidant was determined based on the absorbance of ABTS reduced solution according to the following formula: % of radical scavenging activity = [(Abs control − Abs sample )/Abs control ] × 100

Conclusions
Isolation of secondary metabolites from the dichloromethane and n-butanol extracts of N. oppositifolia led to the identification of two flavonoids (compounds 1 and 2) and three phenylethanoid glycosides (compounds 3-5). This study is the first to report the insecticidal and free radical scavenging activities of compounds from genus, Nuxia, including N. oppositifolia. The results indicate that further phytochemical and biological examination of this plant should be conducted with the aim of developing new classes of environmentally-friendly insecticidal agents. In addition, N. oppositifolia appears to be a rich source of natural antioxidant phenolics.