**1. Introduction**

Insect disease vectors transmit many disease pathogens and are important in global public health. *Aedes aegypti* (L.) and *Ae. albopictus* (Skuse) are the primary and secondary vectors of Zika and dengue as well as other viruses [1]. The use of synthetic insecticides in mosquito control has proven to be one of the major approaches for the prevention and reduction of mosquito-borne disease incidence [2]. Insect repellents also play an important role in the reduction of disease incidence by preventing infected mosquitoes from biting humans [3]. Moreover; repellents have always been used against host-seeking

vectors as they provide immediate; localized; personal protection. *N*,*N*-Diethyl-3-methylbenzamide (DEET) has been in use for more than 60 years and is the gold standard to which all repellents are measured in the marketplace [4]. The discovery of novel insecticides and repellents against disease vectors from non-toxic and biodegradable plant sources continues to be the focus of recent research efforts [5–8].

*Magnolia grandiflora* L. (Magnoliaceae) is a large evergreen tree native to North America [9] that has medicinal and ornamental values. Medicinal use of various parts of *M. grandiflora* is reported in American Indian medicine and also listed as a bitter tonic and antimalarial. Several biologically active compounds have been reported from *Magnolia* species [10–12]. As a part of our natural product screening program for mosquitoes, we tested essential oils from various parts of *M. grandiflora* for their larvicidal and biting deterrent activities. This paper reports insecticidal and biting deterrent activities of essential oils and select pure compounds from various parts of *M. grandiflora* against yellow fever mosquito, *Aedes aegypti*.

#### **2. Results**

Water-distilled essential oils of the leaves, flowers, immature fruits, mature fruits, and seeds of *M. grandiflora* were analyzed by GC-FID and GC-MS. Chemical compositions of the essential oils and total ion current (TIC) chromatogram are given in Table 1 and Figure S1 (Suplementary Material). Chemical profiles of the oils varied among essential oils. Sesquiterpene hydrocarbons (31.5%) were dominant in the seeds and immature fruit essential oils (32%) whereas the leaf and flower oils were rich in monoterpene hydrocarbons (30.9% and 43.8%, respectively). Oxygenated monoterpenes (36.9%) were the major components of the mature fruit oil followed by monoterpene hydrocarbons (15.2%), sesquiterpene hydrocarbons (15.5%), and oxygenated sesquiterpenes (16.2%). The α- and β-pinenes and 1,8-cineole were the major contents of leaf, flower, immature fruit, and mature fruit whereas these compounds were either very low or absent in seed essential oil (Table 1). The seed oil was differentiated from other essential oils because of the presence of fatty acid; hexadecanoic acid (2.9%) and fatty acid esters (2.2%). The saturated aliphatic esters (10.9%) and two phenolic compounds; methyl chavicol (2.6%) and eugenol (1.3%) were also only found in the seed essential oil. Major compounds, α- and β-pinenes and 1,8-cineole present in leaf, flower, immature fruit, and fruit essential oils were either in very low concentration or absent in seed essential oil (Table 1). Fatty acids and esters (5.1%) were high and aliphatic esters (10.9%) were present only in the seed essential oil. Hexadecanoic acid (2.9%), 1-decanol (3.3%), 1-octanol (6.2%), and 1-heptanol were also present only in the seed essential oil.




**Table 1.** *Cont.*


**Table 1.** *Cont.*

RRI: relative retention indices calculated against n-alkanes; %: calculated from FID data; tr: trace (< 0.1 %); IM: identification method based on the relative retention indices (RRI) of authentic compounds on the HP Innowax column; MS, identified based on computer matching of the mass spectra with those of the Wiley and MassFinder libraries and comparison with literature data. % calculated from FID data.; -: not detected.

The essential oils obtained from five different plant parts of *M. grandiflora* were investigated for their biting deterrent activity against *Ae*. *aegypti*. All the essential oils showed biting deterrence above the negative control. Seeds essential oil produced significantly higher biting deterrence than the essential oils from the other parts (Figure 1). Seed essential oil with high minimum effective dose BDI value (0.89) showed biting deterrent activity similar to DEET whereas all the other essential oils had activity lower than DEET. 1-Decanol with PNB value of 0.8 showed biting deterrence similar to DEET (PNB = 0.8) whereas the activity of 1-octanol was above negative control but lower than DEET and 1-decanol (Figure 2).

**Figure 1.** Mean BDI (± SEM) values of the essential oils from different parts of *M. grandiflora* against *Ae. aegypti.* All the essential oils were tested at 10 μg/cm2 whereas DEET (*N*,*N*-diethyl-3-methylbenzamide) was tested at 25 nmol/cm2 and ethanol a solvent control. A BDI value greater than 0 indicates biting deterrence relative to ethanol and a BDI value not significantly different from 1 shows deterrence similar to DEET.

Biting deterrence of 1-heptanol was similar to the negative control. 1-Decanol with 3.3% of seed essential oil appears to be the major compound responsible for the biting deterrent activity of the seed essential oil. In in vitro A & K bioassay, MED value of 1-decanol (6.25) was lower than DEET (12.5) which indicated better repellency of 1-decanol than DEET (Figure 3).

**Figure 3.** Data are %age (mean ± SEM) females biting. Minimum effective dosage (MED) values in this bioassay were ≤ 1% biting in 1 min which is two females out of 200 in this cage.

The toxicity of essential oils from *M. grandiflora* against 1-d-old larvae of *Ae. aegypti* is given in Table 2. In initial screening, essential oils of immature and mature fruits showed high toxicity whereas leaf, flower, and seeds essential oils gave only 20%, 0%, and 50% mortality, respectively, at the highest screening dose of 125 ppm. Therefore dose-response bioassays were not conducted on leaf, flower, and seeds essential oils. Immature fruit and mature fruit essential oils with LC50 values of 49.4 and 48.9 ppm, respectively at 24-h post-treatment showed similar toxicity.


**Table 2.** Toxicity of essential oils from *M. grandiflora* and its select pure compounds against 1-day-old *Ae. aegypti* at 24-h post-treatment.

\* LC50 and LC90 values are in ppm and 95% C.I are confidence intervals. \*\* Leaf, flower and seeds essential oils gave only 20%, 0 and 50% mortality, respectively, at the highest dose of 125 ppm.

Pure compounds 1-decanol, 1-octanol, and 1-heptanol, present in seed essential oil were also screened for larvicidal activity. Both 1-decanol and 1-octanol were active in screening bioassays whereas 1-heptanol did not show any mortality at the highest dose of 125 ppm. 1-Decanol and 1-octanol were further evaluated to observe the dose response. 1-Decanol with LC50 of 4.8 ppm was the most toxic compound followed by 1-octanol (LC50 = 34.3 ppm) at 24-h post-treatment. 1-Decanol was very toxic (LC50 = 4.8 ppm) as a pure compound, the seed essential oil that contained 3.3% of this compound showed 50% mortality at the highest dose of 125 ppm. 1-Decanol amounted to be 4.07 ppm as a part of seed essential oil at 125 ppm which caused mortality similar to the pure compound. 1-Decanol and 1-octanol amounted to be 4.07 and 7.75 ppm, respectively, as a part of the essential oil dose of 125 ppm. Since the toxicity of 1-octanol as a pure compound was low, the main compound responsible for the toxicity of the seed essential oil appears to be 1-decanol.
