Mosquito Larvicidal Activity and Cytotoxicity of the Extracts of Aromatic Plants from Malaysia

Simple Summary Mosquitoes are not only a nuisance, but most of them pose a threat to human beings as they can transmit pathogens that are harmful and may turn fatal, such as dengue, chikungunya, and Zika. This study was carried out to evaluate the larvicidal properties of the solvent extracts from three aromatic plants, Curcuma longa (turmeric), Ocimum americanum (hoary basil), and Petroselinum crispum (parsley)—against the mosquito vector of dengue, Aedes albopictus. Thereafter, the phytochemical profiles and the cellular toxicity of the extracts were evaluated. The results indicate that the hexane extracts of O. americanum and P. crispum had the greatest larvicidal activity. Among these two, O. americanum was shown to be less toxic to African monkey kidney cells and possessed an abundant amount of methyl eugenol, which is a phytochemical with known larvicidal activities. Overall, our findings indicate that these aromatic plants, especially O. americanum, may prove to be highly promising alternatives as bioinsecticides. Abstract Despite ongoing control efforts, the mosquito population and diseases vectored by them continue to thrive worldwide, causing major health concerns. There has been growing interest in the use of botanicals as alternatives to insecticides due to their widespread insecticidal properties, biodegradability, and adaptability to ecological conditions. In this study, we investigated the larvicidal activity and cytotoxicity effects of solvent extracts from three aromatic plants—Curcuma longa (turmeric), Ocimum americanum (hoary basil), and Petroselinum crispum (parsley)—against Aedes albopictus. Subsequently, we examined the phytochemical composition of the extracts through gas chromatography–mass spectrometry (GC–MS) analysis. Results revealed that the hexane extracts of O. americanum and P. crispum exhibited the greatest larvicidal activity with the lowest median lethal concentration (LC50) values (<30 µg/mL) at 24 h post-treatment, with the former found to be significantly less toxic towards African monkey kidney (Vero) cells. The GC–MS analysis of the said extract indicated the presence of different classes of metabolites, including phenylpropanoids, very long-chain alkanes, fatty acids and their derivatives, and terpenes, with the most abundant component being methyl eugenol (55.28%), most of which, have been documented for their larvicidal activities. These findings provide valuable insights into the potential use and development of bioinsecticides, particularly from O. americanum.


Introduction
Dengue fever, Zika, and chikungunya are among the many mosquito-borne diseases that continue to pose significant threats to public health, particularly in tropical and subtropical regions. It was anticipated that close to half of the world's population would be at risk of arboviral infections by the year 2050 [1]. Among all these, dengue stands out as the most rapidly spreading virus globally. little room for the target vectors to develop resistance to such substances [18]. Moreover, botanical insecticides are preferred alternatives due to their rapid degradation and low toxicity [17]. The best approach to controlling mosquito populations is to target the aquatic larvae before to mature into terrestrial adults.
Turmeric (Curcuma longa L.) is a traditional Chinese herb belonging to the Zingiberaceae family [17]. It is a fragrant, perennial rhizomatous herb with large and pointed leaves [19]. The C. longa plant can grow up to 1 to 2 m in height with long, dotted leaves and funnel-shaped flowers. The yellowish egg-or pear-shaped rhizomes, which are grown underground, have a mild aroma with a sharp taste similar to ginger and are often used as a condiment and as coloring agents in medicines and food [20]. The constituents of the leaf, rhizome, and flowers of C. longa have been extensively studied [21][22][23]. The rhizome essential oil has been reported to exhibit antimicrobial, anti-cancer, insecticidal, larvicidal, repellent, and antioxidant activities [24][25][26][27][28][29].
Additionally, the leaf oil has also been reported to exhibit fumigant toxicity against beetles generally found in stored products [30]. The C. longa plant contains tetrahydrocurcumin, an odorless and heat-resistant antioxidant compound, and numerous polyphenolic compounds such as curcumin, eugenol, cinnamic acid, limonene, linalool, turmerone, and vanillic acid [31]. Available scientific evidence suggests that the essential oil and some constituents in the turmeric extracts were reportedly shown to possess potent larvicidal properties against Aedes aegypti [32], Ae. albopictus [33], Anopheles gambiae [19], An. stephensi [34], Culex quinquefasciatus [35], and Cx. pipiens pallens [17]. Nonetheless, the toxicities of these phytochemicals may vary depending on the origins of the plants and their target mosquito species [18,36].
Ocimum americanum L., also known as hoary basil, lime basil, or American basil, is a medicinal and aromatic annual herb in the family Lamiaceae. It has a strong citrus smell, and the plant can grow between 15 and 35 cm in height with elliptic, pointy leaves and white or pale lilac flowers [37]. Unlike O. basilicum (Thai basil), which is a popular aromatic herb incorporated in Thai and Vietnamese cuisines, O. americanum is not frequently used as a culinary herb but rather as herbal medicine [38]. The seeds are considered diuretics and can be used as a tonic. A decoction of the leaves of the plant can be taken for treating coughs, constipation, stomachaches, and dysentery, as well as a mouthwash for relieving toothache [38,39]. The plant has been shown to possess antimicrobial, antifungal, antiulcer, insecticidal, and larvicidal activities as well as wound-healing effects [40][41][42].
Having about 150 members, Ocimum is considered the largest genus in the Lamiaceae family and is known to possess a rich source of essential oils with various biological activities [43]. O. americanum exhibits varying phytochemical compositions depending on the solvent system used [38]. Inevitably, the choice of extraction method would highly depend on the chemical nature of the bioactive compounds of interest [41]. The major compounds found in the essential oil and various solvent extracts of O. americanum are said to be eugenol, methyl chavicol, 1,8-cineole, citral, linalool, camphor, monoterpenoids, flavonoids, polyphenols, and limonene [40,[44][45][46]. The known insecticidal compounds found in the volatile oils of some Ocimum spp. are methyl cinnamate, methyl chavicol, and eugenol. Other chemicals that have been reported to have insect-repellent activity are thymol, carvacrol, camphor, caryophyllene oxide, cineole, limonene, and myrcene. However, the chemical components and essential oil levels of Ocimum may vary between species and cultivars and under different growing conditions [47,48].
Unlike the more pronounced members of the Ocimum genus, for instance, O. basilicum, O. sanctum, and O. tenuiflorum, which are immensely investigated for their mosquitocidal and repellency properties, there is minimal research and evidence to reflect on the larvicidal efficacy of the extracts and essential oils of O. americanum against mosquito vectors. Only recently, Narayanan et al. [49] reported on the bioactive components of the aqueous extracts of O. americanum, which showed significant larvicidal properties against Ae. aegypti, An. stephensi, and Cx. quinquefasciatus. Petroselinum crispum (Mill.) Fuss is a bright green, biennial herb belonging to the family Apiaceae. It is commonly called parsley or garden parsley. The plant can grow up to 30 to 100 cm tall, with aromatic flat or curled leaves and small, yellow-green flowers [50]. Parsley was appreciated for its medicinal properties long before it was accepted as a vegetable, a garnish, and a seasoning and flavoring agent. It has been traditionally used for ailments and complaints of the gastrointestinal tract as well as the kidney and lower urinary tract [51]. Parsley seeds have been claimed to be antimicrobial, antiseptic, and antispasmodic [52]. The leaves possess anticoagulant activity and are used as an antitussive and as a treatment for dermatitis, poor vision performance, hemorrhoids, eczema, nosebleeds, amenorrhea, hypertension, and hyperuricemia [53][54][55][56].
The seeds of P. crispum produce high amounts of essential oils. According to the reports by Farzaei et al. [50] and Agyare et al. [57], the primary components and active compounds that are reportedly found in the essential oils and solvent extracts of parsley consist of myristicin, apiol, monoterpene hydrocarbons, eugenol, coumarins, phenolic compounds, and flavonoids, particularly apigenin, apiin, and 6"-acetylapiin. In another study, four compounds isolated and identified from the methanolic extract of P. crispum, which were pabulenol, oxypeucedanin, oxypeucedanin hydrate, and N-(2-phenylethyl) hexanamide, were shown to possess significant phototoxicity properties [58,59]. The essential oil of P. crispum has long been considered a potential bioinsecticide due to the many reported findings of its significant mosquitocidal, larvicidal, and repellency properties against mosquito vectors, primarily the dengue vector Ae. aegypti [60][61][62] and other less common species such as Cx. pipiens, Culiseta longiareolata [63], and Ochlerotatus caspius [64].
In Malaysia, these three aromatic herbs are commonly used not only in traditional medicine but also as culinary herbs. Despite the known larvicidal potential of these plants against many mosquito vectors, there is a lack of data on their mosquitocidal properties against another equally important dengue vector, Ae. albopictus. Additionally, plants are a rich, untapped pool of phytochemicals that may one day replace synthetic insecticides in mosquito control programs. Although numerous studies have documented the efficacy of plant extracts, most of these do not look into characterizing and determining the structures of the active compounds that are responsible for their larvicidal activity. Hence, there is a need for more isolation and purification work to discover the true potential of these phytochemicals as botanical larvicides. Moreover, it is essential to assess the safety of these plant extracts through cytotoxicity screenings before they are adopted in public health interventions.

Preparation of Plant Extracts
The fresh plant materials were cleaned thoroughly under running tap water to remove dirt and soil. The rhizomes of C. longa (250.89 g), the leaves of O. americanum (786.72 g), and the leaves and stems of P. crispum (603.09 g) were subjected to maceration sequentially using solvents of increasing polarity, i.e., hexane, chloroform (Qrec, Chonburi, Thailand), ethyl acetate, ethanol (Merck, Darmstadt, Germany), methanol (RCI Labscan, Bangkok, Thailand), and distilled water [66]. The macerations were performed at room temperature and with an agitation of 110 rpm for three cycles (one day/cycle). After filtration, the organic solvents were removed using a rotary evaporator, while the water extracts were lyophilized. All dry extracts were kept at −20 • C pending bioassay. The percentage of yield (w/w) was calculated using the following formula: Percentage of yield = (Weight of dry extract)/(Weight of fresh plant material) × 100

Mosquito Sampling and Larvae Culturing
Sixty ovitraps were set up inside the university campus (Kampar Campus) between October 2014 and January 2015 for mosquito egg collection. A hardboard paddle (10.0 cm × 2.5 cm × 0.3 cm) was inserted diagonally into each ovitrap ( Figure 1). The ovitraps were hung on the trees approximately 1 m above the ground. The paddles were replaced, and the water was refilled three times a week. The egg-containing paddles were airdried for two days before being immersed in plastic containers (18.0 cm × 17.0 cm × 7.0 cm) filled with dechlorinated tap water for egg hatching. The emerged larvae were fed with ground cat food (Cuties Catz, Perfect Companion (M) Sdn. Bhd., Kuala Lumpur, Malaysia). The third-instar larvae were then identified using a stereomicroscope based on their morphology [67]. Only larvae of the species Ae. albopictus were used for larvicidal bioassays. tially using solvents of increasing polarity, i.e., hexane, chloroform (Qrec, Chonburi, Thailand), ethyl acetate, ethanol (Merck, Darmstadt, Germany), methanol (RCI Labscan, Bangkok, Thailand), and distilled water [66]. The macerations were performed at room temperature and with an agitation of 110 rpm for three cycles (one day/cycle). After filtration, the organic solvents were removed using a rotary evaporator, while the water extracts were lyophilized. All dry extracts were kept at −20 °C pending bioassay. The percentage of yield (w/w) was calculated using the following formula: Percentage of yield = (Weight of dry extract)/(Weight of fresh plant material) × 100

Mosquito Sampling and Larvae Culturing
Sixty ovitraps were set up inside the university campus (Kampar Campus) between October 2014 and January 2015 for mosquito egg collection. A hardboard paddle (10.0 × 2.5 × 0.3 cm) was inserted diagonally into each ovitrap ( Figure 1). The ovitraps were hung on the trees approximately 1 m above the ground. The paddles were replaced, and the water was refilled three times a week. The egg-containing paddles were air-dried for two days before being immersed in plastic containers (18.0 × 17.0 × 7.0 cm) filled with dechlorinated tap water for egg hatching. The emerged larvae were fed with ground cat food (Cuties Catz, Perfect Companion (M) Sdn. Bhd., Kuala Lumpur, Malaysia). The third-instar larvae were then identified using a stereomicroscope based on their morphology [67]. Only larvae of the species Ae. albopictus were used for larvicidal bioassays.

Larvicidal Bioassay
The larvicidal bioassay was performed according to the World Health Organization (WHO) guidelines [68] with slight modifications. Each extract was dissolved in a dimethyl sulfoxide-ethanol mixture (3:2, v/v), sonicated for 1 min using an ultrasonic bath (S100H Elmasonic, Germany), and filtered using 0.45 µm nylon syringe filters to produce a stock solution of 60 mg/mL. Appropriate volumes of the stock solution were pipetted into round plastic containers (9.2 × 6.0 cm) filled with tap water to produce five different concentrations (50, 100, 200, 400, and 600 µg/mL) for the bioassay. The final volume for each container was 150 mL. Later, 20 third-instar larvae were introduced into each container, and larval mortality was observed and recorded at 2, 24, and 48 h post-treatment. The dimethyl sulfoxide-ethanol mixture in the containers was maintained at ≤1% to avoid any toxicity to the larvae. The larvae were considered dead if they did not move when prodded with a needle in the siphon or cervical region. A 1% dimethyl sulfoxide-ethanol mixture was used as a negative control, while temephos (Vector Control Research Unit, Universiti Sains Malaysia, Penang, Malaysia) at 1 µg/mL was used as a positive control. The bioassay was

Larvicidal Bioassay
The larvicidal bioassay was performed according to the World Health Organization (WHO) guidelines [68] with slight modifications. Each extract was dissolved in a dimethyl sulfoxide-ethanol mixture (3:2, v/v), sonicated for 1 min using an ultrasonic bath (S100H Elmasonic, Germany), and filtered using 0.45 µm nylon syringe filters to produce a stock solution of 60 mg/mL. Appropriate volumes of the stock solution were pipetted into round plastic containers (9.2 × 6.0 cm) filled with tap water to produce five different concentrations (50,100,200, 400, and 600 µg/mL) for the bioassay. The final volume for each container was 150 mL. Later, 20 third-instar larvae were introduced into each container, and larval mortality was observed and recorded at 2, 24, and 48 h post-treatment. The dimethyl sulfoxide-ethanol mixture in the containers was maintained at ≤1% to avoid any toxicity to the larvae. The larvae were considered dead if they did not move when prodded with a needle in the siphon or cervical region. A 1% dimethyl sulfoxide-ethanol mixture was used as a negative control, while temephos (Vector Control Research Unit, Universiti Sains Malaysia, Penang, Malaysia) at 1 µg/mL was used as a positive control. The bioassay was conducted in three replicates. The percentage of mortality was calculated using the following formula: Percentage of mortality = (Number of dead larvae)/(Total number of larvae) × 100

Larvae Morphological Examination
The dead larvae treated with the hexane extracts of O. americanum and P. crispum at 600 µg/mL were examined for morphological abnormalities using a stereomicroscope.

Cytotoxic Activity
Healthy kidney epithelial cells derived from the African monkey (ATCC ® CCL-81™) were used to assess the cytotoxicity of plant extracts. The cells were grown in supplemented Dulbecco's Modified Eagle Medium (DMEM) at 37 • C and 5% carbon dioxide. The supplementation used was 10% fetal bovine serum, 10,000 U/mL of penicillin, 10 mg/mL of streptomycin, and 3.7 mg/mL of sodium carbonate [69]. Cell count was enumerated using a hemacytometer, and a cell concentration of 40,000 per well was seeded in a 96-well microplate. Subsequently, 100 µL of plant extract (5,10,20,40,80,160, 320, and 640 µg/mL) was pipetted to the wells and incubated at 37 • C and 5% carbon dioxide for 72 h. Prior to this, the plant extract was diluted twofold serially in DMEM with 1% fetal bovine serum. Cell control and medium control were included in each microplate. After incubation, the viability of cells was ascertained using the protocol of the neutral red uptake assay, as described by Repetto et al. [70]. The absorbance value was measured at 540 nm using a microplate reader (Tecan, Switzerland) and used to determine the percentage of cell viability. A plot of the percentage of cell viability versus concentration was established for each extract, and the median cytotoxic concentration (CC 50 ) was interpolated from the plot for the extract. The assessment was conducted in three replications.

Profiling of Metabolites Using Gas Chromatography-Mass Spectrometry (GC-MS)
The hexane extract of O. americanum was selected for metabolite profiling due to its potency for larvicidal activity. The qualitative composition analysis of the extract was performed using a gas chromatography-mass spectrometer (QP2010 Plus, Shimadzu Corporation, Kyoto, Japan). The separation of components was achieved using a fused silica capillary column (30.0 m × 0.25 mm × 0.25 µm) with 5% diphenyl-95% dimethylpolysiloxane (SLB-5ms, Supelco, Bellefonte, PA, USA). The column temperature was maintained at 50 • C for 3 min, ramped at a rate of 4 • C/minute to 300 • C, and held for 5 min. The total analysis time for each sample was 70.50 min. The helium gas (mobile phase) flowed at a linear velocity rate of 30 cm/minute. The extract was dissolved in acetone (HPLC grade, Fisher Scientific Limited, Loughborough, UK) at 2 mg/mL and filtered using a 0.22 µm nylon syringe filter prior to injection. The injector port temperature was set at 280 • C. The injection volume was 1 µL, and a split ratio of 10.0 was used for the sample injection. For the mass spectrometer settings, the ion source and interface temperatures were 200 • C and 300 • C, respectively. Electron impact ionization at 70 eV was used. The fragment ions of each component were monitored for an m/z of 40-600. The spectra of each component were compared with the spectra in the NIST 14 Mass Spectral Library and Search Software (National Institute of Standards & Technology, Gaithersburg, MD, USA), and the identity of the component with a matching quality >86% was reported. The analysis was performed in duplicate.

Data Analysis
The larval mortality rate was expressed as the mean ± standard deviation of three replicates. The data were subjected to Probit analysis to determine the median lethal concentration (LC 50 ), 95% lethal concentration (LC 95 ), upper confidence limit, lower confidence limit, and regression coefficient. The percentages of larval mortality and cell viability were tested for significance using one-way analysis of variance (ANOVA), followed by the post-hoc Duncan's multiple range test. The statistical analysis was conducted using IBM SPSS Statistics for Windows, Version 23.0 (IBM Corp., Armonk, NY, USA). The significance level was set at α = 0.05.

Results
In this study, each aromatic plant sample was extracted sequentially using six solvents of increasing polarity, i.e., n-hexane (polarity index: 0.1), chloroform (4.1), ethyl acetate (4.4), ethanol (4.3), methanol (5.1), and water (10.2) [71]. This allows the segregation of plant secondary metabolites according to their polarity and facilitates the identification of an extract with specific biological activity. As shown in Figure 2, the yields for most of the extracts were less than 2% w/w except for the water extract of C. longa and the ethanol extract of P. crispum, which generated a yield of 9.77% and 2.50% w/w, respectively. The total yields for the extracts of C. longa, O. americanum, and P. crispum were 13.11%, 3.57%, and 5.40% w/w, respectively. the post-hoc Duncan's multiple range test. The statistical analysis was conducted using IBM SPSS Statistics for Windows, Version 23.0 (IBM Corp., Armonk, NY, USA). The significance level was set at α = 0.05.

Results
In this study, each aromatic plant sample was extracted sequentially using six solvents of increasing polarity, i.e., n-hexane (polarity index: 0.1), chloroform (4.1), ethyl acetate (4.4), ethanol (4.3), methanol (5.1), and water (10.2) [71]. This allows the segregation of plant secondary metabolites according to their polarity and facilitates the identification of an extract with specific biological activity. As shown in Figure 2, the yields for most of the extracts were less than 2% w/w except for the water extract of C. longa and the ethanol extract of P. crispum, which generated a yield of 9.77% and 2.50% w/w, respectively. The total yields for the extracts of C. longa, O. americanum, and P. crispum were 13.11%, 3.57%, and 5.40% w/w, respectively. The 18 plant extracts were evaluated for larvicidal activity against third-instar larvae of Ae. albopictus at concentrations ranging from 50 to 600 µg/mL and the mortality rate was recorded at 2, 24, and 48 h post-treatment. The evaluation was performed in triplicate, with 20 larvae in each replicate. Since no mortality was observed in the negative control, Abbot's correction was not applied in calculating the percentage of larval mortality. All extracts from the rhizomes of C. longa, except water extract, showed larvicidal activity at 24 h post-treatment. The hexane extract was the only extract that could kill all larvae within 24 h (100% mortality rate) when its concentration exceeded 200 µg/mL ( Figure 3A). Although the water extract displayed larvicidal activity only at 48 h post-treatment, the mortality rate was less than 10%. Interestingly, the mortality rate for the methanol extract increased from 11.7 ± 2.9% to 88.3 ± 12.6% and from 6.7 ± 11.5% to 93.3 ± 5.8% for 400 µg/mL and 600 µg/mL, respectively, after an additional 24 h of treatment. These results suggest that the active larvicidal compounds may have a slow onset of action.
Among the three aromatic plants evaluated, O. americanum can be regarded as the plant with the strongest larvicidal activity, in terms of potency and onset of action, against the larvae of Ae. albopictus. In fact, the hexane extract of the leaves of O. americanum was the only extract found to have larvicidal activity within 2 h of treatment in the present study. It resulted in larval mortality rates of 10.0 ± 0.0% and 23.4 ± 7.6% at 400 and 600 µg/mL, respectively. The larvicidal effect of O. americanum was mainly documented from the hexane, chloroform, and ethyl acetate extracts, suggesting that the larvicidal compounds are non-polar or intermediate polar ( Figure 3B). After 24 h of treatment, the hexane extract (≥50 µg/mL) produced a larval mortality rate of 100%. Consequently, further The 18 plant extracts were evaluated for larvicidal activity against third-instar larvae of Ae. albopictus at concentrations ranging from 50 to 600 µg/mL and the mortality rate was recorded at 2, 24, and 48 h post-treatment. The evaluation was performed in triplicate, with 20 larvae in each replicate. Since no mortality was observed in the negative control, Abbot's correction was not applied in calculating the percentage of larval mortality. All extracts from the rhizomes of C. longa, except water extract, showed larvicidal activity at 24 h post-treatment. The hexane extract was the only extract that could kill all larvae within 24 h (100% mortality rate) when its concentration exceeded 200 µg/mL ( Figure 3A). Although the water extract displayed larvicidal activity only at 48 h post-treatment, the mortality rate was less than 10%. Interestingly, the mortality rate for the methanol extract increased from 11.7 ± 2.9% to 88.3 ± 12.6% and from 6.7 ± 11.5% to 93.3 ± 5.8% for 400 µg/mL and 600 µg/mL, respectively, after an additional 24 h of treatment. These results suggest that the active larvicidal compounds may have a slow onset of action.
Among the three aromatic plants evaluated, O. americanum can be regarded as the plant with the strongest larvicidal activity, in terms of potency and onset of action, against the larvae of Ae. albopictus. In fact, the hexane extract of the leaves of O. americanum was the only extract found to have larvicidal activity within 2 h of treatment in the present study. It resulted in larval mortality rates of 10.0 ± 0.0% and 23.4 ± 7.6% at 400 and 600 µg/mL, respectively. The larvicidal effect of O. americanum was mainly documented from the hexane, chloroform, and ethyl acetate extracts, suggesting that the larvicidal compounds are non-polar or intermediate polar ( Figure 3B). After 24 h of treatment, the hexane extract (≥50 µg/mL) produced a larval mortality rate of 100%. Consequently, further dilution beyond 50 µg/mL was performed for the hexane extract to obtain the LC 50 Table 1, the LC50 and LC95 values for the hexane extract at 24 h post-treatment were 26.60 µg/mL and 51.39 µg/mL, respectively.

LC95 values. As shown in
As for P. crispum, only the hexane extract showed significant larvicidal activity. A larval mortality rate of 100% was observed at 24 h when the hexane extract concentration exceeded 50 µg/mL. The LC50 and LC95 values for this extract were 14.35 µg/mL and 83.66 µg/mL, respectively ( Table 1). The three more polar extracts (ethanol, methanol, and water) were devoid of larvicidal activity ( Figure 3C). As for P. crispum, only the hexane extract showed significant larvicidal activity. A larval mortality rate of 100% was observed at 24 h when the hexane extract concentration exceeded 50 µg/mL. The LC 50 and LC 95 values for this extract were 14.35 µg/mL and 83.66 µg/mL, respectively ( Table 1). The three more polar extracts (ethanol, methanol, and water) were devoid of larvicidal activity ( Figure 3C). Unlike the normal untreated Ae. albopictus third-instar larva, which has a dark brown midgut and a pair of transparent sausage-like anal papillae ( Figure 4A), larvae treated with the hexane extract of O. americanum had a whitish midgut with a shrunken and pigmented cuticle of anal papillae ( Figure 4B). Similar abnormalities were also seen in the larvae treated with the hexane extract of P. crispum ( Figure 4C). Unlike the normal untreated Ae. albopictus third-instar larva, which has a dark brown midgut and a pair of transparent sausage-like anal papillae ( Figure 4A), larvae treated with the hexane extract of O. americanum had a whitish midgut with a shrunken and pigmented cuticle of anal papillae ( Figure 4B). Similar abnormalities were also seen in the larvae treated with the hexane extract of P. crispum ( Figure 4C).  Generally, the chloroform and ethyl acetate extracts of the three aromatic plants displayed higher toxicity towards Vero cells than the other four extracts, as shown in Figure 5. Only the water extract of O. americanum was not toxic to the Vero cells at all concentrations evaluated (p > 0.05). On the other hand, the ethyl acetate extract of C. longa exhibited the lowest CC 50 (12.7 ± 0.6 µg/mL) among the extracts, indicating high toxicity toward the cells (Table 2). Notably, the methanol extract of C. longa also exerted strong toxicity (CC 50 = 23.3 ± 1.5 µg/mL) towards the Vero cells, unlike the methanol extracts of the other two plants, which showed weak cytotoxicity with CC 50 values higher than 640 µg/mL. This finding is also consistent with the significant larvicidal activity shown by the same extract at the 48-h exposure period ( Figure 3A) compared to that of the other two plants. The values are expressed as means ± standard deviations of triplicates. '-' denotes no significant cytotoxicity even at the highest concentration tested (640 µg/mL). Mean values with different superscript letters ( a-d ) denote that the extracts are significantly different (p < 0.05) from each other by a one-way ANOVA or an independent-sample T-test. For the two extracts with strong larvicidal activity in this study, the hexane extract of O. americanum showed much lower toxicity (CC50: >640 µg/mL) against Vero cells than the hexane extract of P. crispum (CC50: 159.3 µg/mL). Therefore, the hexane extract of O. americanum was selected for further analysis using gas chromatography-mass spectrometry to obtain more information on the potential larvicidal components present in the extract. The total ion chromatogram of the extract is shown in Figure 6. A total of 31 peaks were detected from the extract, and 14 of them were successfully identified, representing 92.07% For the two extracts with strong larvicidal activity in this study, the hexane extract of O. americanum showed much lower toxicity (CC 50 : >640 µg/mL) against Vero cells than the hexane extract of P. crispum (CC 50 : 159.3 µg/mL). Therefore, the hexane extract of O. americanum was selected for further analysis using gas chromatography-mass spectrometry to obtain more information on the potential larvicidal components present in the extract. The total ion chromatogram of the extract is shown in Figure 6. A total of 31 peaks were detected from the extract, and 14 of them were successfully identified, representing 92.07% of the total peak area or 91.95% of the total peak height ( Table 3). The major component in the extract, based on the peak area, was methyl eugenol (55.28%), followed by tetrapentacontane (10.77%), eugenol (7.77%), linolenic acid (4.62%), and hexatriacontane (3.25%). The values are expressed as means ± standard deviations of triplicates. '-' denotes no significant cytotoxicity even at the highest concentration tested (640 µg/mL). Mean values with different superscript letters ( a-d ) denote that the extracts are significantly different (p < 0.05) from each other by a one-way ANOVA or an independent-sample T-test.

Curcuma longa
14, x FOR PEER REVIEW 12 of 19 Figure 6. The total ion chromatogram for the hexane extract of Ocimum americanum leaves using gas chromatography-mass spectrometry.  Figure 6. The total ion chromatogram for the hexane extract of Ocimum americanum leaves using gas chromatography-mass spectrometry.

Discussion
Our findings on C. longa are consistent with the results reported in the literature. The essential oils of the rhizome of C. longa possess larvicidal activities against the disease-carrying mosquitoes Ae. aegypti [72,73], An. gambiae [19], An. stephensi, and Cx. quinquefasciatus [74]. The C. longa oil at 200 µg/mL could kill 84% of Ae. albopictus larvae within 24 h [32]. A larvicidal compound named ar-turmerone has been isolated from the ethyl acetate and ethanol extracts of the rhizome and is active against Ae. aegypti and Cx. pipiens [17,75].
To the best of our knowledge, this study is the first report of the larvicidal activity of O. americanum against Ae. albopictus. Several studies have evaluated the essential oils and solvent extracts derived from the leaves and/or stems of O. americanum for larvicidal activity against Ae. aegypti and reported LC 50 values ranging from 15.03 µg/mL to 168 µg/mL for 24 h post-exposure [47,[76][77][78]. Studies of the larvicidal activity of P. crispum have been largely focused on the essential oils of the seeds and fruits. The essential oils are active against the larvae of the mosquitoes Ae. aegypti [73], Cx. pipiens, Cs. longiareolata [63], and Oc. caspius [64]. Our results indicated that the hexane extract of the aerial parts of P. crispum could kill all Ae. albopictus larvae at 50 µg/mL after 48 h post-treatment. Based on the criterion proposed by Pavela [79], both hexane extracts of O. americanum and P. crispum have strong larvicidal activity, as their LC 50 values for 24 h post-treatment were <50 µg/mL.
The morphological changes on the midgut and anal papillae of Ae. albopictus larvae in this study have also been observed when the larvae were exposed to the ethanol extract of papaya seed and the chloroform extract of the seaweed Bryopsis pennata, respectively [80,81]. In contrast, deformed larvae of Ae. albopictus with a blackish midgut and an extended cephalo-thoracic junction was found following treatment with the ethanolic leaf and fruit extracts of Piper nigrum [82] and the hexane leaf extract of O. basilicum [83]. These suggest that the midgut of mosquito larvae serves as a common target organ for the larvicidal effect of plant extracts, and the toxicity effects depend on the plant species, which is consistent with the findings reported by David et al. [84]. The morphological deformities are likely caused by the interference of hormonal control or interruption of chitin synthesis by the bioactive components in the extracts [85].
The cytotoxicity results of the present study were comparable to those of other published studies. Grover et al. [86] reported that the CC 50 values of the ethanol and hydroalcoholic (methanol-water, 3:2 v/v) extracts for C. longa rhizomes were 525.0 and 16.8 µg/mL, respectively. Berrington and Lall [87] extracted the aerial parts of P. crispum using acetone solvent and obtained a CC 50 value of 105 µg/mL against Vero cells. Although the cytotoxicity of the methanol extract of O. americanum using the same Vero cell line was similar, the CC 50 values obtained for the chloroform (509.1 µg/mL), ethyl acetate (430.7 µg/mL), and ethanol (>640 µg/mL) extracts were much higher than those of our previous study, which recorded 86.3, 60.8, and 226.3 µg/mL, respectively [66]. This could be attributed to the source of origin of the O. americanum used in this study. The O. americanum in the present study was sourced from the Ipoh area, while the sample used in the previous study was obtained from the Kampar area. More studies are needed to confirm this observation.
Methyl eugenol and eugenol belong to the phenylpropanoids class of natural products, which are synthesized from primary metabolites, phenylalanine or tyrosine amino acids, via the shikimic acid pathway in plants [88]. They are common and major essential oil constituents of many Ocimum species, such as O. basilicum, O. campechianum, O. gratissimum, O. sanctum, and O. urticifolium [89,90]. Being the major component in the extract, methyl eugenol is likely to account for the potent larvicidal activity observed in this study. Methyl eugenol has been shown to have a biocidal effect against the mosquito larvae of Ae. aegypti, Anopheles spp., Cx. pipiens pallens, and Ochlerotatus togoi [91,92], albeit its activity against Ae. albopictus larvae is yet to be studied. On the other hand, eugenol has been reported to have larvicidal activity against Ae. albopictus with LC 50 values of 28.1-67.4 µg/mL [93][94][95].
The gas chromatography-mass spectrometry analysis revealed that the leaves of O. americanum contained a substantial quantity of very long-chain hydrocarbons or alkanes, which are tetrapentacontane (C 54 H 110 ) and hexatriacontane (C 36 H 74 ), with abundances of 10.77% and 3.25%, respectively (Table 3). To the best of our knowledge, this is the first report of the presence of these alkanes in Ocimum species. Very long-chain alkanes are essential for the protection of plants from drought stress [96]. As the molecules only contain carbon and hydrogen atoms, they barely serve as bioactive metabolites, and hence, they may not account for the larvicidal activity of the extract.
The leaves of O. americanum also contained different types of fatty acids and their methyl or ethyl derivatives, which are linolenic acid (C18:3n3), palmitic acid (C16:0), linoleic acid (C18:2n6), methyl linolenate, and ethyl linolenate (Table 3). Similar fatty acids have been detected in the ethyl acetate or methanol extract of the leaves or aerial parts of O. americanum [41,97]. Among these components, the three fatty acids have been assessed against larvae of Ae. albopictus, and their LC 50 values after 24 h of exposure were 71.34 µg/mL for linolenic acid, 40.96-85.61 µg/mL for palmitic acid, and 7.19-68.92 µg/mL for linoleic acid [98,99]. This indicates that the fatty acids could also contribute to the larvicidal property of O. americanum leaves.
Several terpenes were found in the hexane extract of O. americanum leaves as well, including elemol and β-elemene (sesquiterpenes), limonene (a monoterpene), and squalene (a triterpene), depending on the number of isoprene units present in the molecules. The two sesquiterpenes have been documented in the essential oils obtained from the leaves of O. americanum [100]. The β-elemene isolated from the essential oil of Syzygium zeylanicum is effective against Ae. albopictus larvae, with an LC 50 value of 11.15 µg/mL after 24 h post-treatment [101]. In contrast, elemol was regarded as inactive against the larvae of Ae. aegypti and Ae. albopictus [102]. Despite being a minor compound with an abundance of <1%, limonene is known to have larvicidal activities against many mosquito species, i.e., Ae. aegypti [103], Ae. albopictus, Cx. pipiens molestus [104], An. stephensi, Cx. quiquefasciatus [105], An. funestus, and An. arabiensis [92]. Like limonene, squalene is only a minor compound in the hexane extract of O. americanum leaves. Nonetheless, it is not surprising to find the presence of squalene, as it is a metabolic intermediate of sterol biosynthesis in plants [106]. Squalene has been shown to have many biological properties, such as antioxidative, antibacterial, antifungal, anticancer, anti-inflammatory, cardioprotective, etc., as reviewed by Lozano-Grande et al. [106] and Lou-Bonafonte et al. [107]. However, further study is required to ascertain whether squalene possesses any mosquito larvicidal activity.

Conclusions
This present study showed that the rhizomes of C. longa, the leaves of O. americanum, and the leaves and stems of P. crispum possessed larvicidal activity against the third-instar larvae of Ae. albopictus. Among the aromatic plant extracts evaluated, the hexane extracts of O. americanum and P. crispum displayed the strongest larvicidal activity with the lowest LC 50 values (<30 µg/mL) at 24 h post-treatment. Between these two hexane extracts, O. americanum was found to be significantly less toxic toward Vero cells than P. crispum. Gas chromatography-mass spectrometry analysis on the hexane extract of O. americanum indicated the presence of different classes of metabolites, including phenylpropanoids, very long-chain alkanes, fatty acids and their derivatives, and terpenes, with the most abundant component being methyl eugenol (55.28%). Some of the major components and minor components in the extract are known to be larvicidal compounds. Based on the findings from this study, further toxicity assessments of the potential active components, such as methyl eugenol, could be extended to aquatic organisms. This study also highlighted that aromatic plants are rich sources of botanical insecticides, which may play an important role in the ongoing efforts to reduce the mosquito population and the transmission of mosquito-borne diseases to humans. Hence, there is a possibility of exploring the potential of O. americanum leaves to be developed as a source of bioinsecticides.