Repellency Potential, Chemical Constituents of Ocimum Plant Essential Oils, and Their Headspace Volatiles against Anopheles gambiae s. s., Malaria Vector

African malaria mosquitoes ( Anopheles gambiae sensu stricto) transmit a malaria parasite ( Plasmodium falciparum ) to humans. Te current control strategies for the vector have mainly focussed on synthetic products, which negatively impact the environment and human health. Given the potential use of environmentally friendly plant-derived volatiles as a control, this work aims to examine and compare the repellency potential of essential oils and headspace volatiles from Ocimum gratissimum, Ocimum tenuiforum, and Ocimum basilicum and their chemical compositions . Te repellency potential and chemical composition of the plants were achieved by using the protected arm-in-cage method and gas chromatography-mass spectrometry (GC-MS) analysis. Among the three Ocimum species, both the essential oils and the headspace volatiles from O . tenuiforum achieved the longest repellency time lengths of 90–120minutes. One hundred and one (101) chemical constituents were identifed in the headspace volatiles of the three Ocimum spp. Nonetheless, ( − )-camphor, ( E )- c -bisabolene, terpinolene, β -chamigrene, cubedol, ( E )-farnesol, germacrene D-4-ol, viridiforol, c -eudesmol, tetracyclo [6.3.2.0 (2,5).0(1,8)] tridecan-9-ol, 4,4-dimethyl, α -eudesmol, iso-longifolol, and endo-borneol were unique only to O. tenuiforum headspace volatiles. Either essential oils or headspace volatiles from O. tenuiforum could ofer longer protection time length to humans against An. gambiae . Tough feld studies are needed to assess the complementarity between the chemical constituents in the headspace volatiles of O. tenuiforum , our observations provide a foundation for developing efective repellents against An. gambiae .


Introduction
Malaria remains one of the most critical public health problems in sub-Saharan Africa (SSA) [1,2]. In 2021, 247 million malaria cases were recorded globally, with more than mosquitoes) vectors are two microparasites, namely, Wuchereria bancrofti and Plasmodium falciparum [6]. Te former is a zooparasitic nematode that causes a flarial disease commonly known as elephantiasis [7], whereas the latter is a parasitic sporozoan that causes malaria in humans widely across SSA [3,[8][9][10]. In Ghana, the Plasmodium parasite accounts for more than 85% of all malaria cases [2]. Millions of people are infected daily by P. falciparum through mosquito bites [11]. Although progress has been made to combat malaria through many interventions such as prompt and accurate diagnosis, single and combination therapy with known antiplasmodial drugs, insecticidetreated nets, and malaria vaccines, there are still signifcant morbidity and mortality primarily due to the inherent difculties associated with the vector control [12].
Unfortunately, none of the control methods have effectively reduced malaria transmission rates among people in SSA [13]. Te majority of the control strategies to reduce malaria have largely been vector-based interventions which include reducing mosquito breeding sites, use of screens against mosquitoes, application of N, N-diethyl-3-methylbenzamide (DEET), a synthetic repellent, genetic control methods through the release of mosquitoes carrying a lethal gene to suppress target populations, application of humanderived volatiles as a trap, and application of indoor residue spray [2,8,12,14]. However, there have been numerous reported limitations associated with the current control strategies [3,[15][16][17]. For example, the population control of the vector through reducing breeding sites presents a great challenge due to mosquitoes' ability to breed wherever stagnant water is available [18]. Although providing screens such as insecticide-treated nets are excellent means of personal and community protection against malaria disease [19,20], there has been a decline in the use of treated nets mainly due to skin irritation and demand for sufcient indoor air circulation [2]. Te DEET developed over 6 decades ago is still the most widely used synthetic mosquito repellent [15,21]. Unfortunately, accessibility and undesirable properties such as an unpleasant odor and skin irritation have limited its use. Also, DEET has been observed to inhibit the function of ion channels and acetylcholinesterase in humans and other mammals [22]. Te active ingredients of most indoor mosquito sprays belong to the pyrethroid, organochlorine, organophosphate, and carbamate classes of pesticides [9]. However, considering the environmental impact, health implications, and insecticide resistance development in mosquitoes to these synthetic sprays [23,24], it suggests the need for study on (i) environmentally friendly vector management approaches and (ii) people's views on the use of these vector control tactics.
In Africa, traditional medicinal plants and/or their derived products continue to be vital against malaria vectors [25]. Tese plants are usually burnt to generate smoke or hung in houses to repel mosquitoes [13]. Te most cited genera of plant species with promising mosquito repellent activities have been Cymbopogon, Eucalyptus, and Ocimum [25,26].
Ocimum gratissimum L., Ocimum tenuiforum L., and Ocimum basilicum L. are aromatic medicinal plants, belonging to the family Lamiaceae, that are found in Africa, Asia, and Mediterranean countries [31]. Tese plants possess antimicrobial, antioxidant, repellent, larvicidal, and insecticidal properties [32,33]. Traditionally in Ghana, these plants are burnt or placed in rooms to serve as mosquito repellents, though the practice is yet to be investigated. Most mosquito repellency studies on these plants have focused on plants' essential oils, usually obtained through hydrodistillation. However, the heat in the hydrodistillation process can decompose some constituents [34]. For a more realistic plant volatile profle, headspace volatile collection is appropriate, especially when it involves ecological application [35]. To the best of our knowledge, however, there has been no study on the headspace volatile composition of O. gratissimum, O. tenuiforum, and O. basilicum isolated by dynamic headspace volatile techniques and their mosquito repellency activity. Given the potential use of these environmentally friendly plant-derived volatiles as Anopheles mosquito repellents, the aim of the current study was in twofolds: (1) compare the mosquito repellency activity of essential oils or headspace volatiles from the three Ocimum plants to commercially available, mosquito repellent used in Ghana; (2) investigate the chemical composition of the headspace volatiles from these Ocimum species. About 100 g of fresh Ocimum plant leaves were hydrodistilled for their essential oil using the Clevenger-type apparatus for 3 hrs. Te extracted oil was dried over anhydrous sodium sulphate to eliminate hydrosols and then stored in a refrigerator at −4°C until used for repellency assay. Te percentage oil yield was calculated using equation (1) percentage oil yield � volume (v) of oil obtained weight (w) of plant used × 100.

(1) Organoleptic Tests for Essential Oils from Ocimum Plants.
Te smells of the essential oils were evaluated by randomly selected 30 people to grade the smell of the oil from 0 to 10, as previously described by Buckle [36]. Oils were graded as follows: 10 corresponding to excellent; 7 to 9 ranked as very good; 5 to 6 as good; 4 as acceptable; and ≤4 as ofensive.
Colour of the essential oil was done by physical examination using the eyes.
(2) Refractive Index of Essential Oils from Ocimum Species. Refractive index of the essential oil was evaluated using an Abber refractometer (A. KRÜSS Optronic GmBH-DR6300-TF, Hamburg USA) at 25°C.  [6,7]. Colonies of An. gambiae were reared in cages (sizes were similar to those used by [27]) under dark laboratory conditions (27°C; photoperiod = 12L: 12D; RH = 70 ± 5%), using the procedures in the report of Das et al. [37]. Five-day-old female An. gambiae adults from the colonies were used for repellency assays.

Dilution of Essential Oils in 70% Ethanol for Repellency
Assays. Seventy percent ethanol was used to dilute essential oil from each Ocimum plant to adjust the ratios between the volume of essential oil and that of 70% ethanol to be 1 : 0, 1 : 1, and 1 : 9. Tus, the corresponding percentages of the amount of an essential oil present in the mixtures (i.e., oil + 70% ethanol) were undiluted at 50% and 10%, respectively. Tese mixtures were separately labelled and used for repellency assays.

Treatments with Essential Oils from Ocimum Plants against Anopheles gambiae (s.s.).
Te oil was tested as undiluted, 50%, and 10% 'diluted in 70% ethanol for the essential oil repellency assays. A 0.1 mL of the undiluted essential oil was applied to protect the frst author's forearms (i.e., about 30 cm from wrist to elbow; plastic hand gloves were used to cover hands). A treated forearm was then exposed to 25 blood-starved 5-day-old female An. gambiae in a cage (30 × 30 × 30 cm 3 ) under laboratory conditions (27°C; photoperiod = 12L: 12D; RH = 70 ± 5%). Te timelength observed for the frst mosquito bite during exposure of the treated forearm to the insects was recorded as the repellency time of the plant essential oil. Similar treatments to the author's forearms were done for 50 and 10% diluted oils in 70% ethanol. For reference, 0.1 mL of commercially available mosquito repellent (i.e., Odomos; Dabur Limited, India) was used as a positive control, whereas 0.1 mL of 70% ethanol only was used as a negative control treatment to the section of the author's forearm, which was exposed to 25 female An. gambiae in a cage. In each case of a treatment, three replicates were made.

Assays with Headspace Volatiles from Ocimum Plants against Anopheles gambiae (s.s.).
Te repellency activity of headspace volatiles from each Ocimum species was assessed according to the method described by Logan et al. [38]. A 0.1 mL of the eluted headspace volatile in 750 μL of diethyl ether was applied to protect the author's forearms; the procedures and conditions applied in the present study were similar to those mentioned above in the case of the undiluted essential oils and the Odomos. However, only the redistilled diethyl ether was used as the negative control. Te time (in minutes) for the frst mosquito bite was used as the repellency time of the plant's headspace volatiles.

Assay Data
Analysis. Te R software (R.v.4.1.1∼Rstudio.v.1.4.1717) developed by the R Core Team [39] was used to analyze the repellency time lengths (RTLs) of the essential oils or the headspace volatiles. Having applied the Shapiro-Wilk test, normality in the data for treatments at 10 and 50% dilution levels was signifcant (P < 0.05) for lack of normal distribution, whereas data for treatments of essential oils or headspace volatiles were normally distributed. Moreover, all transformation techniques applied to data using formulae did not improve outputs. Terefore, we compared the P values of nonparametric analyses (Wilcoxon rank-sum, Kruskal-Wallis rank-sum, and Dunn's tests) to those of parametric tests (two-sample (or paired) t-tests, analysis of variance (ANOVA), and Tukey's Honestly Signifcant Diference (HSD)), respectively, similar to the analyses in the report of Heve et al. [40]. Because all the parametric statistics generated the lowest P values for better conclusions in this study [40], a paired t-test at P values ≤0.05 was used to compare the "mean ± standard error (SE)" values of two variables [41,42]. On the other hand, Tukey's HSD test at P values ≤0.05 was used to compare "mean ± SE" values of RTLs among variables that were more than two [41].  [27]. Similarly, (E)-Ocimene was synthesized via the synthetic route previously reported by Hassemer et al. [43].

GC/GC-MS Analytical Procedures.
Constituents of the essential oils hydrodistilled from the Ocimum species considered in this study have been reported [13,33,[44][45][46][47][48]. As a result, we focused on the identifcation of compounds that are present in the headspace volatiles isolated from the three Ocimum spp. Analysis of the headspace plant volatile was done according to the method previously described by Osei-Owusu et al. [27]. Volatile extracts were analyzed on a GC (Agilent Technologies, 6890N, Stockport, UK), equipped with a fame ionization detector (FID) and an HP-1 capillary column (50 m × 0.32 mm i.d., 0.52 μm flm thickness). Te oven temperature was maintained at 30°C for 1 min and programmed at 5°C/min to 150°C, where it was held for 0.1 min, then at 10°C/min to 230°C and held for 27 min. Te carrier gas was nitrogen. One (1) μL of the sample was injected into the injection port of the equipment manually. GC-MS analysis of eluted volatiles was performed using a Hewlett-Packard 5880A gas chromatograph (HP-5880A) with an HP-1 capillary column (50 m × 0.32 mm id, 0.52 μm flm thickness). Ionization was by electron impact (70 eV, source temperature 250°C). Helium was the carrier gas. Te oven temperature was maintained at 30°C for 5 min and then programmed at 5°C/min to 250°C. Tentative identifcations were made by comparison of mass spectra with the NIST 2005 mass spectral database and Adam's library. Confrmation of peak identity was made by comparing their Kováts index (KI) values and GC peak enhancement with authentic compounds. Te KIs of the compounds were calculated based on the homologue series of n-alkane (C7-C22) in the following equation: where I � Kováts retention index, n � number of carbon atoms in the smaller n-alkane, N � number of carbon atoms in the larger n-alkane, and t r � retention time.  (Table 2). However, a decrease in the dilution level of essential oil against An. gambiae was a signifcant source of variation (F (2,18) � 3.7; P � 0.04508) in RTLs (in rows of Table 2). In the case of essential oil from O. gratissimum or O. tenuiforum, the RTL decreased as the dilution level of the oil was decreased (Table 2). However, in the case of essential oil from O. basilicum against An. gambiae, a decrease in the dilution level of the oil caused an inconsistent trend in RTL values: 0.1 mL of 50% essential oil from O. basilicum signifcantly reduced its RTL value to 10 ± 10 minutes, whereas the 10% essential oil from the same Ocimum species rather caused an increase in the RTL value ( Table 2). Comparing the performance of each essential oil to that of Odomos, no signifcant diference was observed in RTLs between the undiluted oil from O. tenuiforum and Odomos (Table 2). Similarly, the RTL values of 50% essential oil from O. tenuiforum were also not signifcantly diferent from those observed for Odomos (  Table 3).

Discussion
Te control strategy of mosquitoes using conventional synthetic insecticides has been challenging due to resistance, environmental impact, and health implications. Tus, more environmentally friendly vector management strategies are warranted. Using plant extracts provides a promising strategy for controlling the malaria vector [13,49,50]. Extracts from several plant species worldwide have been tested against diferent Anopheles spp. [51]. In feld conditions, essential oils from Pinus spp., lemon grasses (i.e., citronella oil from Cymbopogon spp.), and Dalbergia sissoo (Roxburgh) have shown high repellency potential, thereby ofering a longer efective protection time between 6 and 11 hours (hrs) against Anopheles culicifacies (Giles), Note: values were hedonically generated and then compared to ranges for their meanings, according to Buckle [36]. SD: standard deviation. RI: refractive index.    Journal of Chemistry

Journal of Chemistry
Anopheles subpictus, and Anopheles annularis [51][52][53]. Laboratory trials have also revealed that essential oils from Ligusticum sinense (Umbell.) showed a remarkable repellency time over 11.5 hrs against An. minimus and Ae. aegypti (L.) [54], whereas essential oils from stephensi and An. sundaicus [51]. Nonetheless, essential oils from the majority of plants tested under laboratory conditions could only achieve a repellency time <5 hrs against An. albimanus, An. dirus, and An. stephensi [51]. In the case of laboratory trials involving essential oils from the majority of plant species against An. arabiensis and An. gambiae, the observed repellency time seems to be ≤1.5 hrs [51].
To the best of our knowledge, there are no previous reports on the headspace volatiles of O. tenuiforum, O. gratissimum, and O. basilicum against mosquitoes. In the current study, we explored the mosquito repellency properties and chemical composition of the headspace volatile isolated from the three Ocimum species. We observed that the undiluted essential oils from the three Ocimum plants tested against An. gambiae achieved a repellency time of 90 ± 17 minutes. Moreover, our laboratory trials involving headspace volatiles from these Ocimum plants proved that the repellency time-lengths observed for the headspace volatiles were within the range of those observed for the essential oils. Tus, both the undiluted essential oils and the headspace volatiles achieved similar repellency time lengths, which were not signifcantly diferent from those of a commercially available repellent. Our observations in this study suggest that repellency time-lengths achieved by either essential oils or headspace volatiles from Ocimum plants against An. gambiae were similar to those observed for essential oils from Ocimum spp. tested against Ae. aegypti [48] or the list of several plant species tested on culicids in the report of Asadollahi et al. [51]. Although complementarity between oils and headspace volatiles may be more efective, essential oils or headspace volatiles from the O. tenuiforum may be an alternative to protect humans against An. gambiae. Tis is because oils or headspace volatiles from O. tenuiforum consistently outperformed the remaining Ocimum plants used in this study, possibly because of some unique constituents.

Conclusions
When the performancesof essential oils from O. gratissimum, O. basilicum, and O. tenuiforum against An. gambiae in assays, the latter achieved the longest repellency time comparable to the commercially sourced repellency. In similar assays involving headspace volatiles from the three Ocimum species against An. gambiae, O. tenuiforum outperformed and again achieved the longest repellency time again. Using GC/GC-MS analysis, 101 chemical constituents were identifed in the headspace volatiles from the three Ocimum species. However, (−)-camphor, (E)-c-bisabolene, and endo-borneol were present in very low quantities in the headspace volatiles from O. tenuiforum only, suggesting that longer repellency time lengths achieved by O. tenuiforum could hypothetically be linked to these three unique chemical constituents. Tus, further studies are required to investigate the usefulness of the complementary roles of chemical compounds identifed in the more repelling O. tenuiforum.

Data Availability
Te data used to support the fndings of this study are made available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.