Introduction
Plant secondary metabolites or bioactive compounds are products secreted by plants for defense against plant natural enemies. The products serve as repellents, antifeedants, growth and mating inhibitors, anti-moulting, and insecticides [1, 2] against these enemies. The products, however, are environmentally friendly [3] and serve as food and preservatives [4]. The bioactive compounds are diverse with different modes of action. This property gives them great potential for novel biological products [5] and makes it challenging for diseases and pests to develop resistance against them [6]. The tragedy is that most products remain unexplored and serve merely as repository sources [7]. 
Urtica massaica Mildbr (hereafter U. massaica) is a perennial herb [8] belonging to the Urticaceae family [9]and, although considered a weed, is among the most used wild plant species in the world [10, 11]. It is rich in proteins [12], vitamins [13], and polyphenols [14], and it has pharmaceutical [15], antimicrobial [16], and fungicidal [17] potential. It has, therefore, been used as vegetables with high potential to manage food insecurity [18-19] and as an herb to treat various microorganism-caused infections [20-23], stomach ulcers, hypertension, nerve disorders, diabetes, and rheumatism [4, 24].
Though the toxic effect of the extracts has been reported on immature Anopheles gambiae (hereafter A. gambiae) [25], the chemical profile and toxicity have not been studied. We, therefore, report herein on the characterization and potency of crude methanol and hexane extracts of U. massaica against laboratory-cultured A. gambiae larvae under controlled conditions.

Materials and Methods
Study site, sourcing for experimental mosquitoes and study design
This research was a laboratory-based experimental bioassay using crude U. massaica extracts on A. gambiae larvae. We extracted and characterized chemical constituents from parts of U. massaica using methanol and hexane in the University of Eldoret’s chemistry laboratories. Third larval instars (L3s) of A. gambiae mosquitoes were obtained from a laboratory stock at the Centre for Global Health Research/Kenya Medical Research Institute (CGHR/KEMRI), Kisian, Kisumu. Since the effect of treatment meted on the larvae was measured only after the bioassay, an informal ‘after-only with control’ experimental design [26] was used to determine the larvicidal effect of crude methanol and hexane extracts of U. massaica on the larvae.

Plant materials
Fresh leaves, stems, and roots of U. massaica (stinging nettle) were sourced from 350 16’ 46” E, 00 31’ 41” N in Eldoret. The plant was identified, and a voucher specimen number JOY2021/001 was issued and later deposited at the School of Biological Sciences, University of Nairobi, herbarium.

Methanol and hexane extracts of U. massaica
Two hundred grams of ground leaves of U. massaica were soaked in 400 mL of absolute methanol for 1 hour. The suspension was then filtered using Whitman’s No. 1 filter paper, and the filtrates were freeze-dried using the Edwards Modulyo freeze-drying machine. The result is a paste taken as stock material [27]. This procedure was repeated for ground stem and roots and hexane solvent. 

Gas chromatography-mass spectrometry (GC-MS) analysis
Sample preparation
This procedure was done as described elsewhere [28]. Briefly, 1 mg of each crude extract was weighed (in triplicates) and dissolved in 1 mL dichloromethane. The samples were vortexed for 10 s, ultra-sonicated for 1 hour, and centrifuged at 14000 rpm. The supernatant was dried using anhydrous Na2SO4 and centrifuged at 14000 rpm before GC-MS analysis.

Instrument conditions
This activity was conducted as described elsewhere [29]. Briefly, the samples were analyzed using a 7890A gas chromatograph connected to a 5975C mass selective detector. A temperature of 270 °C was set as the inlet and 280 °C as the transfer line. The oven temperature was programmed at between 35 °C and 285 °C, with the initial temperature being maintained for 5 minutes, adjusted to 10 °C/minute, and progressively brought up to and held at 280 °C for half an hour. A low bleed capillary column (HP-5 MS, 30 m × 0.25 mm i.d., 0.25 µm) and helium were employed as the carrier gas with a 1.25 mL/min flow rate. The detector’s ion source and quadrupole temperatures were maintained at 230 °C and 180 °C, respectively. Electron impact of mass spectra was obtained at 70 eV. About 1.0 µL and the analyte injected in split/splitless mode and fragmented ions analyzed, in full scan mode, over 40 to 550 m/z range. The filament delay time for the analytes was set at 3.3 min. The compounds were identified by comparing their fragmentation patterns with reference spectra from Library–MS databases. This process also included the National Institute of Standards and Technology (NIST) 11, 08, and 05, Adams and Chemical mass spectral databases.

Liquid chromatography with tandem mass spectrometry analysis
The analysis was conducted as described elsewhere [30]. Three replicates of 1 mg of each crude methanolic extract were weighed and constituted in 1 mL methanol. The samples were then vortexed for 10 s ultrasonicated for 1 h before centrifugation at 14000 rpm. Thereafter, the supernatant was filtered and analyzed qualitative tandem liquid chromatography quadrupole time of flight mass spectrometry (MeOH) under the following conditions: Ultra-performance liquid chromatography (UPLC) (Waters ACQUITY I-Class system); UPLC column (Waters ACQUITY UPLC BEH C18 column, 2.1×50 mm, 1.7-μm particle size Waters Corporation, Dublin, Ireland); Column temperature of 25 °C; mobile phase of water (A) and methanol (B), each with 0.01% formic acid; flow rate of 0.3 mL/min, gradient from 95% A to 100% B and back to starting solvent proportion; with the run time being 25 min. A positive Q-tof ion mode with a nitrogen desolvation flow rate of 500 L/h and an accuracy of <5 ppm was used. The quantitative analysis of the secondary metabolites present was based on a standard curve of apigenin (y=10288x–11117; R2=0.999). 

Larvicidal bioassay
Preparation of serial dilutions 
The dilutions were prepared according to Vloemans et al. [31]. Briefly, 1 g of crude methanol stock’s extracts of U. massaica leaves were weighed and dissolved in 1% of dimethyl sulfoxide (100 mL) and then serially diluted. Also, 80 mL of this solution was then topped up with 20 mL of distilled water to make 100 mL. The mixture was then serially diluted to 40, 20, 10, 5, and 2.5 mL/100 cm3 (e/w) of distilled water. 

Larviciding
One hundred newly transformed L3s were placed in 3 plastic containers, each measuring 6×5.7×3.5 cm, using a plastic pipette. Two sets of the containers held 33, while the third had 34 larvae each. All the containers contained similar doses of a particular treatment. Each container held approximately 33 mL of a particular dose. Doses were either 80, 40, 20, 10, 5, or 2.5 mL/100 mL (e/w). The larvae were left exposed overnight, after which the experiment was stopped, and the number of dead or live individuals was noted and recorded. All larvae (live, moribund, and dead) were then put in a pail of hot water and dispensed in a septic tank. The experiment was replicated four times. Larvicidal activities were tested following the World Health Organization (WHO) procedure [32] and standards [33] for insecticidal effectiveness. Larval mortality was calculated using the Equation 1: 


Furthermore, observed larval mortality of between 5% and 20% was corrected using Abbot’s formula [34] (Equation 2):


The extract dose was taken as the independent variable, while observed mortalities were the dependent variable. Dimethyl sulfoxide and distilled water were used as negative controls. The temperature and humidity regime at the laboratory was maintained at 28 – 30 °C and 70% - 80%, respectively, and a photoperiod at 12 h light (06.30 – 18.30 hours) alternated with 12 h darkness (18:30 – 06:30 hours).

Statistical analysis 
Data on characterization and bioassay on the effect of the crude extracts of U. massaica on larvae of A. gambiae were entered in Excel spreadsheets and appropriately organized for processing. Descriptive statistics were used to determine the quantity of bioactive compounds and the effect of the solvent of extraction, dose, and part of the plant used on exposed larvae. A one-way analysis of variance was used to determine the significance of the impact of extract as a larvicide. All statistical analysis was performed using IBM SPSS statistics software for Windows, version 22 (IBM Corp, Armonk, NY, 2013).

Results
Five major chemical constituents were extracted from U. massaica parts. Those that could not be classified were placed under “other groups.” The order of abundance was phenolic > amino acids and nitrogen derivatives > steroids and terpenoids > flavonoids, nitrogen derivatives, and other groups (Figure 1).  Roots produced the highest quantity of constituents [12], followed by leaves [16] and then stems [12]. All the observed abundance, however, did not significantly differ (P<0.05) except for the flavonoids (Table 1). 


Individual chemical constituents, however, differed in abundance per chemical group and plant part. Luteolin 6-C-glucoside (flavonoids), neoergosterol and β-sitosterol (steroids and terpenoids), isoleucine (amino acid and nitrogen derivatives), 3,4-diferuloylquinic acid (phenolic compounds) and p-isopropyl aniline (nitrogen derivatives) (Figure 2) were abundant per group and catechin (10.00) in roots, β-sitosterol (7.84) in leaf and 3,4-diferuloylquinic acid (11.43) in stem (Figure 3).  Interestingly, extracts from stems were not only more potent than those from leaves or roots, but their effects equaled the WHO threshold of >80% mortality for insecticidal effectiveness for doses of 10 mL/100 cm3 (e/w) and above (Figure 4).  Observed mortalities, however, were not significantly different (P>0.05) irrespective of dose or control (Table 2).



Discussion
Different parts of plants (leaves, fruits, seeds, roots, and bark) contain polyphenols or secondary metabolites (flavonols, anthocyanins, and phenolic acids) that are responsible for mosquitocidal properties [35]. Indeed, much of the research on malaria intervention is on the search for novel chemical agents that have equivalent potency as synthetic insecticides but whose activity is specific and friendly to humans and the environment [35, 36]. The study reported herein was one of such, and herein we demonstrate that U. massaica contains several chemical agents that are toxic to A. gambiae larvae. The findings are similar to Justicia adhatoda L. [37] and Acacia nilotica [38] against different mosquito species.
In this study, the roots contained more bioactive compounds than the leaves or stem, though extracts from the stem were more potent than those of the leaves or roots. These findings were similar to those of Baz et al. [39], Anupam et al. [40], and Yugi and Kiplimo [41], who reported on the differential distribution and concentration of bioactive compounds in different plant parts. These findings come when insecticide resistance and environmental challenges [42] posed by synthetic insecticides are a thorn in the flesh against the race to reduce, and if not pacified, malaria infection must be welcomed. Indeed, it adds to the cumulated stock and use of natural products that are not only rich in bioactive compounds but are target-specific and safe for the environment [3, 36]. As such, the findings of this study put extracts of U. massaica among plants that can provide alternative sources of green insecticides [43] for mosquito-borne disease control [33]. 
The study showed that U. massaica contains secondary metabolites identified as tannins, terpenes, saponins, flavonoids, alkaloids, and phenols. A total of 47 bioactive compounds were extracted in different quantities (roots, 19; leaves, 16; and stem, 12) of the U. massaica plant. This amount was moderate but still higher than recently reported elsewhere [14]. This finding is promising as we believe that with refining, we are likely to realize more compounds. Though the exact nature and the specific bioactive compound responsible for the observed activity were not expressly determined, it is evident from an earlier demonstration that phenolic compounds, flavonoids, and tannins possess insecticidal properties [44] and may have been responsible for the observed toxicity. These findings are comparable with earlier works by Hoesain et al. [45] of plant metabolite potential against Spodoptera litura, Hillary et al. [46] of efficacy of plant products, Bassey et al. [47] of Allium sativum and Murraya koenigii, and Folawewo et al. [48], Oboho et al. [49] of Hippocratea africana with demonstrable mosquitocidal [50] activities. The bioactive compounds’ toxicity was believed to be due to their impairing mitochondrial function in the exposed insect vectors [51]. 
The present study demonstrates the toxicity of extracts of U. massaica against A. gambiae larvae with activity being dependent on dose, solvent, and part of plant extracted. Though the time of exposure was not tested, the observations reported here agree with those reported by Ubulom et al. [51], Opara et al. [52], and Ghosh et al. [40] on the count of increasing concentration of bioactive compounds in different parts of the plant. 

Conclusion
U. massaica bioactive compounds are diverse and have toxicants against A. gambiae larvae. They qualify for exploitation as green insecticides to target and protect against the anthropophilic, endophilic, and endophagic mosquitoes in areas where they are endemic and resist synthetic insecticides.

Study limitations 
The study was limited to L3 of laboratory-reared A. gambiae mosquitoes.


Ethical Considerations
Compliance with ethical guidelines
This research was a laboratory-based experimental bioassay. It involved the use of laboratory-sourced and membrane-fed mosquitoes only. There was, therefore, no need for ethical consent. 

Funding
The University of Kabianga, Kericho, Kenya funded the project.

Authors contributions
Conceptualization, data analysis, and writing the original draft: Jared Yugi Owiti, supervision, methodology, investigation, data collection, review and editing: All authors.

Conflict of interest
The authors declared no conflict of interest. 

Acknowledgements
The authors thank Richard Amito, Harnel Versey, and Gayle Aurelia for processing and culturing the experimental mosquitoes. Also, the authors appreciate the Centre for Global Health Research (CGHR), Kisumu, for providing laboratory space, experimental mosquitoes, and equipment, respectively.

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