Bio-active principles in Commiphora africana resin dichloromethane extract and their insecticidal activity against bedbugs

doi:10.21203/rs.3.rs-3228063/v1

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Bio-active principles in Commiphora africana resin dichloromethane extract and their insecticidal activity against bedbugs

https://doi.org/10.21203/rs.3.rs-3228063/v1

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Bedbugs (Cimex lecturalius Linnaeus) are ecto-parasite pests that wholly feed on human and domestic animals’ blood causing anemia to the host on excessive feeding. Bedbug control has proved futile due to various challenges including; development of insecticide resistance, high associated cost and environmental pollution. Natural herbal-based phytochemicals remain unexploited and we focused on Comiphora africana (A. Rich.) Engl. resin traditsionally used bedbug control. We previously showed that dichloromethane extract of C. africana resin is highly repellent and toxic against bedbugs. In this study, we isolated compounds from the dichloromethane extract using column chromatographic techniques. The isolated compounds were evaluated for repellency and toxicity against bedbugs; and characterized using conventional spectroscopic techniques. Five compounds (taraxasterol, pseudo-taraxasterol, beta-sitosterol, fungisterol and guggusterol) were isolated and characterized for the first time in this plant. Fungisterol had the highest repellency (75%) against bedbugs which was not significantly different (P > .05) to the positive control (neocidol) (74%) after > one-hour exposure. Fungisterol also elicited highest toxicity against bedbugs with LC₅₀ of 25.73 mg/L after 24 h exposure. Blending fungisterol with other identified active terpenes did not synergize the overall repellent/toxic responses. This study identifies active compounds in C. africana resin and therefore lays a solid background in bedbug control using isolated compounds.

Commiphora africana

bedbugs

resin

toxicity

repellency

Bedbugs (Cimex lectularius Linnaeus) are ecto-parasite pests of significant public health importance that pose a great threat to humans and domesticated animals. They cause anemia upon heavy feeding (blood-sucking) on human or animal host (Goddard and Deshazo 2009). They also leave physical marks on human skin impacting them negatively on their social life (Reinhardt et al. 2009). Several control measures of bedbugs including the use of insecticides have become unsuccessful due to development of resistance to commonly used insecticides that are additionally harmful to the environment (Dang et al. 2017). Moreover, these control methods require a heavy capital investment (Rukke et al. 2022).

There is a growing interest in exploitation of natural products as a substitute tool in control and management of bedbugs and other related arthropods. This is due to the fact that, they are believed to be environmentally benign (Gondhalekar et al. 2021). Several parts and products of selected plant species are used in control and management of various arthropods. For instance, Commiphora africana (A. Rich.) Engl. resin in its crude form is locally used in control of bedbugs, lice, ticks and fleas in various parts of Kenyan coastal region. In our previous study on evaluation of aqueous extract, polar organic solvent extracts, and non-polar organic solvent extracts of C. africana resin against bedbugs, we found that dichloromethane extract was the most potent repellent and toxic extract against bedbugs (Wairagu et al. 2022). In this follow-up study, it is necessary to isolate pure compounds from the dichloromethane extract that are of biological importance against bedbugs. We hypothesized that; i) the dichloromethane extract of C. africana resin contain compounds that are isolable, purifiable and stable ii) the isolated compounds have repellency and toxicity responses against bedbugs and iii) blending of active isolated compounds will lead to a synergistic repellency and toxicity effects against bedbugs.

We extracted crude C. africana resin using dichloromethane solvent, isolated and purified compounds from the extract using Column Chromatography techniques, and evaluated their repellency and toxicity alongside with positive control (neocidol) and selected blends against bedbugs under laboratory conditions. This is the first study to be carried out to identify bioactive principles of C. africana resin. This study will lay a strong background in the development of an effective and safe bedbug control measures by formulating an eco-friendly insecticide as alternative to commonly used commercial insecticides.

General Experimental Procedures

Solvents, silica gel 60G (70–230 mesh) and sephadex LH-20 were bought from Sigma-Aldrich (Tauschem, Germany) with percentage purity of > 98%. Instruments used in this study included FTIR, GC-MS and NMR.

Sample collection

C. africana resin was harvested from mature trees in Taita Taveta County after identification and confirmation by a botanist from the National Museums of Kenya. The resin of the tree was harvested, packed and transported to KEFRI laboratory as previously reported by Wairagu et al. (2022). The samples were dried in an oven (Sanyo, model MOV-212f) at 50 C for five days. Aerial parts of the authenticated C. africana tree were pressed and archived at East African Herbarium in the National Museums of Kenya where it was assigned a voucher reference (NWWairagu 001).

Rearing of bedbugs

Adult bedbugs of mixed sexes were collected, reared and multiplied according to a protocol of Araujo et al. (2009) as briefly described by Wachira et al. (2020). This was carried out under laboratory conditions of 28 ± 2℃ and 78 ± 3% RH.

Solvent extraction of C. africana resins

One kilogram (1 kg) of dried C. africana resin was extracted with 4 L dichloromethane for 72 h in a 10 L flask. The content was filtered and rinsed through a muslin cloth of pore size 0.7 mm, followed by a Whatman filter paper No 1. The solvent from the filtrate was removed in-vacuo using a rotary evaporator.

Column separation

A hundred grams (100 g) of powdered crude extract was subjected to separation with a column (100 cm long by 6 cm internal diameter) using Kieselgel silica gel 60G (70–230 mesh). Silica gel (450 g) was first packed in the separation column as we previously described by Wachira et al. (2020) before introducing the extract on top of silica gel level. The crude extract was first eluted with hexane, followed by a slow gradient of hexane-ethyl acetate, and finally with ethyl acetate. Fractions with similar Thin Layer Chromatography profiles were pooled together, concentrated in vacuo using a rotary evaporator at 45°C, and further separated and purified in a smaller column (2 cm diameter and 30 cm long) to afford pure compounds.

Identification of compounds

Compounds with a single spot under TLC were also run with GC to confirm their purity. Those with a single sharp peak were considered pure and further analysed using FTIR (Bruker, model Alpha - laser class 1), GC-MS (Shimadzu QP 2010-SE, Japan) and NMR spectrometer (Brucker-600, Germany). For FTIR analysis, a mixture of sample and KBr (1:100) was finely ground in a mortar, pelleted and mounted onto the FTIR sample cell for analysis. Data was generated and recorded in absorbance mode. In GC-MS analysis, ultrapure helium was used as the carrier gas at 1 mL/min in a column (A BPX5: 30 m long; 0.25 mm internal diameter; and 0.25 µm film thickness). The GC program was set as: 50 ˚C (1 min); 5 ˚C /min to 250 ˚C (9 min) with a total run-time of 50 min. 1H-NMR and 13C-NMR analysis absorption peaks were recorded on an NMR spectrometer using deuterated chloroform (CDC1₃) as the solvent and Trimethylsilane (TMS) as an internal standard.

Bioassay tests

Repellency of bedbugs on treatment with isolated compounds

The isolated compounds from the most active dichloromethane extract, neocidol (positive control), selected blends (active pure compounds mixed in equal proportions as previously described by Wachira et al. (2020) and blank (solvent) were evaluated for repellency against bedbugs using the filter paper method of Araujo et al. (2009) as we briefly described in Wachira et al. (2020). In each assay, ten bedbugs were introduced in a line cutting through the middle of a Whatman filter paper. One half was treated with test compounds/blends dissolved in acetone (solvent) while the other with the solvent alone (control). These assays were carried out in triplicates with different bedbugs in each test. The distribution of bedbugs in the two halves of the filter paper was separately observed and recorded after ½, 1, 2, 6 and 24 h. These assays were carried out at 25 ± 2°C and 75 ± 2% RH. Percentage repellency was calculated as (C-T)/(C + T)*100 (where C and T represent the number of bedbugs in the untreated (control) and treated halves of the filter paper, respectively.

Mortality of bedbug on exposure to isolated compounds

Bedbug mortality assays were carried out using the method of Ojianwuna et al. (2016) where circular filter papers (Whatman No. 2) with a diameter of 90 mm were treated by spraying 2 mL of pure compounds at varying concentrations (.0, .25, .5, .75, 1, 1.25 and 1.4% v/v in acetone). The filter papers were air dried for 30 minutes at 25^oC and thereafter placed into a clean petri dish. Ten bedbugs were introduced separately into the dishes with the compounds, blends, solvent and neocidol (positive control) and mortality rates were recorded after 24, 48 and 72 h exposure. Assays were conducted in triplicates at 25 ± 2°C and 75 ± 2% RH. The number of dead bedbugs was counted and mortality rates calculated as a ratio of D/T (where; D and T represent the number of dead bedbugs and the total number of bedbugs introduced, respectively), expressed as a percentage.

Data analysis

NMR spectra of isolated compounds were interpreted using MestreNova (spectral data analyzing software); GC-MS and FTIR used fragmentation patterns and absorption bands respectively from a library. Repellency and mortality rate data were rank-transformed and subjected to one-way Analysis of Variance (ANOVA). Means were separated using Student Newman-Keuls’ post-hoc analysis test. Bedbug mortality data was subjected to dose response analysis to determine LC₅₀ of the compounds/blends at a 95% confidence level. Mean values with P < .05 were considered statistically significant. SPSS version 20 (Chicago, IL, USA) software was used in analysis.

Purification and isolation of compounds

Column separation using silica gel (70–230 mesh) and sephadex LH-20, successfully yielded five compounds that were purified and characterized using FTIR, GC, HR-MS, 1H-NMR and 13C-NMR spectroscopic techniques. Physical and spectroscopic characteristics identified the compounds as taraxasterol (1), pseudo-taraxasterol (2), beta(β)-sitosterol (3), fungisterol (4) and guggulsterol (5). These compounds were isolated from C. africana resin for the first time. They eluted with 90 − 70% hexane in ethyl acetate. The yields afforded from the isolated pure compounds were 50, 35, 45, 28 and 15 mg for taraxasterol, pseudo-taraxasterol, β-sitosterol, fungisterol and guggulsterol, respectively.

Characterization of isolated compounds

Pseudo-taraxasterol isolated as white crystals with a melting point of 216–218°C was soluble in organic solvents. The IR spectrum (Fig. 2A-supplementary) showed characteristic absorption peaks at 3608.8–3329.3cm^− 1 (O-H stretch), 2939.3 cm^− 1 (C-H stretch of CH₃), 1589.6cm^− 1 (cyclic C = C stretch), 1422.6cm^− 1 (cyclic methylene groups (CH₂)_n vibrations), and 1067.33 cm^− 1 (C–OH stretch vibrations of secondary alcohols). These absorption peaks are characteristic of triterpenes isolated from Anthemis mirheydari Iranshahr as described by Jassbi et al. (2016). 1H-NMR (500 MHz, CDCl₃) spectrum (Fig. 2B-supplementary) showed unique peaks at δ 5.30 (dd, J = 9.93 and 9.2 Hz) and δ 3.51 (dd, J = 10.1 and 3.5 Hz) attributed to alkenyl and to carbinolic hydrogens, respectively. The spectrum confirmed presence of 8 methyl groups, of which seven were singlets [δ 1.01, 1.01, 0.90, 0.89, 1.56, 0.88, 0.90 and 0.93) and one doublet at δ 1.01 (J = 6.93 Hz). The presence of an axial hydroxymethine (δ 3.51, dd, J = 10.06 and 3.49) and an exomethylene proton δ 5.30, dd (J = 9.93 and 9.20) were also observed. These chemical shifts agreed with those of pseudo-taraxasterol isolated from Anthemis mirheydari as described by Jassbi et al. (2016). 13C-NMR (500 MHz, CDCl₃) spectrum (Fig. 2C-supplementary) gave 30 carbon signals with six quaternary δ 38.7, 41.6, 42.4, 34.5, 37.0 and 140.6 (alkenyl carbon), seven (-CH) methines (one oxygen bearing carbon at δ 78.0), 9 methylenes (- CH₂), and 8 methyl (δ 23.0, 23.0, 16.8, 16.8, 16.2, 17.7, 22.0 and 15.9) groups. These chemical shifts agree with those of pseudo-taraxasterol as reported by Abreu et al. (2013). TIC of GC showed a single peak at RT of 37.10 min. The HR-MS (Fig. 2D-supplementary) gave an [EI (+)]: m/z ratio for this compound as 426.386 while the m/z ratio calculated for molecular ion, C₃₀H₅₀O [M⁺] was 426. The spectroscopic data from FTIR, GC, HR-MS, 1H-NMR and 13C-NMR confirmed the compound to be pseudo-taraxasterol(2).

Fungisterol isolated as a white powder with a melting point of 132–133.8°C was soluble in dichloromethane and ethyl acetate. The IR spectrum (Fig. 4A-supplementary) indicated a broad absorption peak 3496 − 3312 cm^− 1 (O-H stretch), 2948.61 cm^− 1 (C-H stretch), 1596.77 cm ^− 1 (C-C stretch vibrations), 1406.25 (O-H bending vibrations) and 1115.10 cm^− 1 (C-O vibrations) as previously reported by Sen et al. (2012). 1H-NMR (500 MHz, CDCl₃) indicated the presence of six methylene groups at δ 0.52, 0.77, 0.78, 0.79, 0.83 and 0.90 (Fig. 4B-supplementary). The two protons appearing at δ 3.61 and 5.15 for carbon number 2 and 7 represented protons of a hydroxyl group and a double bond proton respectively. These chemical shifts are characteristic to a modified triterpenoid as earlier reported by Nazifi et al. (2008). 13C-NMR (500 MHz, CDCl₃) spectrum (Fig. 4C-supplementary) showed 28 carbon peaks showing presence of a double bond at δ 117.4 and 139.6 suggesting that the possible structure of the compound is fungisterol. This compound was previously identified by Nazifi et al. (2008) from the bulbs of Ornithogalum cuspidatum Bertol.through GC-MS. TIC of GC showed a single peak at RT of 26.8 min. HR-MS spectrum (Fig. 4D-supplementary) gave an [EI (+)]: m/z ratio for this compound as 400.371 while the m/z ratio calculated for molecular ion, C₂₈H₄₈O [M⁺] was 400.68 (Nazifi et al., 2008). The spectroscopic data from FTIR, GC, HR-MS, 1H-NMR and 13C-NMR confirmed the compound to be fungisterol (4).

Guggulsterol isolated as a white powder with a melting point of 165.2–168.5°C was soluble in dichloromethane and ethyl acetate. The IR spectrum (Fig. 5A-supplementary) showed a major broad absorption peak between 3691.7 and 3206.4cm^− 1 (OH stretching vibrations), 2923.1 cm^− 1 (C-H stretch for CH₃), 1595.7 cm^− 1 (C = C stretch), 1426.8 cm^− 1 (-CH₂ vibrations), and 1076.7 cm^− 1 (C-OH vibrations of secondary alcohols) (Sultana and Jahan, 2005). These absorption bands were characteristic of triterpenes similar to those derived from the gum-resin of Commiphora mukul (Hook. ex Stocks) Engl (Sultana and Jahan, 2005). 1H-NMR (500 MHz, CDCl₃) indicated two double bond protons at δ 4.30 and 5.3ppm, multiple hydroxyl protons at δ 3.57, 3.60, 3.69 and 3.74 ppm, methyl protons at δ 0.89, 1.31, 1.45, 1.52, 2.02 and 2.20 ppm (Fig. 5B-supplementary). These chemical shifts are characteristic of guggulsterol isolated from the gum-resin of Commiphora mukul (Sultana and Jahan, 2005). 13C-NMR (500 MHz, CDCl₃) spectrum indicated 27 carbon peaks with four olefinic carbon atoms whose chemical shifts were δ 129.9, 171.6, 172.3 and 174.4 ppm (Fig. 5C-supplementary). These chemical shifts agreed with those of guggulsterol isolated from the gum-resin of Commiphora mukul (Sultana and Jahan, 2005). TIC of GC showed a single peak at RT 27.9 min that corresponded to guggulsterol (Sultana and Jahan, 2005). HR-MS spectrum (Fig. 5D-supplementary) gave an [EI⁺)]: m/z ratio for this compound as 418.34 while the m/z ratio calculated for molecular ion, C₂₇H₄₆O₃ [M⁺] was 418.66 (Sultana and Jahan, 2005). The fragmentation pattern was similar that of guggulsterol isolated from Commiphora mukul by Sultana et al. (2005). The spectroscopic data from FTIR, GC, HR-MS, 1H-NMR and 13C-NMR confirmed the compound to be guggulsterol (5).

Mean repellency of isolated pure compounds against bedbugs

The repellency of bedbugs on treatment with isolated compounds were evaluated separately at half-, one-, six- and twelve- hours exposure times and results summarized in Table 1. Generally, the mean percentage repellencies of taraxasterol, pseudo-taraxasterol, β-sitosterol, fungisterol and guggulsterol were significantly (P < .05) higher than that of acetone solvent. The mean repellencies of bedbugs on treatment with the test compounds significantly (P < .05) increased with increase in time of exposure (Table 1). For instance, fungisterol had mean repellencies of 71% and 89.1% after half- and twelve- hour exposure times, respectively. Fungisterol showed significantly higher (P < .05) mean repellency against bedbugs than all the other tested compounds (Table 1). The repellencies of bedbugs on treatment with the compounds increased with an increase in time of exposure due to the saturation of the bedbug odor receptors with the test compounds (Hansen et al. 2014). In all the exposure times, the higher mean repellency of fungisterol was not significantly (P > .05) different from that of neocidol (Table 1). For instance, after 1 h exposure of bedbugs to fungisterol and neocidol, their mean repellencies were 75% and 74%, respectively.

Table 1

Mean percentage repellency of bedbugs on treatment with isolated compounds at 1.25% w/v in acetone
	Repellency (%) Mean ± SD
	Time of exposure (hours)
Compound ID	½	1	6	12
Taraxasterol	50.0 ± 1.1^Ab	65.0 ± 1.1^Bc	70.0 ± 0.5^Cb	77.0 ± 0.7^Dbc
Pseudo-taraxasterol	55.0 ± 1.1^Ac	61.0 ± 0.8^Bb	69.0 ± 0.4^Cb	75.0 ± 0.9^Db
β-Sitosterol	65.0 ± 0.4^Ad	70.0 ± 0.8^Bd	73.0 ± 0.9^Cc	76.0 ± 1.0^Db
Fungisterol	71.0 ± 0.8^Ae	75.0 ± 1.0^Be	84.4 ± 0.8^Cd	89.1 ± 0.2^Dd
Guggusterol	65.0 ± 1.0^Ad	70.0 ± 0.3^Bd	75.0 ± 1.1^Cc	78.0 ± 0.6^Dc
Neocidol	73.0 ± 0.5^Ae	74.0 ± 1.0^Ae	85.0 ± 0.1^Bd	91.3 ± 0.5^Cd
^£Acetone	0.0 ± 0.0^Aa	0.0 ± 0.0^Aa	0.0 ± 0.0^Aa	0.0 ± 0.0^Aa
^£ solvent; Means followed by different small and capital letters in a column and row respectively indicate significant difference (P < .05)

Taraxasterol, pseudo-taraxasterol, β-sitosterol, fungisterol and guggulsterol indicated that they are potent repellents against bedbugs. Some Commiphora species such as Commiphora holtziana Engl. have previously been identified to have larvicidal properties, lowering oviposition and repellent activity against arthropods species such as mosquitoes (Deepa et al. 2015). Commiphora swynnertonii Burtt. extract has been shown to possess anti-ectoparasitic and repellency activities against lice, mosquito, ticks, fleas, trypanosome and mites (Edwin et al., 2017; Nagagi et al. 2016). Its stem bark exudate extract has high acaricidal activity against the brown ear tick (Rhipicephalus appendiculatus Neumann) and can be used in tick control (Edwin et al. 2017). The biological effects are due to the presence of triterpenes in greater amount which are responsible for repellency and toxicity activities (Birkett et al. 2008).

Mean mortality of isolated pure compounds against bedbugs

The mortality of bedbugs on exposure to the pure isolated compounds was evaluated at varying concentrations (Gaire et al. 2019) and their LC₅₀ values summarized in Table 2. Generally, all the tested compounds showed significantly (P < .05) lower mortality than neocidol indicated by their higher LC₅₀ values than that of neocidol (Table 2). Higher LC₅₀ values indicate lower mortality, the vice versa is true. Generally, the LC₅₀ of the tested compounds significantly (P < .05) decreased with increase in exposure time. For instance, the mean LC₅₀ of taraxasterol when exposed to bedbugs for 24 and 72 hours was 38.72 and 22.48 mg/L, respectively. Fungisterol had significantly (P < .05) lower LC₅₀ values (indicating higher mortality) than all the other tested compounds (Table 2). The LC₅₀ values of fungisterol were not significantly different (P > .05) to those of neocidol at various exposure times.

Table 2

**Mean LC**₅₀**of bedbugs on exposure to isolated pure compounds ofC. africanaresin CH**₂Cl₂**extract**
	Mean LC₅₀ ± SE (mg/L)
Test compound	Time of exposure (h)
	24	48	72
Taraxasterol	38.72 ± 0.45^Cc	30.02 ± 0.38^Bc	22.48 ± 0.28^Ac
Pseudo-taraxasterol	31.44 ± 0.27^Cb	27.72 ± 0.34^Bbc	20.33 ± 0.33^Ac
β-Sitosterol	45.10 ± 0.12^d	33.93 ± 0.17^d	27.71 ± 0.20^d
Fungisterol	9.93 ± 0.18^Ca	6.13 ± 0.22^Ba	9.40 ± 0.27^Aa
Guggusterol	30.37 ± 0.34^Cb	24.31 ± 0.43^Bb	17.26 ± 0.54^Ab
Neocidol	9.81 ± 0.05^Ca	6.02 ± 0.02^Ba	5.38 ± 0.04^Aa
Means followed by different small and capital letters in a column and row respectively indicate significant difference (P < .05)

Taraxasterol, pseudo-taraxasterol, β-sitosterol, fungisterol and guggulsterol were isolated for the first time from C. africana resin. However, previous studies have reported wide range of medicinal properties other than insecticidal activity. For instance, Sharma and Zafar (2014) reported that taraxasterol possesses anti-tumor, anti-allergy, anti-oxidant and anti-inflammatory actions and also acts as a control against snake venom. Pseudo-taraxasterol on the other hand was shown to exhibit anti-inflammatory and anti-nociceptive activity in a combination with other triterpenoids (Da-Silva et al. 2017). Beta-sitosterol isolated from Cestrum diurnum Linn. leaves was reported to exhibit toxicity against larval forms of Aedes aegyptica Linn. and Anopheles stephensi Theobald (Mabrouk et al. 2011). A study by Mabrouk et al. (2011) screened activity of fungisterol from Penicillium brevicompactum Lindley and was confirmed to have antibacterial activity against gram-positive and gram-negative bacterial pathogens. It also demonstrated anticancer activity against breast and cervix carcinoma cell (Mabrouk et al. 2011).

Blending studies

The most potent repellent/toxic compounds were blended in equal proportions (Wachira et al., 2020) and evaluated for repellency and toxicity against bedbugs. The results of the selected blends are summarised in Table 3. The resultant mean percentage repellency of bedbugs on exposure to a two- or three- constituent blend of fungisterol (most repellent and toxic compound) with other active compounds (β-sitosterol and guggusterol), was not significantly different (P > .05) to that of fungisterol as an individual compound (Table 3). On the other hand, there was also no significance difference (P > .05) in LC₅₀ values of fungisterol (9.93 mg/L) and its blend of either β-sitosterol or guggusterol or both (Table 3).

Table 3

Mean repellency and toxicity of selected blends
Blend	Mean % repellency ± SE after 1/2 h exposure	Mean LC₅₀ ± SE (mg/L) after 24 h
1 + 2 + 3	70.2 ± 0.2^b	9.69 ± 0.29^a
1 + 2	69.5 ± 0.3^b	9.72 ± 0.09^a
1 + 3	64.3 ± 0.5^a	9.61 ± 0.11^a
2 + 3	69.0 ± 0.1^b	9.54 ± 0.03^a
2	71.0 ± 0.8^bc	9.93 ± 0.08^a
Neocidol	73.0 ± 0.5^c	9.81 ± 0.05^a
Numbers 1, 2 and 3 represents β-sitosterol, fungisterol and guggusterol, respectively. Means followed by different letters in a column indicate significant difference (P < .05)

These three- or two- constituent blends are in part behaviorally redundant, since repellency and mortality of bedbugs to a 2- or 3-component blend of fungisterol, β-sitosterol and guggusterol was similar to that of fungisterol as individual compound. A similar pattern was previously observed by Tasin et al. (2007) where attraction of grapevine moth to a 3-component blend of b-caryophyllene, (E)-b-farnesene and (E)-4,8-dimethyl-1,3,7-nonatriene was not significantly different from a 10-component blend. There was no synergistic repellent or toxic effect of bedbugs observed after exposure to blends of fungisterol with β-sitosterol or/and guggusterol. This was attributed to all the compounds which belonged to one homologous class of compounds (terpenoids) competing for the same receptors (Dambolena et al., 2016) where their modes of action leading to mortality is the similar.

Compounds in C. africana resin responsible for bedbug repellency and mortality have been identified and includes taraxasterol, pseudo-taraxasterol, β-sitosterol, fungisterol and guggulsterol. Although these compounds have been previously reported in other plant species, they have been isolated from C. africana resin for the first time. Fungisterol was the most potent repellent and toxic compound and can be applied in bedbug control upon further research on toxicology to humans and the environment. There was no synergistic repellent or toxic effects upon exposure of bedbugs to blends of fungisterol with β-sitosterol or/and guggulsterol since they compete for the same receptor neurons and associated genes in bedbug odor stimuli pathway. We anticipate that blending active terpenoids with other active compounds from other classes such as flavonoids, fatty acids, steroids and esters will lead to a synergistic/additive effect. Since bedbugs have developed a mechanism in resisting the commonly used insecticides and are, therefore, resurgent, a bio-pesticide product from fungisterol can be developed and used as a substitute in control of bedbugs and thereafter near eradication.

Acknowledgments

We appreciate the support received from Prof. Lugemwa F. Nelson (University of Nairobi) in Spectroscopic analyses and compound elucidation; and Mr. Richard Odera from the Department of Plant Sciences of Kenyatta University for technical support in bedbug rearing.

Conflict of interest: The authors declared no conflict of interest

Compliance with Ethical Standards: The study followed the guidelines of animal care and husbandry as stated by IUCAC committee of Kenyatta University.

Funding: NWW, National Forest Products Research Programme, Kenya Forestry Research Institute (KEFRI), 2018/2019.

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Bio-active principles in Commiphora africana resin dichloromethane extract and their insecticidal activity against bedbugs

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Abstract

Figures

Introduction

Materials and methods

General Experimental Procedures

Rearing of bedbugs

Column separation

Identification of compounds

Bioassay tests

Repellency of bedbugs on treatment with isolated compounds

Mortality of bedbug on exposure to isolated compounds

Data analysis

Results and discussion

Purification and isolation of compounds

Characterization of isolated compounds

Mean repellency of isolated pure compounds against bedbugs

Mean mortality of isolated pure compounds against bedbugs

Blending studies

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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