Chemical composition of essential oil in Piper nigrum from Nigeria and its bioactivity against stored-grain insects

. Experiments were carried out on the essential oil (EO) and its bio-efficacy against rice weevil ( Sitophilus oryzae ) and cowpea seed beetle ( Callosbruchus maculatus ). The EO from Piper nigrum was extracted by using steam distillation. Thirty-nine components were identified by using GC-MS, and the major components are linalool (21.73%), γ - bisabolene (8.75%), and β -caryophyllene (7.35%). The insects were exposed to the EO. At p < 0.05, the efficacy depends on the dose and time of exposure. S. oryzae is more resistant to the oil than C. maculates . The essential oil of P. nigrum may be used as a botanical insecticide against insect pests.


Material
Filter paper (Whatman No. 1) and acetone (100 %) were purchased from RIRO Ltd., Nigeria.Piper nigrum (black pepper) was obtained from the Agbalata market in Badagary, Lagos, Nigeria.The Federal Research Institute of Nigeria (FRIN), Ibadan, Nigeria, identified the seeds.The seeds were ground to powder after being air-dried.

Culture of insects
Rice weevil (Sitophilus oryzae) and cowpea seed beetle (Callosbruchus maculatus) founding insect cultures were taken from beans and rice that were already infested.The founding insect culture was kept in a 5 L plastic container at 24 °C and 70% humidity.

Essential oil distillation
The P. nigrum ground powder was subjected to hydro distillation in a modified Clevenger-type apparatus for six hours.The EO was dried after extraction with sodium sulphate (anhydrous) and kept at 4 °C in an air-tight container.

Gas-chromatography and mass-spectrometry (GC-MS) analysis
An Agilent 6890N instrument equipped with a flame ionisation detector and an HP-5MS (30 m × 0.25 mm × 0.25 μm) capillary column was used for the GC-MS analysis.An Agilent Technologies 5973N mass spectrometer was used to identify the components of the EO.First, the GC was set at an oven temperature of 60 °C and ramped at 10 °C per minute to 180 °C.This heating was followed by another ramping at 20 °C per minute to 280 °C.
The sample (1 μL) was injected with a 1:10 split ratio with helium as a carrier gas with a flow rate of 1 mL per minute.Prior to injection, the injector was set at 270 °C.The spectra were measured over

Contact effect
The essential oil contact activity for the samples prepared against cowpea seed beetle (C. maculatus) was assessed on a filter paper disc.The sample was allowed to flow on a filter paper disc placed in a Petri dish on a regular basis.After the solvent was eliminated, ten bean weevils were placed in the Petri dish, which was then sealed.
Every six hours, the percentage mortality of insects was measured.Insects that did not respond to a mild touch with a little probe were deemed dead.Each experiment was carried out in triplicate, except for the control experiment, which utilised only acetone.A similar test was carried out for S. oryzae with the samples prepared for the rice weevil.

Vapour effect
The insects were directly exposed to EOs vapour in a 1.5 L air-tight glass jar.The filter paper was placed upward on the top cover of the jar; then, ten bean weevils were introduced into the jar.
Every six hours, the percentage mortality of insects was measured.Each experiment was carried out in triplicate, except for the control experiment, which utilised only acetone.A similar test was carried out for S. oryzae with the samples prepared for the rice weevil.

Repellent effect
The area preference method was used to evaluate the EO repellent effects against C. maculatus.The filter paper was divided into two halves, and one half was used as the tested area.Afterward, the treated half was attached to the untreated half with a clear adhesive tape, and the re-made filter paper was put in a Petri dish.This procedure was repeated for all the samples.For each sample, ten mature insects from each species were separately placed in the centre of the re-made filter paper; then, each Petri dish was capped and preserved in an incubator at 27 ± 2 °C and relative humidity of 75 ± 5%.

Statistical analysis
The probit analytical method was used to evaluate the data.The LD50 and LD95 values of the EOs against each stored-product insect species were estimated by using the IBM SPSS software, version 21.0.The percentage mortality estimates for different exposure times were determined by using an analysis of variance (one-way ANOVA).

Gas chromatography-mass spectroscopy
The with what Ashouri and Shayesteh reported [22].

Fumigating
Methyl bromide is a well-documented environmental pollutant known as one of the causes of ozone layer depletion [23].Likewise, it is also known as a fumigant in the protection of insect pests.It is imperative to look for a possible alternative to it.Essential oils, owing to their high insecticidal activity, could be a viable alternative to methyl bromide [20].An application of a 5 mL•L -1 EO formulation on C. maculates and S.
oryzae shows 100% mortality in five days in both treatments (Tables 4 and 5).The mortality is dosedependent.The probit analysis reveals that the LD50 and LD95 for the fumigating activity of the essential oil on C. maculates are 4.36 and 6.95 mL•L -1 after 48 h of exposure, while those of S.
oryzae are 6.23 and 15.86 mL•L -1 after the same exposure time.Thus, it can be deduced from the observed fumigant activity that the essential oil of P. nigrum has an active vapour that could be deployed as an efficient alternative insecticide.
Therefore, further probing these natural fumigants' usage is necessary to control insects in stored products.
Pérez et al. [24] reported that the EOs from the analysed three piper species display varying inhibition levels against S. zeamais.As the oil dose increases, the percentage of mortality also increases.This finding is similar to what was reported in our work.However, the result from the probit analysis (LD50 and LD95) shows that the contact activity exhibits more toxicity against both insects than the fumigating activity.Note: The result shows the mean ± SD of three replicates.Data within a row followed by the same letter are not significantly different at p < 0.05.

Repellant
Tables 6 and 7  C. maculates at a lower exposure dose and a smaller duration.The ease of handling and accessibility to farmers, and the toxicity against stored-grain insects make this insecticide product a potential bio-insecticides in Africa.
in the environment and end up as a part of the food chain.Synthetic agrochemicals form part of the ecologically unsafe agricultural chemicals [3].The havoc caused by synthetic insecticides increases because of the insecticide residues left on food and eventually got into man's body via the food chain.There is a need to regulate the use of these pesticides based on the documented environmental risk of the use of synthetic chemicals [4-6].There is an increasing demand for biopesticides because of the growing need to regulate the use of these pesticides.Biodegradable plant extracts are environmentally friendly and safe compared with most widely used synthetic chemicals.Therefore, it is necessary to explore these plant extracts to develop safe alternatives to synthetic pesticides [7].Essential oils (EOs) from plant or their fractions outperform synthetic insecticides regarding local availability, minimal mammalian toxicity, and quick degradation [8, 9]).Several studies are carried out on essential oils' insecticidal activities on store pests [1, 10-13].These essential oils are potential insecticides; therefore, several plants grown in Africa are investigated for possible pesticide use.Callosbruchus maculatus and Sitophilus oryzae are huge pests that affect various grains, such as dried beans, wheat, corn, and rice, in Africa [14].The infestation of this pest results in enormous economic damage to farmers.Although several studies were conducted on P. nigrum, research shows that the plant's chemical composition varies with location [15-18].Khan et al. [2] suggested examining the extracts collected from different locations under various climatic conditions to determine their effectiveness for pest management.It is then vital to know the EO chemical composition of P. nigrum from Nigeria and investigate its insecticidal activity against rice weevil and cowpea seed beetle with regard to the exposure time at various doses of the formulation.

20 -
550 m/z; the components were classified from retention indices and compared with those found in the literature.The retention indices were also examined in reference to a homologous sequence of n-alkanes (C8-C24).The mass spectra from both columns were classified further by comparing them with those found in the literature.Without the use of correction factors, GC peak areas were used to evaluate component relative percentages.Insecticide formulationFor C. maculatus, five different concentrations of EOs were prepared.A volume of 2, 4, 6, 8, and 10 µL of the EO was picked out with a micropipette, then diluted with 2 mL of 100% acetone to obtain concentrations of 1, 2, 3, 4, and 5 mL•L -1 , respectively.Similarly, four different concentrations of EOs were formulated for rice weevil.A volume of 5, 10, 15, and 20 µL of the EO was diluted with 2 mL of 100% acetone to obtain 2.5, 5, 7.5, and 10 mL•L -1 concentrations, respectively.

Table 2 .
Percentage contact mortality of P. nigrum EO against C. maculatus (concentration 1-5 mL•L -1 ) The result shows the mean ± SD of three replicates.Data within a row followed by the same letter are not significantly different at p < 0.05.Table3.Percentage contact mortality of P. nigrum EO against S. oryzae (concentration 2.5-10 mL•L -1 The result shows the mean ± SD of three replicates.Data within a row followed by the same letter are not significantly different at p < 0.05.Table4.The percentage fumigative mortality of P. nigrum EO against C. maculatus (concentration 1-5 mL•L -1 ) The result shows the mean ± SD of three replicates.Data within a row followed by the same letter are not significantly different at p < 0.05.Table 5. Percentage fumigative mortality activity of P. nigrum EO against S. oryzae (concentration 2.5-10 mL•L -1 ) show the essential oil average repellancy values of P. nigrum at different concentrations and exposure times against C. maculates and S. oryzae.All formulations were repellent to C. maculates and S. oryzae.The 5 mL•L -1 formulation entirely evokes repellent action against C. maculates in 20 min and S. oryzae in 50 min.When the formulation concentration increases, the percentage of repellency also increases.The formulation has a higher repellency against C. maculates than against S. oryzae, and this repellency is similar to what was reported by Park [25].Table 6.Percentage repellant activity of P. nigrum EO against C. maculatus (concentration 1-5 mL•L -1 ) Table 7. Percentage repellant activity of P. nigrum EO against S. oryzae (concentration 2.5-10 mL•L -1 ) 4 Conclusion In this study, linalool is the major component in the P. nigrum essential oil, and it can be responsible for the contact and fumigating activities against C. maculates and S. oryzae.The P. nigrum essential oil has toxic effects on the studied insect pests.The essential oil is more toxic against

Table 1 .
The chemical composition of essential oil of P. nigrum from Nigeria GC-MS analysis indicates 39 components in the P. nigrum EO, which agrees with what Fan et al. reported [15].In corroboration with other researchers, the major components (Table 1) are linalool (21.73%), γ-bisabolene (8.75%), and βcaryophyllene (7.35%) [15-17].Fan et al. reported limonene as the major compound presenting 35.06% of total oil from the fruits of black pepper, of the EO of P. nigrum could be a result of the soil composition, climate, and vegetative conditions [21].analysis; the LD50 and LD95 for C. maculates are 4.37 and 6.07 mL•L -1 , while those of S. oryzae are 4.80 and 12.98mL•L -1 .This information shows that the essential oil is more toxic to C. maculates than to S. oryzae.The percentage of mortality increases with the concentration of formulation and exposure time; this mortality change is consistent