Skip to main content

Larvicidal activity of the black pepper, Piper nigrum (Fam: Piperaceae) extracts on the cattle tick, Rhipicephalus australis (Acari: Ixodidae)



The cattle farming parasite Rhipicephalus australis is the main tick and one of the most important in the world from an economic point of view. Various studies have been developed in order to find plant extracts with effective acaricidal properties and environmentally friendly. Studies involving plant extracts for parasite control on commercial animal herds is a developing area in New Caledonia. Bioactive natural products play an important role as lead compounds in the development of new pesticides.


The ethanolic extract of Piper nigrum L. dried fruits as well as the ethyl acetate extract and the methanolic extract of stems exhibited 100% larvicidal activity (50 mg/mL) against Rh. australis larvae, the cattle tick, an hematophagous parasite. Bioguided fractionation of the ethanolic extract of dried mature fruits using the same assay led to the isolation of five compounds belonging to piperamide family. The structures of isolated compounds were elucidated using spectroscopic methods: ESI-HRMS, 1H- and 13C-NMR spectral data, including DEPT and 2D-NMR experiments (COSY, HSQC, HMBC, and NOESY). These include 1 compound described for the first time in P. nigrum, homopellitorine (2) and 4 known compounds, namely pellitorine (1), pipyaqubine (3), 2-methylpropylamide (4), and N-isobutyl-2,4-eicosadienamide (5).


This first report on the larvicidal activity of P. nigrum extract and pure compounds on this tick species suggests that P. nigrum could be a natural biosourced alternative for the control of the larval stage of Rh. australis.

Graphical Abstract


The cattle tick, Rhipicephalus australis (Canestrini 1887) was introduced in New Caledonia in 1942 and is the main health problem in cattle farming. Locally, Rh. australis was synonymized with the southern cattle tick, Rhipicephalus microplus, until Rh. australis was reinstated as a separate cattle tick species in 2012 [15]. This tick is the main species and one of the most important in the world from an economic point of view. It has been estimated that 80% of the world’s cattle population is at risk from tick and tick-borne diseases (TBDs) causing estimated annual losses of US$ 22–30 billion [37]. The principal control method involves the use of synthetic acaricides by dip, spray, injection, or pour-on treatments [43, 48]. The continuous use of these chemical compounds has led to the selection and development of strains of Rh. australis resistant to organophosphates, pyrethroids and formamidine, a phenomenon that is a major concern for worldwide cattle breeders [14, 29, 43]. Moreover, such chemical control causes meat and milk contamination that can also have undesirable effects on other organisms and the environment [14, 17, 18, 27, 44].

The need of new scientific investigations for alternative ways to control this tick is related to the evolution of resistance of Rh. australis to synthetic acaricides. As a matter of fact, various studies have been performed in order to find plant extracts with acaricidal properties [3, 6, 7, 10, 14, 22, 26, 28, 32, 33, 40, 44, 65] to discover natural compounds at least as effective as classic treatments but also environmentally friendly and susceptible to be produced on a large, commercial scale [30].

In a first study, Borges et al. [6] inventoried 55 plants belonging to 26 families tested against Rh. australis [6]. In a second review in 2016, Benelli et al. describe the results of 62 extracts on this parasite excluding essential oils [3]. In 2020, Quadros et al. listed 27 plants-derived substances with potential for tick control and prevention on Rh. australis [44].

In New Caledonia, several works have been devoted to the acaricidal activity of natural substances on Rh. australis larvae, but mainly concerned essential oils. Lebouvier et al. [32, 33], showed that essential oils from endemic trees of New Caledonia could provide natural acaricides for the control of the cattle tick Rh. australis. Nevertheless, the development of an alternative tick control strategy must be associated with a high safety profile as well as availability and remanence. Indeed, the potential toxicity of essential oils, their low extraction yield and their volatile nature reduce their valorization and application potential despite the many biological activities they may present [32, 35]. Therefore, it seems that organic solvent extracts from plants have many positive aspects for valorization in the control of cattle ticks and the Piperaceae family is a very interesting example.

Piperaceae family is represented by at least 3000 species and is known to have acaricidal compounds, such as monoterpenes, sesquiterpenes, alkaloids, and phenylpropanoids [4, 11, 12, 24, 38, 39, 42, 45, 49, 52, 66]. The genus Piper is very large, and several species of Piper have been used as spice and in traditional medicine and bear an immense commercial, economical, and medicinal importance. Some Piper species have simple chemical profiles, while others, such as Piper nigrum contain diverse suites of secondary bioactive metabolites [49]. Piper nigrum L., commonly known as black pepper, is a climber originally native to India. The acrid and pungent taste of P. nigrum fruits attracted the attention of chemists as early as 1819 when Oestred isolated piperine. Since that time, the search for active constituents from different Piper species is being continued and this has been intensified in the recent years, particularly because of interesting biological activities of various chemicals from several Piper species [12, 20, 34, 49, 52, 56,57,58,59,60, 66]. Indeed, some Piper species are listed as remedies for stomach pain, asthma, bronchitis, fever, abdominal pain, hemorrhoidal afflictions, rheumatism, as anti-inflammatory and stimulant agents, but also as insect repellents, insecticidal, acaricidal, antifungal, and antibiotic [16, 23, 28, 34, 47, 52, 56,57,58, 67]. The chemistry of Piper species has been widely investigated, and phytochemical investigations from all parts of the world have led to the isolation of several physiologically active compounds, including alkaloids, amides, propenylphenols, lignans, neolignans, terpenes, steroids, pyrones, piperolides, chalcones, flavones, and flavanones [8, 12, 23, 31, 34, 45, 50, 54,55,56,57,58,59,60, 61,62,63, 66].

Several Piperaceae have also been studied against cattle ticks [7, 10, 20, 28] and their major characteristic and active constituents could be attributed to a considerable variety of amide alkaloids [2, 51, 52, 66] and to a possible synergistic action [5, 11, 40, 41, 50, 52]. Therefore, an investigation on either major or minor compounds seems to be of particular interest.

In this context, we evaluated the larvicidal activity of the black pepper, Piper nigrum (Fam: Piperaceae) extracts on the cattle tick, Rhipicephalus australis (Acari: Ixodidae).

Materials and methods

Plant material and extraction procedure

Fruits and aerial parts (leaves and stems) of Piper nigrum were collected at the IAC SRA (Agronomic Research Station of Institut Agronomique néo-Calédonien) of Pocquereux (La Foa, New Caledonia, 21°44′10,338’’ S 165°53′44,296’’ E). The IAC SRA extends over 90 hectares and hosts numerous experimental orchards devoted to tropical fruit crops as well as many plants of food interest and this is where the pepper plants are grown.

Dried fruits, dried leaves, and dried stems were grounded into powder (Grinder Moulin IKA® M20 250 mL) and extracted (30 g of dry matter in 400 mL of solvent macerated at room temperature for 48 h then put in ultrasound for 20 min before filtration). Three different solvents were used for the extraction viz. ethanol 95%, ethyl acetate, and methanol (solvents from Univar Solutions, Manchester, USA) and filtered under vacuum using a Buchner funnel (Buchner funnel JIPO). The filtrates were concentrated under reduced pressure at 40 °C. The dried crude extracts were stored at 4 °C in the IAC extracts collection.

Biological test

Preparation of tick

A Rh. australis breeding was ongoing in IAC on calves. The strain was sensible to deltamethrin and resistant to amitraz. In the final stage of engorgement, female ticks were collected and used in the biological test within 24 h. Ticks were rinsed by water and dried using filter papers. The female ticks were incubated at 27 °C and 85% relative humidity for 1 week. Eggs were then collected and placed in the same conditions until larvae were 2 weeks old.

Larval packet test

The FAO modified LPT method (Larval Packet Test; Stone 1962 [64]) was used to assess the acaricidal effect of samples on 14 days old larvae. Nylon papers (Anowo LTD) were impregnated with 50 mg of extracts (1 mL at 50 mg/mL per paper) and placed during one hour in a fume hood to allow the solvent (EtOH 95%) to evaporate before being folded into packets using bulldog clips. Approximately 100 Rh. australis larvae were placed into each treated nylon paper packet, which was then sealed with additional bulldog clips and placed in an incubator (27 °C; 85% RH) for 24 h. Two replicates and a control (nylon paper with solvent) for each sample were used. After exposure to the sample, the numbers of live and dead larvae were counted to calculate the percentage of larval mortality. To determine the LC50, six successive dilutions were tested, with the initial concentration depending on mass of extract available.

Statistical analysis

For each extract, the lethal concentration 50% (LC50), 90% (LC90), 99% (LC99), slope, chi-square, heterogeneity and the 95% confidence intervals (CI95%) were calculated according to Probit analysis [21] using Poloplus® program, Le Ora Software [36].

Compounds isolation

Chromatographic and spectroscopic methods for compounds isolation

The chromatography columns were performed on silica gel (Sigma-Aldrich, 70–230 mesh). Thin-layer chromatography were carried out on aluminum plates (aluminum sheet silica gel SIL/UV254; 0.20 mm; 20 × 20 cm) and visualized with UV light (254 and 366 nm) then sprayed with vanillin−H2SO4.

The semi preparative chromatography isolations were performed with Waters Deltaprep instrument. An HPLC column (Thermo Scientific Hypersil ODS C18 HPLC Preparative column, 250 × 10 mm; particle size 5 µm) was used for the analysis. The mobile phase consisted of milliQ water (solvent A) and acetonitrile (HPLC supra gradient grade) purchased from Unichrom (Ajax Finechem Pty. Ltd., New Zealand) (solvent B), and the flow rate was set to 2 mL.min−1. The column oven was set at 35 °C. The injection volume was 100 µL. UV–Visible spectra were recorded between 200 and 400 nm.

The ESI-HRMS spectra were recorded on a QToF instrument (Agilent 6530, Les Ulis, France) in infusion mode. Ionization source conditions were drying gas temperature 325 °C, drying gas flow rate 11 L/min, nebulizer 35 psig, fragmentor 175 V, skimmer 65 V. Range of m/z was 200–1700. Purine ion C5H4N4 [M + H]+ (m/z 121.050873) and the hexakis (1H,1H,3H-tetrafluoropropoxy)-phosphazene ion C18H18F24N3O6P3 [M + H]+ (m/z 922.009798) were used as internal lock masses. Full scans were acquired at a resolution of ca 11 000 (at m/z 922).

The 1H and 13C NMR 1D spectra as well as 2D spectra (COSY, HSQC, HMBC and NOESY), were recorded in CDCl3 on a Bruker Avance 400 spectrometer (Sarrebourg, France) operating at 400 MHz for 1H spectra and 100 MHz for 13C.

Successive fractionations for compounds isolation

Fractionation 1

The ethanol crude extract (45 g; 7% yield), active at 100% against Riphicephalus australis, was chromatographed on normal phase silica gel column (particle size 0.060–0.200 mm). The mobile phase consisted of a stepwise gradient of acetone in CH2Cl2: 0% (4L), 0.25% (1.5L), 1% (2L), 2.5% (1L), 3% (2L), 4% (2L), 5% (2L), 8% (2L), 10% (4L) to end with 100% MeOH. The fractions were combined based on the thin-layer chromatography (TLC) profiles to give 17 different fractions A-Q. Among the 17 fractions obtained, two of them (Fractions G and H) showed 100% activity, and were selected for further purifications.

Fractionation 2

Further purification of fraction G (818 mg) by semi preparative chromatography on reverse phase silica gel—60 C8 rp perfluorinated (27 × 1.5 cm; particle size 0.040–0.063 mm) eluted with MeOH/H2O (90/10—100/0 in 15 min and 100/0 for 15 min) provided 5 subfractions and compound 5.

Fractionation 3

Further purification of fraction H (5 g) by normal phase silica gel column (particle size 0.063–0.100 mm) was carried out. The mobile phase consisted of a gradient of petroleum ether and ethyl acetate from 90/10 (2L), 80/20 (4L) and 70/30 (2L) to provide 16 subfractions FH.F1 to FH.F16.

Fractionation 4

Fraction FH.F6 was subjected to further chromatography.FH.F6 (510 mg) was chromatographed by semi preparative chromatography on reverse phase silica gel – 60 C8 rp perfluorinated (27 × 1.5 cm; particle size 0.040–0.063 mm) eluted with ACN/H2O (70/30 during 15 min; to 100/0 in 30 min and 100/0 for 15 min) to provide compounds 1, 2, 3, and 4.


Acaricidal effect of crude extracts

Table 1 shows the acaricidal activities of Piper nigrum crude extracts against Rh. australis. The leaves EtOAc extract showed only 19% of larval mortality and so no lethal concentrations were calculated. The stems extracts (EtOAc and MeOH) and the fruits EtOH extracts (mature and green/unripe) showed 100% activity against 14 day-old larval Rh. australis so their LC50, LC90, and LC99 were calculated and as compared to the literature.

Table 1 Acaricidal activities of Piper nigrum crude extracts at 50 mg/mL

The dried mature fruits ethanolic extract showed the best extraction yield (7%) and was selected for bioguided fractionation. Among the 17 fractions obtained, two of them (Fraction G, fraction H, and fraction H.F6) showed 100% activity and were selected for further purification of their major compounds (Fig. 1).

Fig. 1
figure 1

Fractionation steps of P. nigrum dried mature fruits ethanolic extract

Isolation and identification of pure compounds (1)−(5)

Bioassay-guided fractionation of the Piper nigrum dry mature fruits EtOH extract afforded five pure compounds (Fig. 1) identified by spectroscopic analysis (HRMS and NMR, Table 2) and by comparison to the published data. Structures of compounds (1)−(5) are presented in Fig. 2.

Table 2 1H NMR (400 MHz) and 13C NMR (100 MHz) for compounds 1–5 (CDCl3)
Fig. 2
figure 2

Structures of compounds (1)−(5) Pellitorine (1), homopellitorine (2), pipyaqubine (3), 2-methylpropylamide (4) and N-isobutyl-2,4-eicosadienamide (5)

Compound (1)—Pellitorine ((2E,4E)-N-isobutyldecadienamide)

For 1H and 13C NMR spectra, see Table 2; HRMS m/z (%): 224.2024 [M + H]+ (60), 246.1848 [M + Na]+ (100), 287.2124 (25), 469.3787 [2 M + Na]+ (100), 692.5723 [3 M + Na]+ (10); (corresponding to a formula C14H25NO, calculated 223.1936). The spectroscopic data matched to those found in literature [34, 38, 63].

Compound (2)—Homopellitorine (N-2'-methylbutyl-2E,4E-decadienamide)

For 1H and 13C NMR spectra, see Table 2; HRMS m/z (%): 260.2004 [M + Na]+ (60), 274.2165 (50), 344.1552 (40), 413.2689 [M + Na + C9H15ON]+ (100), 734.4350 [3 M + Na]+ (2); (corresponding to a formula C15H27NO, calculated 237.2092). The spectroscopic data matched to those found in literature [5, 54, 63].

Compound (3)—Pipyaqubine or pirrollidide (N-pyrrolidyl-2,4-octadecadienamide)

For 1H and 13C NMR spectra, see Table 2; HRMS m/z (%): 290.2706 [M-C3H7]+ (70), 356.1535 [M + Na]+ (100), 476.3711 [MH + 2(C5H11)]+ (5), 691.3224 [2 M + H + Na]+ (2); (corresponding to a formula C22H39NO, calculated 333.3031). The spectroscopic data matched to those found in the literature [23].

Compound (4)—N-isobutyl-2E,4E,12Z-octadecatrienamide (2-Methylpropylamide)

For 1H and 13C NMR spectra, see Table 2; HRMS m/z (%): 334.3131 [M + H]+ (30), 356.2960 [M + Na]+ (100), 689.5981 [2 M + Na]+ (15); (corresponding to a formula C22H39NO, calculated 333.5512). The spectroscopic data matched to those found in literature [31].

Compound (5)—N-isobutyl-2E,4E-eicosadienamide (N-isobutyleicosa-trans-2,trans-4-dienamide)

For 1H and 13C NMR spectra, see Table 2; HRMS m/z (%): 364.3589 [M + H]+ (50), 386.3404 [M + Na]+ (100), 727.7101 [2 M + H]+ (2), 749.6904 [2 M + Na]+ (5); (corresponding to a formula C24H45NO, calculated 363.6202). The spectroscopic data matched to those found in the literature [1].


Piper nigrum is already known for its insecticidal and acaricidal activities. Extracts from different parts were shown to be toxic for houseflies (Musca domestica L.), rice weevils (Sitophilus oryzae L.), cowpea weevils (Callosobruchus maculatus F.), Aedes aegypti and for several more lepidopteran and hymeopteran herbivorous insects [16, 23, 25, 34, 41, 47, 50,51,52, 54, 56,57,58, 61].

Godara et al. [22] observed that methanolic extract of dried fruits of P. nigrum significantly affected mortality rates of adults engorged females of Rh. australis in a dose-dependent manner with an additional effect on the reproductive physiology of ticks by inhibiting oviposition and the LC50 value of methanolic extract was calculated as 0.48% (0.46–0.49) [22]. Nonetheless, no researches have been done on the larvicidal activity of Piper nigrum against Rh. australis although [23] demonstrated that P. nigrum could induce mortality on Culex pipiens pallens and Aedes egypti larvae. The research studies on larvae stage could emphase a more strategic and preventive control. This is why in this study we focused on the larvicidal activity against Rh. australis and our results are thus the first to report the larvicidal activity of P. nigrum fruit extracts on this tick species.

The activity of several Piperaceae extracts on cattle ticks larval stage have also been studied (Table 3).

Table 3 Larvicidal activity of different Piperaceae extracts against Rhipicephalus microplus*

Da Silva Lima et al. [10] showed that fruits hexane extract of P. tuberculatum showed the greatest efficacy (LC50 = 0.004%) against tick larvae followed by the ethyl ether, ethanol and methanol extracts with LC50 of 0.008%, 0.273%, and 0.449%, respectively [10]. However, hexane and ethyl ether being apolar solvents, we were more interested in alcoholic extracts in comparison with our results and in a development context for more adaptable and reproducible application tests. Therefore, in this work, we found ethanolic extracts (50 mg/mL) of Piper nigrum dried mature and unripe (green) fruits showing LC50 of 2.499 mg/mL and 1.533 mg/mL (0.25 and 0.15%), respectively, on Rh. australis larvae. We can therefore, cite that Jyoti et al. [28] reported a dose-dependent mortality response on larval stages for both Piper longum extracts and higher acaricidal property was exhibited by the alcoholic extract with LC50 and LC95 values of 0.488% (0.48–0.49) and 1.39% (1.35–1.44), respectively [28]. Braga et al. [7] showed that the LC50 of Piper tuberculatum extracts after 24 h of exposure were 3.62, 3.99 and 5.30 mg/mL (0.36, 0.40 and 0.530%) for fruit, stem and leaf extracts, respectively.

It can thus be considered that the ethanolic extracts of the fruits of Piper nigrum show the best LC50 of 0.2% on average for the fruits (ripe and green) as compared to the ethanolic extract of Piper tuberculatum fruits having an LC50 of 0.3% on average (0.3% [7] and 0.27% [10]) and the ethanolic extract of Piper longum fruits having an LC50 of 0.5% [28] on tick larvae (Table 3).

Furthermore, in our work, the ethyl acetate extract and the methanol extract from P. nigrum stems showed a LC50 of 0.34 mg/mL and 4 mg/mL (0.034% and 0.4%), respectively, on the larval stage of Rh. australis. Braga et al. [7] showed that the LC50 of Piper tuberculatum extracts on Rh. microplus larvae after 24 h of exposure were 3.62, 3.99 and 5.30 mg/mL (0.36, 0.40, and 0.530%) for fruit, stem and leaf extracts, respectively [7]. Ferraz et al. [20] observed that the essential oil of aerial parts of Piper mikanianum had a LC50 = 0.233% on tick larvae and that the essential oil of aerial parts of P. xylosteoides had a LC50 = 0.615% while the essential oil of aerial parts of P. amalago was inactive [20].

Moreover, Barrios et al. [2] highlights that the acaricidal property of P. tuberculatum can be attributed to the fact that its leaves and stems contain a considerable variety of amides and other compounds active against ectoparasites [2]. Indeed, according to Yu et al. [66], the major characteristic and active constituents of P. nigrum fruits are amide alkaloids [66].

In our study, the structures of isolated compounds were elucidated using spectroscopic methods: ESI-HRMS, 1H- and 13C-NMR spectral data, including DEPT and 2D-NMR experiments (COSY, HSQC, HMBC, and NOESY). These include one compound described for the first time in P. nigrum, homopellitorine (2) and 4 compounds previously described in P. nigrum, namely pellitorine (1), pipyaqubine (3), 2-methylpropylamide (4), and N-isobutyl-2,4-eicosadienamide (5). Moreover, the chromatographic profiles showed that piperine was the major compound of the fruit and stem extracts. The final quantities of the isolated compounds did not allow to determine their LC50 and LC90. However, in an application context, we would like to highlight the use of crude extracts obtained with waste from pepper cultivation. Our isolated piperamides are the major compounds of the bioactive subfractions (100% activity on larvae) from which they are derived, and we can therefore hypothesize that they are partly responsible for the acaricidal activity observed with probably a synergistic action with other compounds. Da Silva et al. suggested that berberine and piperine alkaloids have an in vitro acaricidal action on Rh. australis larvae [9]. In 2002, Scott et al. already concluded that the biological activity of P. tuberculatum may be due to compounds present in smaller proportion with a synergic effect of several piperamides [50]. Indeed, Rodrigues et al. [47] showed that pellitorine, pipyaqubine, and piperine had LC50 of 20, 31 and 10 µg/mL, respectively, on Aedes aegypti larvae [23, 47]. Ee et al. [13] showed that pellitorine could be a potential anticancer hit compound [13] and we can find in literature that pellitorine and piperine exhibited also antibacterial [46] and insecticidal [53] activities. Furthermore, Miyakado et al. [40, 41] highlighted the insecticidal effect of different piperamides: pellitorine, pipercide, dihydropipercide, and guineensine. They attributed the high toxicity of the crude extracts of P. nigrum to a synergistic action carried by the different Piperaceae amides [40, 46]. In 2005, Lee et al. pointed out bioactive constituents (fungicidal, insecticidal, and mosquito larvicidal activities) derived from Piperaceae fruits to be pipernolanine, piperoctadecalidine, pellitorine, guineensine, pipercide, and retrofractamide A [34]. One important fact is that the efficacity of Piper extracts as botanical insecticides has been correlated with the concentration of piperamides present [51, 52]. Moreover in 2015, Ramesh et al. showed that sesamin, piperine, guineensine, pellitorine, trichostachine, and 4,5-dihydropiperlonguminine were considered to be the six marker compounds in Piper nigrum L. [45].

Piperamides can thus be considered as important bioactive compounds having a synergistic action [5, 11, 50, 52]. It also seems that piperine inhibits several metabolic enzymes and increases the oral bioavailability of many drugs and nutrients. Piperine enhances therapeutic effects and helps digestion by stimulating the intestinal and pancreatic enzymes [49]. As a matter of fact, our results are very interesting as Piper nigrum L. fruits are commonly cultivated, used and available worldwide.

The crops of Piper nigrum for the food industry generate a lot of waste (pericarp, stems and leaves usually considered as wastes during making of pepper) that can become sustainable sources in circular bioeconomy. Dried fruits, leaves and stems, as renewable parts of the plant, could be waste materials to recycle. Indeed, many studies have shown that P. nigrum is valued for its medicinal properties for treating pain, chills, rheumatism, flu, muscular aches and that its fruits shown antibacterial, antioxidant, anticancer, antimutagenic, antidiabetic, anti-inflammatory, analgesic, anticonvulsant, or neuroprotective effects [66, 67]. Thus, the acaricidal properties and the medicinal properties of the different parts of P. nigrum lead us to think that it is a plant to be valued for various applications. Indeed, as Yu et al. point out, we can consider that all this knowledge contributed to maximizing the use of different parts of P. nigrum as added-value resources for the food and pharmaceutical industries application [66]. Finally, as Quadros et al. [44] point out, for the development of commercial natural organic biopesticides it is important to consider the availability of the plant resource, the need for chemical standardization and quality control, the long-term stability, storage and transportation [44]. Finally, as Salehi et al. [49] highlighted, most of the studies were performed using in vitro models, so in vivo experimental approaches are needed to validate Piper spp extracts as acaricides [49].


The EtOH extracts of Piper nigrum dried fruits were the most active extracts (50 mg/mL) against Rh. australis. The dried mature fruits ethanolic extract showed the best extraction yield (7%). and was selected for bioguided fractionation that led to the isolation and structure elucidation of 5 major compounds involved in the acaricidal activity, including one compound described for the first time in P. nigrum. Furthermore, as adult ticks are the main problem for livestock in terms of damages, the research studies on tick’s larvae emphasis a more strategic and preventive control. Phytochemical studies on Piper spp. have been conducted to find potential pharmaceuticals or pesticides, but the most interesting investigations pertain on the synergy interactions.

Overall, the research findings clearly explain the feasibility of Piper nigrum aerial parts (fruits and stems) as potent naturals acaricides for the cattle tick control but also highlight the need for more investigations on the synergistic effects of phytochemical compounds.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. All data generated or analyzed during this study are included in this published article [and its supplementary information files].


CH2Cl2 :


CDCl3 :

Deuterated chloroform


Confidence intervals


Correlated spectroscopy


Electrospray ionization


Ethyl acetate




Heteronuclear multiple bond correlation


High-resolution mass spectrometry


Heteronuclear single quantum coherence


Institut Agronomique néo-Calédonien


Lethal concentration


Larval packet test




Nuclear magnetic resonance


Nuclear Overhauser effect spectroscopy


Relative humidity


Resistance ratio


Agronomic research station


Tick-borne diseases


  1. Addae-Mensah I, Torto FG, Oppong IV, Baxter I, Sanders JKM. N-Isobutyl- trans -2-trans-4-eicosadienamide and other alkaloids of fruits of Piper guineense. Phytochemistry. 1977;16:483–5.

    Article  CAS  Google Scholar 

  2. Barrios H, Flores B, Duttmann C, Mora B, Sheleby-Elías J, Jiron W, Balcazar JL. In vitro acaricidal activity of Piper tuberculatum against Rhipicephalus (Boophilus) microplus. Int J Acarol. 2022;48(3):1–5.

    Article  Google Scholar 

  3. Benelli G, Pavela R, Canale A, Mehlhorn H. Tick repellents and acaricides of botanical origin: a green roadmap to control tick-borne diseases? Parasitol Res. 2016;115(7):2545–60.

    Article  PubMed  Google Scholar 

  4. Bernard CB, Krishanmurty HG, Chauret D, Durst T, Philogene BJR, Sanchez-Vindas P, Hasbun C, Poveda L, San RL, Arnason JT. Insecticidal defenses of Piperaceae from the neotropics. J Chem Ecol. 1995;21(6):801–14.

    Article  CAS  PubMed  Google Scholar 

  5. Boonen J, Bronselaer A, Nielandt J, Veryser L, De Tré G, De Spiegeleer B. Alkamid database: Chemistry, occurrence and functionality of plant N-alkylamides. J Ethnopharmacol. 2012;142(3):563–90.

    Article  CAS  PubMed  Google Scholar 

  6. Borges LMF, Sousa LAD, Barbosa CS. Perspectives for the use of plant extracts to control the cattle tick Rhipicephalus (Boophilus) microplus. Revista brasileira de parasitologia veterinaria. 2011;20(2):89–96.

    Article  PubMed  Google Scholar 

  7. Braga AGS, de Souza KFA, da Barbieri FS, de Fernandes CF, Rocha RB, Vieira Junior JR, Lacerda CL, Celestino CO, Facundo VA, Brito LG. Acaricidal activity of extracts from different structures of Piper tuberculatum against larvae and adults of Rhipicephalus microplus. Chem Pharmacol Acta Amazonica. 2018;48(1):57–62.

    Article  Google Scholar 

  8. Chandra P, Bajpai V, Srivastva M, Kumar RKB, Kumar B. Metabolic profiling of Piper species by direct analysis using real time mass spectrometry combined with principal component analysis. Anal Methods. 2014;6(12):4234–9.

    Article  CAS  Google Scholar 

  9. da Silva GD, de Lima HG, de Freitas HF, da Rocha PSS, Luz YDS, de Figueiredo MP, Uzêda RS, Branco A, Costa SL, Batatinha MJM, Botura MB. In vitro and in silico studies of the larvicidal and anticholinesterase activities of berberine and piperine alkaloids on Rhipicephalus microplus. Ticks Tick Borne Dis. 2021;12(2):101643.

    Article  PubMed  Google Scholar 

  10. da Lima AS, do Filho JGNS, Pereira SG, Guillon GMSP, da Santos LS, Costa Júnior LM. Acaricide activity of different extracts from Piper tuberculatum fruits against Rhipicephalus microplus. Parasitol Res. 2014;113(1):107–12.

    Article  PubMed  Google Scholar 

  11. Dyer LA, Richards J, Dodson CD. Isolation, synthesis, and evolutionary ecology of Piper amides. In: Dyer LA, Palmer ADN, editors. Piper: a model genus for studies of phytochemistry, ecology, and evolution. Boston, MA: Springer; 2004. p. 117–39.

    Chapter  Google Scholar 

  12. Ee GC, Lim CM, Lim CK, Rahmani M, Shaari K, Bong CF. Alkaloids from Piper sarmentosum and Piper nigrum. Nat Prod Res. 2009;23(15):1416–23.

    Article  CAS  PubMed  Google Scholar 

  13. Ee GCL, Lim CM, Rahmani M, Shaari K, Bong CFJ. Pellitorine, a potential Anti-cancer lead compound against HL60 and MCT-7 cell lines and microbial transformation of piperine from Piper Nigrum. Molecules. 2010;15(4):2398–404.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Espinoza J, Flores B, Jerez J, Sheleby-Elías J. Short communication: in vitro evaluation of resistance of Rhipicephalus (Boophilus) microplus against three widely used ixoidicides. Veterinarija ir Zootechnika. 2022;80(1):65–9.

    Google Scholar 

  15. Estrada-Peña A, Venzal JM, Nava S, Mangold A, Guglielmone AA, Labruna MB, de la Fuente J. Reinstatement of Rhipicephalus (Boophilus) australis (Acari: Ixodidae) with redescription of the adult and larval stages. J Med Entomol. 2012;49:794–802.

    Article  PubMed  Google Scholar 

  16. Fan LS, Muhamad R, Omar D, Rahmani M. Insecticidal properties of Piper nigrum fruit extracts and essential oils against Spodoptera litura. Int J Agric Biol. 2011;13(4):517–22.

    CAS  Google Scholar 

  17. FAO—Food and Agriculture Organization of the United Nations. Resistance management and integrated parasite control in ruminants—guidelines, 2004; Module I–ticks: acaricide resistance: diagnosis, management and prevention. 2004.

  18. FAO—Food and Agriculture Organization of the United Nations. Ticks and tick-borne disease control: a practical field manual. Rome, Italy: FAO; 1984. p. 621.

    Google Scholar 

  19. Fernandez CMM, Lorenzetti FB, Bernuci KZ, Iwanaga CC, de Campos Bortolucci W, Romagnolo MB, Simões MR, Cortez DAG, de Lima SRB, Gazim ZC, Filho BPD. Larvicidal potential of piperovatine in the control of cattle tick. Vet Parasitol. 2018;263:5–9.

    Article  CAS  PubMed  Google Scholar 

  20. de Ferraz ABF, Balbino JM, Zini CA, Ribeiro VLS, Bordignon SAL, von Poser G. Acaricidal activity and chemical composition of the essential oil from three Piper species. Parasitol Res. 2010;107(1):243–8.

    Article  Google Scholar 

  21. Finney DS. Probit analysis. 3rd ed. Cambridge: Cambridge University Press; 1971. p. 333.

    Google Scholar 

  22. Godara R, Verma MK, Katoch R, Yadav A, Dutt P, Satti NK, Katoch M. In vitro acaricidal activity of Piper nigrum and Piper longum fruit extracts and their active components against Rhipicephalus (Boophilus) microplus ticks. Exp Appl Acarol. 2018;75(3):333–43.

    Article  CAS  PubMed  Google Scholar 

  23. Gulzar T, Uddin N, Siddiqui BS, Naqvi SNH, Begum S, Tariq RM. New constituents from the dried fruit of Piper nigrum Linn., and their larvicidal potential against the Dengue vector mosquito Aedes aegypti. Phytochem Lett. 2013;6(2):219–23.

    Article  CAS  Google Scholar 

  24. Gupta A, Gupta M, Gupta S. Isolation of piperine and few sesquiterpenes from the cold petroleum ether extract of Piper nigrum (Black Pepper) and its antibacterial activity. Int J Pharmacognosy Phytochem Res. 2013;5(2):101–5.

    Google Scholar 

  25. Harvill EK, Hartzell A, Arthur JM. Toxicity of pipeline solutions to houseflies. Contributions Boyce Thompson Inst Plant Res. 1943;13(2):87–92.

    CAS  Google Scholar 

  26. Hüe T, Cauquil L, Fokou JBH, Dongmo PMJ, Bakarnga-Via I, Menut C. Acaricidal activity of five essential oils of Ocimum species on Rhipicephalus (Boophilus) microplus larvae. Parasitol Res. 2015;114:91–9.

    Article  PubMed  Google Scholar 

  27. Jonsson NN, Piper EK. Integrated control programs for ticks on cattle. QLD, Australia: The University of Queensland; 2007. p. 163.

    Google Scholar 

  28. Jyoti, Saini SPS, Singh H, Rath SS, Singh NK. In vitro acaricidal activity of Piper longum L against amitraz resistant Rhipicephalus microplus (Acari: Ixodidae). Exp Parasitol. 2022;241:108356.

    Article  CAS  PubMed  Google Scholar 

  29. Klafke GM, Sabatini GA, de Albuquerque TA, Martins JR, Kemp DH, Miller RJ, Schumaker TTS. Larval immersion tests with ivermectin in populations of the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) from State of Sao Paulo, Brazil. Vet Parasitol. 2006;142(3–4):386–90.

    Article  CAS  PubMed  Google Scholar 

  30. Katoch R, Yadav A, Vohra S, Khajuria JK. Recent trends in herbal ectoparasitical drugs. Palampur, India: Communication of the Himachal Pradesh Agricultural University; 2006.

    Google Scholar 

  31. Kikuzaki H, Kawabata M, Ishida E, Akazawa Y, Takei Y, Nakatani N. LC-MS analysis and structural determination of new amides from javanese long pepper (Piper retrofractum). Biosci Biotechnol Biochem. 1993;57(8):1329–33.

    Article  CAS  Google Scholar 

  32. Lebouvier N, Hüe T, Brophy J, Hnawia E, Nour M. Chemical composition and acaricidal activity of Nemuaron vieillardii essential oil against the cattle tick Rhipicephalus (Boophilus) microplus. Nat Prod Commun. 2016;11(12):1919–22.

    PubMed  Google Scholar 

  33. Lebouvier N, Hüe T, Hnawia E, Lesaffre L, Menut C, Nour M. Acaricidal activity of essential oils from five endemic conifers of New Caledonia on the cattle tick Rhipicephalus (Boophilus) microplus. Parasitol Res. 2013;112(4):1379–84.

    Article  PubMed  Google Scholar 

  34. Lee HS. Pesticidal constituents derived from Piperaceae fruits. J Appl Biol Chem. 2005;48(2):65–74.

    CAS  Google Scholar 

  35. Lee KA, Harnett JE, Cairns R. Essential oil exposures in Australia: analysis of cases reported to the NSW poisons information centre. Med J Aust. 2020;212:132–3.

    Article  PubMed  Google Scholar 

  36. Le Ora Software. A userÕs guide to probit or logit analysis. Berkeley, CA: Le Ora Software; 1987.

    Google Scholar 

  37. Lew-Tabor AE, Rodriguez VM. A review of reverse vaccinology approaches for the development of vaccines against ticks and tick-borne diseases. Ticks Tick Borne Dis. 2016;7:573–85.

    Article  CAS  PubMed  Google Scholar 

  38. Lim CM, Ee GCL, Rahmani M, Bong CFJ. Alkaloids from Piper nigrum and Piper betle. Pertanika J Sci Technol. 2009;17(1):149–54.

    Google Scholar 

  39. Liu H-L, Luo R, Chen X-Q, Ba Y-Y, Zheng L, Guo W-W, Wu X. Identification and simultaneous quantification of five alkaloids in Piper longum L. by HPLC–ESI-MSn and UFLC–ESI-MS/MS and their application to Piper nigrum L. Food Chem. 2015;177:191–6.

    Article  CAS  PubMed  Google Scholar 

  40. Miyakado M, Nakayama I, Yoshioka H, Nakatani N. The Piperaceae Amides I: structure of Pipercide, a new insecticidal amide from Piper nigrum L. Agric Biol Chem. 1979;43(7):1609–11.

    Article  CAS  Google Scholar 

  41. Miyakado M, Nakayama I, Yoshioka H. Insecticidal joint action of Pipercide and co-occurring compounds isolated from Piper nigrum L. Agric Biol Chem. 1980;44(7):1701–3.

    Article  CAS  Google Scholar 

  42. Navickiene HMD, de Morandim AA, Alécio AC, Regasini LO, Bergamo DCB, Telascrea M, Cavalheiro AJ, Lopes MN, da Bolzani VS, Furlan M, Marques MOM, Young MCM, Kato MJ. Composition and antifungal activity of essential oils from Piper aduncum, Piper arboreum and Piper tuberculatum. Quim Nova. 2006;29(3):467–70.

    Article  CAS  Google Scholar 

  43. Patarroyo JH, Vargas MIV, González CZ, Guzmán F, Martins-Filho OA, Afonso LCC, Valente FL, Peconick AP, Marciano AP, Patarroyo AM, Sossai S. Immune response of bovines stimulated by synthetic vaccine SBm7462® against Rhipicephalus (Boophilus) microplus. Vet Parasitol. 2009;166(3–4):333–9.

    Article  CAS  PubMed  Google Scholar 

  44. Quadros DG, Johnson TL, Whitney TR, Oliver JD, Chávez ASO. Plant-derived natural compounds for tick pest control in livestock and wildlife: pragmatism or Utopia? Insects. 2020;11:490.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Ramesh B, Sarma VUM, Kumar K, Suresh BK, Sita DP. Simultaneous determination of six marker compounds in Piper nigrum L and species comparison study using high-performance thin-layer chromatography-mass spectrometry. J Planar Chromatogr. 2015;28(4):280–6.

    Article  CAS  Google Scholar 

  46. Reddy PS, Jamil K, Madhusudhan P, Anjani G, Das B. Antibacterial activity of isolates from Piper longum and Taxus baccata. Pharm Biol. 2001;39(3):236–8.

    Article  CAS  Google Scholar 

  47. Rodrigues AM, Martins VP, Morais SM. Larvicidal efficacy of plant extracts and isolated compounds from Annonaceae and Piperaceae against Aedes aegypti and Aedes albopictus. Asian Pac J Trop Med. 2020;13(9):384–96.

    Article  CAS  Google Scholar 

  48. Rodriguez-Vivas RI, Alonso-Díaz MA, Rodríguez-Arevalo F, Fragoso-Sanchez H, Santamaria VM, Rosario-Cruz R. Prevalence and potential risk factors for organophosphate and pyrethroid resistance in Boophilus microplus ticks on cattle ranches from the State of Yucatan, Mexico. Vet Parasitol. 2006;136(3–4):335–42.

    Article  CAS  PubMed  Google Scholar 

  49. Salehi B, Zakaria ZA, Gyawali R, Ibrahim SA, Rajkovic J, Shinwari ZK, Khan T, Sharifi-Rad J, Ozleyen A, Turkdonmez E, Valussi M, Tumer TB, Fidalgo LM, Martorell M, Setzer WN. Piper species: a comprehensive review on their phytochemistry, biological activities and applications. Molecules. 2019;24:1364.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Scott IM, Puniani E, Durst T, Phelps D, Merali S, Assabgui RA, Sánchez-Vindas P, Poveda L, Philogène JR, Arnason JT. Insecticidal activity of Piper tuberculatum Jacq. extracts: synergistic interaction of piperamides. Agric Forest Entomol. 2002;4(2):137–44.

    Article  Google Scholar 

  51. Scott IM, Puniani E, Jensen H, Livesey JF, Poveda L, Sánchez-Vindas P, Durst T, Arnason JT. Analysis of Piperaceae germplast by HPLC and LCMS: a method for isolating and identifying unsaturated amides from Piper spp extracts. J Agric Food Chem. 2005;53(6):1907–13.

    Article  CAS  PubMed  Google Scholar 

  52. Scott IM, Jensen HR, Philogène BJ, Arnason JT. A review of Piper spp. (Piperaceae) phytochemistry, insecticidal activity and mode of action. Phytochem Rev. 2008;7(1):65–75.

    Article  CAS  Google Scholar 

  53. Seo S-M, Shin J, Lee J-W, Hyun J, Park I-K. Larvicidal activities of Piper kadsura (Choisy) Ohwi extract and its constituents against Aedes albopictus, toxicity to non-target organisms and development of cellulose nanocrystal-stabilized Pickering emulsion. Ind Crops Prod. 2021;162:113270.

    Article  CAS  Google Scholar 

  54. Shi YN, Liu FF, Jacob MR, Li XC, Zhu HT, Wang D, Cheng RR, Yang CR, Xu M, Zhang YJ. Antifungal amide alkaloids from the aerial parts of Piper flaviflorum and Piper sarmentosum. Planta Med. 2017;83(1–02):143–50.

    Article  CAS  PubMed  Google Scholar 

  55. Siddiqui BS, Begum S, Gulzar T, Noor F. An amide from fruits of Piper nigrum. Phytochemistry. 1997;45(8):1617–9.

    Article  CAS  Google Scholar 

  56. Siddiqui BS, Gulzar T, Begum S, Rasheed M, Sattar FA, Afshan F. Two new insecticidal amides and a new alcoholic amide from Piper nigrum Linn. Helv Chim Acta. 2003;86(8):2760–7.

    Article  CAS  Google Scholar 

  57. Siddiqui BS, Gulzar T, Begum S, Afshan F. Piptigrine, a new insecticidal amide from Piper nigrum Linn. Nat Prod Res. 2004;18(5):473–7.

    Article  CAS  PubMed  Google Scholar 

  58. Siddiqui BS, Gulzar T, Begum S, Afshan F, Sattar FA. Insecticidal amides from fruits of Piper nigrum Linn. Nat Prod Res. 2005;19(2):143–50.

    Article  CAS  PubMed  Google Scholar 

  59. Siddiqui BS, Gulzar T, Mahmood A, Begum S, Khan B, Rasheed M, Afshan F, Tariq RM. Phytochemical studies on the seed extract of Piper nigrum Linn. Nat Prod Res. 2005;19(7):703–12.

    Article  CAS  PubMed  Google Scholar 

  60. Siddiqui BS, Gulzar T, Begum S, Afshan F, Sultana R. A new natural product and insecticidal amides from seeds of Piper nigrum Linn. Nat Prod Res. 2008;22(13):1107–11.

    Article  CAS  PubMed  Google Scholar 

  61. Silva DR, Endo EH, Filho BPD, Nakamura CV, Svidzinski TIE, de Souza A, Young MCM, Ueda-Nakamura T, Cortez DAG. Chemical composition and antimicrobial properties of Piper ovatum Vahl. Molecules. 2009;14(3):1171–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Silva WC, de Souza Martins JR, de Souza HEM, Heinzen H, Cesio MV, Mato M, Albrecht F, de Azevedo JL, de Barros NM. Toxicity of Piper aduncum L (Piperales: Piperaceae) from the Amazon Forest for the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Vet Parasitol. 2009;164(2):267–74.

    Article  PubMed  Google Scholar 

  63. Stöhr JR, Xiao P-G, Bauer R. Isobutylamides and a new Methylbutylamide from Piper sarmentosum. Planta Med. 1999;65(2):175–7.

    Article  PubMed  Google Scholar 

  64. Stone BF, Haydock KP. A method for measuring the acaricide susceptibility of the cattle tick Boophilus microplus (Can.). Bull Entomol Res. 1962;53(3):563–78.

    Article  CAS  Google Scholar 

  65. Vinturelle R, Mattos C, Meloni J, Lamberti HD, Nogueira J, da Júnior ISV, Rocha L, Lione V, Folly E. Evaluation of essential oils as an ecological alternative in the search for control Rhipicephalus microplus (Acari: Ixodidae). Vet Parasitol Reg Stud Rep. 2021;23:100523.

    Article  Google Scholar 

  66. Yu L, Hu X, Xu R, Ba Y, Chen X, Wang X, Cao B, Wu X. Amide alkaloids characterization and neuroprotective properties of Piper nigrum L.: a comparative study with fruits, pericarp, stalks and leaves. Food Chem. 2022;368:130832.

    Article  CAS  PubMed  Google Scholar 

  67. Zahin M, Bokhari NA, Ahmad I, Husain FM, Althubiani AS, Alruways MW, Perveen K, Shalawi M. Antioxidant, antibacterial, and antimutagenic activity of Piper nigrum seeds extracts. Saudi J Biol Sci. 2021;28:5094–105.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references


The authors also wish to thank Dr Vincent Dumontet (Centre National de Recherche Scientifique, CNRS-UPR2301) for all his advice concerning the interpretation of 1H and 13C NMR analysis. The authors also would like to thank Anowo Ltd. for providing nylon paper.


This study was carried out with funding from the IAC, a public institute and in collaboration with Université Paris-Saclay, CNRS, BioCIS, 91400, Orsay, France.

Author information

Authors and Affiliations



MT analyzed and interpreted all the data, carried out the bibliographical research, wrote the draft, performed the HRMS, 1H and 13C NMR interpretation and compounds identification. Writing—review and editing. PC performed the plants collection, extractions and chromatographic analyses and bioguided fractionations for compounds isolation. TH performed the acaricidal activities, as determined by the FAO modified method (LPT) and the statistical analysis using Poloplus® program and participated in the writing of the paper. AM performed the HRMS, 1H and 13C NMR analysis for compounds identification and reviewed the draft. VK supervised the work as team leader and participated in the bibliographic research as well as the writing of the paper. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Valérie Kagy.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Toussirot, M., Coulerie, P., Hüe, T. et al. Larvicidal activity of the black pepper, Piper nigrum (Fam: Piperaceae) extracts on the cattle tick, Rhipicephalus australis (Acari: Ixodidae). Chem. Biol. Technol. Agric. 10, 23 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: