Southeast Asian J Trop Med Public Health

INSECTICIDAL ACTIVITY OF THAI BOTANICAL EXTRACTS AGAINST DEVELOPMENT STAGES OF GERMAN COCKROACH, BLATTELLA GERMANICA (L.) (ORTHOPTERA: BLATTELLIDAE)

Soraya Saenmanot1, Ammorn Insung2, Jarongsak Pumnuan2, Apiwat Tawatsin3 Usavadee Thavara3, Atchara Phumee4, Fréderick Gay5, Wanpen Tachaboonyakiat6 and Padet Siriyasatien7

1Medical Science Program, Faculty of Medicine, Chulalongkorn University, Bangkok; 2Department of Pest Management Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Lat Krabang, Bangkok; 3National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi; 4Thai Red Cross Emerging Infectious Health Science Centre, King Chulalongkorn Memorial Hospital, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; 5Université Pierre et Marie Curie-Paris 6, CHU Pitié-Salpêtrière, AP-HP, Groupe Hospitalier Pitié-Salpêtrière, Service Parasitologie-Mycologie, Paris, France; 6Department of Materials Science, Faculty of Science, 7Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

Abstract. The German cockroach, Blattella germanica, is considered an important medical and economic pest in Thailand. The insecticidal activities of hexane, acetone and ethanol extracts derived from six Thai botanicals, namely, Piper ret- rofractum Vahl, Stemona tuberosa Lour, Derris elliptica (Wall.) Benth., Rhinacanthus nasutus (L.), Butea superba Roxb., and Foeniculum vulgare Mill. were used to evaluate their toxicities against a variety of developmental stages of B. germanica including nymphs, adults and gravid females. All botanical extracts showed high insecticidal activity (up to 100%) after a 24-hour exposure at 200 µg/insect, using 2 µl/insect 1% Tween-20 in distilled water as negative control. Extracts from P. retrofractum gave the highest mortality to all B. germanica developmental stages, with the acetone µ extract showing maximal insecticidal activity against both gravid (LD50 = 39 g/ µ µ µ insect; LD90 = 52 g/insect) and adult (LD50 = 53 g/insect; LD90 = 104 g/insect) stages after a 6-hour exposure. Isolation and purification of the main components of P. retrofractum acetone extract by column chromatography led to a fraction with highest insecticidal activity against nymphs. Subsequent purification by thin layer chromatography led to identification of as the major bioactive compound. This is the first report of insecticidal activity fromP. retrofractum extracts against B. germanica and holds promise as a potential biopesticide. Keywords: Blattella germanica, botanical extract, German cockroach, insecticidal activity

Correspondence: Padet Siriyasatien, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, 10330, Thailand. E-mail: [email protected]

46 Vol 49 No. 1 January 2018 Thai Botanical Extracts against Blattella germanica

INTRODUCTION are more environmental friendly and less toxic (Kamaraj et al, 2009; Koodalingam Cockroach is considered an important et al, 2009; Elango et al, 2010). Moreover, pest of concern to medical and public several Thai botanicals have long been health. Cockroaches are commonly found employed as insecticides or repellents in homes, hospitals, hotels and restaurants for pest and insect control (Promsiri et al, (Yeom et al, 2012). The insect is used as 2006; Tawatsin et al, 2006; Phasomkusolsil a key indicator of insanitary conditions and Soonwera, 2010). For example, Chang as they are able to carry various human and Ahn (2002) reported that methanol pathogens, including bacteria, viruses and extract of fruit from has protozoa, moreover, they contribute to the insecticidal activity against adults of B. cause of allergies and asthma (Slater et al, germanica when used as a fumigation 2007; Mindykowski et al, 2010). agent. Thavara et al (2007) demonstrated In Thailand, more than ten species of that essential oils of Citrus hystrix (Kaffir cockroaches have been described, includ- lime) exhibit complete repellency against ing the American cockroach (Periplaneta P. americana and B. germanica and also americana), Australian cockroach (P. aus- show a high repellency (87.5%) against tralasiae) and German cockroach (Blattella Neostylopyga rhombifolia under laboratory germanica) (Tawatsin et al, 2001; Sriwichai conditions. Ling et al (2009) stated that et al, 2002; Chompoosri et al, 2004). How- essential oils of Piper aduncum (spike pep- ever, B. germanica is considered as an per) can have insecticidal activity against important medical and economic pest adults and nymphs stage of P. americana. because it has a shorter generation time Tine et al (2011) revealed that azadirachtin and higher fecundity than the other cock- from kernel of neem tree (Azadirachta roaches, which makes it difficult to con- indica) has an insecticidal effect against trol. Conventional synthetic insecticides, newly emerged adults of Blatta orientalis such as pyrethroids, carbamates and or- under laboratory conditions. According to ganophosphates are very popularly used Sittichok et al (2013) essential oils derived compounds for controlling cockroaches from Thai citatus (lem- (Lee et al, 1996; Syed et al, 2014). However, on grass), C. nardus (citronella grass) and these insecticides have several adverse ef- Syzygium aromaticum () had highly fects including acute or chronic toxicity to repellent effects on adult P. americana. humans and animals. They can also have Thus Thai botanicals contain insecti- an adverse impact on the food chain of cidal or repellent substances and, impor- the ecosystem (Govindarajan et al, 2011; tantly, there has been no report, to the Amerasan et al, 2012; Muthukumaran best of our knowledge, of resistance by et al, 2015). Furthermore, cockroaches can pests and insects against Thai botanicals, develop resistance to these insecticides highlighting the attractiveness of Thai bo- (Pai et al, 2005). Due to these problems, tanicals as alternative substances for con- an alternative approach is to search for trolling German cockroaches. Hence, the effective botanical extracts, which are safe present study determined the insecticidal to humans and the environment. activity of organic solvent extracts derived Thailand has an abundance of plants, from six Thai botanicals, namely, Butea su- extracts of which are used in many ap- perba Roxb. (red ‘kwao krua’), Derris elliptica plications in place of chemicals as they (Wall.) Benth. (Derris), Foeniculum vulgare

Vol 49 No. 1 January 2018 47 Southeast Asian J Trop Med Public Health

Mill. (), Piper retrofractum Vahl (Java prepared for a topical application bioassay ), Rhinacanthus nasutus (L.) test (Rahman and Akter, 2006). (white crane flower), and Stemona tuberosa Cockroach Lour. (wild asparagus), against various B. germanica was obtained from the developmental stages of B. germanica in- Biology and Ecology Section, Medical cluding nymph, adult and gravid stages Entomology Group, National Institute of under laboratory conditions. In addition, the Health, Department of Medical Sci- the main component of Thai botanical ence, Ministry of Public Health, Thailand extracts with highest insecticidal effect and reared according to standard protocol against B. germanica was identified. (Thavara et al, 2007). Cockroaches were maintained in the insectary of the Labora- MATERIALS AND METHODS tory Animal Center, Faculty of Medicine, Chulalongkorn University, Bangkok un- Botanical material der ambient temperature (27-30ºC) and Thriving roots of B. superba, D. ellip- humidity (70-90%) and photoperiod of tica, R. nasutus and S. tuberosa, fruits of P. 12:12 hour light:dark cycle. Cockroaches retrofractum and seeds of F. vulgare (1 kg were fed dry dog food and water ad li- each) were purchased from a traditional bitum. The upper inside surfaces of the herbal medicine store, Bangkok, Thailand rearing container were smeared with pe- (Table 1). The plants were identified at troleum jelly to prevent cockroaches from the Production Technology Section, escaping (Habes et al, 2006). B. germanica Faculty of Agricultural Technology, King final instar nymphs (3-4 weeks old), adult Mongkut’s Institute of Technology Lat males, non-gravid females (6-8 weeks Krabang, Bangkok, Thailand. old) and gravid females (with egg case) Preparation of botanical extract were employed for toxicity testing under The botanical materials were washed laboratory conditions. thoroughly with distilled water and cut Topical application bioassay into pieces (<2 cm in size), then left to A group of 10 each of unsexed nymph, dry in the shade and ground into fine gravid female and adult cockroaches (five powder using an electric blender. The males and five non-gravid females) were fine powders were extracted using a se- used in each experiment. Cockroaches quential extraction procedure with 1,000 were anesthetized briefly (<5 minutes) ml aliquots of three different solvents with carbon dioxide to facilitate handling with increasing polarity, namely, hexane, (Tunaz et al, 2009; Liu et al, 2011). Each bo- acetone and ethanol, by maceration at tanical extract of different concentrations room temperature (30 ± 2ºC) for three (2 µl/insect) were topically applied with a days with frequent agitation. The solvents micro applicator (Burkard Manufacturing, were removed by evaporation (AhbiRami Rickmansworth, United Kingdom) onto et al, 2014; Govindarajan and Sivakumar, the first abdominal segment of each cock- 2015). The extracts were weighed, dried roach (Ferrero et al, 2007). Cockroaches in at room temperature (30 ± 2ºC) and stored the negative control group were treated in brown bottles at 4ºC until used. Ex- with vehicle (2 µl/insect). All cockroaches tracts (40, 80, 120, 160 and 200 µg/µl 1% were kept under the same conditions and Tween-20 in distilled water/insect) were mortality was assessed at 1, 2, 4, 6, 24 and

48 Vol 49 No. 1 January 2018 Thai Botanical Extracts against Blattella germanica

Table 1 Thai botanicals used in the study.

Scientific name Common name Thai name Family Part used

Piper retrofractum Vahl. Java long pepper Di-pli Fruit Stemona tuberosa Lour. Wild asparagus, stemona Non-tai-yak Stemonaceae Root Derris elliptica (Wall.) Benth. Derris, tuba root Hang-lai-deang Fabaceae Root Rhinacanthus nasutus (L.) White crane flower Thong-phan-chang Acanthaceae Root Butea superba Roxb. Red ‘kwao krua’ Kwao-khrua-deang Papilionaceae Root Foeniculum vulgare Mill. Fennel Tian-kao-pleuak Umbelliferae Seed

48 hours post-treatment. Each experiment (LCL) confidence limits using SPSS ver- was carried out in quadruplicate. Death sion 23 software (IBM, Armonk, NY). was determined based on the following Results with p-value <0.05 are considered criteria: (i) inability to move when prod- statistically significant. ded with forceps, and (ii) inability to right to a normal posture within two minutes RESULTS (Tungtrongchitr et al, 2012). Isolation and purification of bioactive Yield of botanical extracts component One kg of each botanical produced Extract showing highest insecticidal the following yields: acetone, ethanol and effect againstB. germanica was subjected to hexane extract of B. superba of 0.63%, 1.74% purification by column chromatography and 0.27%, respectively; D. elliptica of 0.74%, and 10 ml aliquot of each fraction was 2.22% and 0.13%, respectively; F. vulgare analyzed by thin layer chromatography of 3.52%, 4.98% and 3.31%, respectively; (TLC) (Dua et al, 2009; Deng et al, 2012; P. retrofractum of 2.86%, 3.15% and 1.18%, Kongkiatpaiboon and Gritsanapan, respectively; R. nasutus of 0.70%, 1.18% 2012). Fractions with the same TLC pro- and 0.19%, respectively; and S. tuberosa of file were combined and tested against B. 0.88%, 4.73% and 0.29%, respectively. Due to germanica. The fraction with the highest their low yields, the hexane extracts, except efficacy againstB. germanica nymphs was those of F. vulgare and P. retrofractum, were analyzed further by two-dimensional TLC not selected for insecticidal activity tests on using hexane per ethyl acetate (40: 60 v/v) B. germanica. solvent system and Dragendorff’s reagent Insecticidal activity of botanical extracts (Kolhe et al, 2011) to identify the main against B. germanica bioactive compound. Among the extracts of the six botani- Statistical analysis cals, P. retrofractum extracts showed the Percent mortality data were subjected highest mortality when compared to other to probit analysis for calculating LD50, extracts of the other five botanicals at ev- LD90, and other statistics at 95% confi- ery experimental time points for gravid dence limit of upper (UCL) and lower (Fig 1A), adult (Fig 1B) and nymph (Fig

Vol 49 No. 1 January 2018 49 Southeast Asian J Trop Med Public Health

Fig 1-Histogram showing the percentage mortality of Thai botanical extract at concentration 200 µg/ insect on gravid (A), adult (B) and nymph (C) stage of B. germanica after 6 hours post-exposure.

1C) stages, or at the highest concentration (Table 5) and among the six botanicals, (200 µg/insect) at 6 hours post-exposure P. retrofractum acetone extract exhibited with extracts from B. superba, F. vulgare highest efficacy against the nymph stage and R. nasutus being the least effective followed by ethanol and hexane extract, µ (Tables 2-4). The acetone, ethanol and (LD50 = 69.3, 89.1 and 91.4 g/insect; LD90 hexane extract of P. retrofractum showed = 129.7, 181.3 and 174.3 µg/insect, respec- 100% lethality against gravid stage at 6 tively) (Fig 2).

hours post-exposure (LD50 = 39.4, 41.2 µ Identification of the major bioactive com- and 42.5 g/insect; LD90 = 51.9, 53.9 and 55.1 µg/insect, respectively). With adult ponent stage, only P. retrofractum acetone extract As the acetone extract from P. retro- showed 100% lethality at 6 hours post- fractum had the highest efficacy against µ exposure (LD50 = 53.2 g/insect; LD90 = all various developmental stages of B. 103.7 µg/insect), followed by ethanol and germanica, this extract was subjected to µ hexane extract (LD50 = 62.4 and 80.1 g/ column chromatography and each frac- µ insect; LD90 = 131.9 and 159.7 g/insect, tion subsequently was tested against the respectively). However, all extracts of nymph stage of B. germanica. The results the six botanicals were unable to produce found that, fractionation of extract of P. 100% killing of B. germanica nymph stage retrofractum by using silica gel column

50 Vol 49 No. 1 January 2018 Thai Botanical Extracts against Blattella germanica

Table 2 Insecticidal activity of organic solvent extracts from Thai botanicals against gravid stage of Blattella germanica at 6 hours post-exposure.

Thai botanical Extraction Vehiclea Percent mortality ± SD solvent Concentration (µg/insect)

40 80 120 160 200

Piper retrofractum Hexane 0 40.0 ± 0.8 100 100 100 100 Acetone 0 52.0 ± 1.0 100 100 100 100 Ethanol 0 45.0 ± 0.6 100 100 100 100 Derris elliptica Acetone 0 27.5 ± 0.5 47.0 ± 1.0 50.0 ± 0.8 52.0 ± 1.0 75.0 ± 1.0 Ethanol 0 27.5 ± 0.5 47.0 ± 1.0 50.0 ± 0.8 55.0 ± 0.6 77.5 ± 0.5 Stemona tuberosa Acetone 0 20.0 ± 0.8 45.0 ± 0.6 47.5 ± 0.5 50.0 ± 0.8 65.0 ± 1.0 Ethanol 0 27.0 ± 1.0 47.5 ± 0.5 50.0 ± 0.8 55.0 ± 0.6 70.0 ± 0.8 Foeniculum vulgare Hexane 0 12.5 ± 0.5 10.0 ± 0.8 12.5 ± 0.5 17.0 ± 1.0 30.0 ± 0.8 Acetone 0 5.0 ± 0.6 15.0 ± 0.6 22.0 ± 1.0 47.0 ± 1.0 47.5 ± 0.5 Ethanol 0 5.0 ± 0.6 7.0 ± 1.0 27.0 ± 1.0 47.0 ± 1.0 50.0 ± 0.8 Rhinacanthus nasutus Acetone 0 5.0 ± 0.6 7.0 ± 1.0 10.0 ± 0.8 30.0 ± 0.8 40.0 ± 0.8 Ethanol 0 2.5 ± 0.5 5.0 ± 0.6 12.5 ± 0.5 35.0 ± 0.6 47.5 ± 0.5 Butea superba Acetone 0 7.5 ± 0.5 10.0 ± 0.8 12.0 ± 1.0 45.0 ± 0.6 47.5 ± 0.5 Ethanol 0 2.5 ± 0.5 5.0 ± 0.6 10.0 ± 0.8 42.0 ± 1.0 47.5 ± 0.5 a1% Tween-20 in distilled water.

Table 3 Insecticidal activity of organic solvent extracts from Thai botanicals against adult stage of Blattella germanica at 6 hours post-exposure.

Thai botanical Extraction Vehiclea Percent mortality ± SD solvent Concentration (µg/insect)

40 80 120 160 200

Piper retrofractum Hexane 0 40.0±0.8 45.0±1.0 80.0±0.8 92.5±0.5 92.5±0.5 Acetone 0 50.0±0.8 77.5±0.5 95.0±0.6 97.5±0.5 100 Ethanol 0 45.0±1.0 75.0±1.0 83.0±1.0 95.0±0.6 97.5±0.5 Derris elliptica Acetone 0 5.0±0.6 22.0±1.0 27.5±0.5 48.0±1.0 53.0±1.0 Ethanol 0 15.0±1.0 25.0±1.0 32.5±0.5 45.0±0.6 60.0±1.0 Stemona tuberosa Acetone 0 2.5±0.5 22.5±0.5 25.0±0.6 43.0±1.0 52.5±0.5 Ethanol 0 7.5±0.5 25.0±0.6 27.5±0.5 45.0±0.6 57.5±0.5 Foeniculum vulgare Hexane 0 0 0 2.5±0.5 3.3±0.6 12.5±0.5 Acetone 0 0 0 2.5±0.5 12.5±0.5 17.5±0.5 Ethanol 0 0 0 5.0±0.6 10.0±0.0 15.0±1.0 Rhinacanthus nasutus Acetone 0 0 0 2.5±0.5 10.0±0.8 25.0±0.6 Ethanol 0 0 0 2.5±0.5 8.0±1.0 27.5±0.5 Butea superba Acetone 0 0 0 2.5±0.5 15.0±1.0 25.0±0.6 Ethanol 0 0 0 2.5±0.5 8.0±1.0 23.0±1.0 a1% Tween-20 in distilled water.

Vol 49 No. 1 January 2018 51 Southeast Asian J Trop Med Public Health

Table 4 Insecticidal activity of organic solvent extracts from Thai botanicals against nymph stage of Blattella germanica at 6 hours post-exposure.

Thai botanical Extraction Vehiclea Percent mortality ± SD solvent Concentration (µg/insect)

40 80 120 160 200

Piper retrofractum Hexane 0 40.0±0.8 43.0±1.0 48.0±1.0 95.0±1.0 95.0±0.6 Acetone 0 43.0±1.0 45.0±0.6 95.0±0.6 97.5±0.5 97.5±0.5 Ethanol 0 45.0±0.6 48.0±1.0 48.0±0.6 93.0±1.0 93.0±1.0 Derris elliptica Acetone 0 15.0±0.0 20.0±0.0 22.5±0.5 32.5±0.5 45.0±1.0 Ethanol 0 28.0±1.0 30.0±0.8 38.0±1.0 45.0±0.6 45.0±1.0 Stemona tuberosa Acetone 0 10.0±0.8 18.0±1.0 20.0±0.8 48.0±1.0 52.5±0.5 Ethanol 0 10.0±0.5 12.5±0.5 28.0±1.0 48.0±1.0 47.5±0.5 Foeniculum vulgare Hexane 0 2.5±0.5 5.0±0.6 8.0±1.0 10.0±0.8 15.0±0.6 Acetone 0 0 2.5±0.5 5.0±0.6 10.0±0.0 20.0±0.8 Ethanol 0 0 2.5±0.5 5.0±0.6 15.0±1.0 15.0±0.6 Rhinacanthus nasutus Acetone 0 2.5±0.5 5.0±0.6 8.0±1.0 10.0±0.8 15.0±0.6 Ethanol 0 0 2.5±0.5 5.0±0.6 10.0±0.8 13.0±1.0 Butea superba Acetone 0 0 2.5±0.5 5.0±0.6 15.0±0.6 12.5±0.5 Ethanol 0 0 5.0±0.6 8.0±1.0 13.0±1.0 25.0±1.0 a1% Tween-20 in distilled water. chromatography led to separation of 14 P. retrofractum have been used bioinsecti- fractions (F1-F14), with F5 having the cides against various insects. For example, highest lethality against the nymph stage, Chansang et al (2005) reported larvicidal with 100% mortality achieved at 200 µg/ activity of nine Thai botanical aqueous insect at 4 hours post-exposure (Table 6). F5 extracts including those from Allium was further purified by one-dimensional sativum, Cleome viscosa, Curcuma longa, followed by two-dimensional TLC, which Gynura pseudochina, Spilanthes paniculata, resulted in the identification of piperine, a P. retrofractum, Rhinacanthus nasutus, Syz- major alkaloid in the fruit of P. retrofractum, pgium aromaticum, and Zingiber monta- as the main bioactive compound (Fig 3). num. They also found that P. retrofractum extract has the highest larvicidal activity rd th DISCUSSION against 3 and 4 instar larvae of Culex quinquefasciatus and Aedes aegypti with

This present study indicates that LD50 value of 135 mg/l and LD90 value of organic solvent extracts of six Thai 79 mg/l. Kraikrathok et al (2013), evaluat- botanicals had toxic effects on various ing extracts of P. interruptum, P. nigrum, P. developmental stages of B. germanica. retrofractum, and P. sarmentosum against Among the six Thai botanicals, extracts diamondback moth, Putella xylostella third of P. retrofractum proved to be the most instars, reported extract of P. retrofractum insecticidal activity against B. germanica. having the highest larvicidal activity with

Several reports have demonstrated that an LD50 value of 237 ppm. In the study of

52 Vol 49 No. 1 January 2018 Thai Botanical Extracts against Blattella germanica

Parmar et al (1997) and Taufiq-Ur-Rahman et al (2005) reported the principle active con- stituent of the fruit of P. retrofractum is pip- erine. Bao et al (2014) explored piperidine alkaloids in family Piperaceae collected in different areas and reported piperine as the major component of dried fruits of P. longum, P. nigrum and Fig 2-Graph showing the LD50 and LD90 value of P. retrofractum extract P. retrofractum. Faba- on various developmental stage of B. germanica after 6 hours post rani (2012) reported exposure. crude methanol ex- tract of P. retrofractum fruit contains methyl piperate, N-isobutyl- 2E, 4E, 14Z-elcosa- trienamide piperine, and oleic acid. Piperine is dark yellow in color, aro- matic and pungent in odor, and has a biting taste. It is soluble in chloroform, ethanol and methanol, but in- soluble in water. Its Fig 3-Two dimension development thin layer chromatography (TLC) of (a.) piperine analytical standard and (b.) fraction 5 from acetone Rf value is 0.33-0.55 extract of P. retrofractum. demonstrating good separation by TLC (Banerji et al, 2002; Ra- P. retrofractum toxicity on mammals by man and Galkar, 2002; Kolhe et al, 2011). Jaijoy et al (2010), aqueous extract of the Previous studies have suggested piperine fruits do not produce acute or subchronic as a potential bioinsecticide. For example, toxicity in either female or male rats at Samuel et al (2016) demonstrated that P. dose of 5,000 mg/kg body weight for acute nigrum and piperine cause mortality to toxicity and 300-1,200 mg/kg body weight third and fourth instars of An. arabiensis, for subchronic toxicity. An. coluzzii, An. funestus, An. gambiae, The presence of piperrine in P. ret- and An. quadriannulatus; in addition, a rofractum has been reported by others. mixture of P. nigrum and piperine causes

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Table 5

Fifty (LD50) and 90 (LD90) percent lethal dose of Piper retrofractum organic solvent extracts against nymph, adult and gravid stages of Blattella germanica at 6 hours post- exposure.

Stage Extraction solvent LD50 (LCL-UCL) LD90 (LCL-UCL) (µg/insect) (µg/insect)

Nymph Hexane 91.4 (30.3-146.2) 174.3 (127.5-388.9) Acetone 69.3 (16.3-111.5) 129.7 (93.8-272.5) Ethanol 89.1 (12.6-150.3) 181.3 (129.6-468.1) Adult Hexane 80.1 (37.7-114.5) 159.7 (123.1-259.1) Acetone 53.2 (20.0-79.4) 103.7 (77.9-175.5) Ethanol 62.4 (18.2-93.9) 131.9 (99.2-221.3) Gravida Hexane 42.5 55.1 Acetone 39.4 51.9 Ethanol 41.2 53.9 a Due to high frequency of complete mortality in gravid stage, LD50 and LD90 values are estimation by probit analysis. LCL, lower confidence limit; UCL upper confidence limit.

high mortality of An. gambiae. Freire-de- rotoxic compounds such as carbamates, Lima et al (2008) reported piperine caused organophosphates and pyrethroids to morphological alteration of Trypanosoma reduce insect resistance to these chemical cruzi epimastigotes. Piperine has insec- insecticide, and to decrease environmental ticidal activity to eggs of Spodoptera fru- exposure to humans and animals (Jensen giperda and Diatraea saccharalis (Tavares et al, 2006; Scott et al, 2008). Based on the et al, 2011), and to Musca domestica and aforementioned, piperine from fruit of P. promastigotes of Leishmania donovani both retrofractum might be the main toxic agent in vitro and in vivo (Kapil, 1993; Raay et al, against the various developmental stages 1999; Veerareddy et al, 2004; Bodiwala et al, of B. germanica. 2007). Three Piper amines isolated from In conclusion, this study clearly P. nigrum, namely, pellitorine, pipercid shows that organic solvent extracts of P. and piperine, show toxicity to male Cal- retrofractum and their crude fraction have losobruchus chinensis ranging from 0.15 to highest insecticidal activity against B. 20 µg/insect (Dev and Koul, 1999). germanica compared to extracts from the Piper amines including dihydro- other five Thai botanicals. The active com- piperine, dihydropiperlonguminine, pound was identified as piperine from the pipercide, piperine, piperlonguminine fruit. Thus, extracts of P. retrofractum have of family Piperaceae are known to act as the potential to be used as bioinsecticides neurotoxins in insects. The mode of ac- that are inexpensive and safe to humans tion in insect is as a contact poison acting and the environment for the controlling on the central nervous system (de Paula of B. germanica instead of current chemi- et al, 2000). Therefore, Piper amines of cal insecticides in areas where the latter family Piperaceae could replace contact insecticides are not appropriate, and insecticides, specifically chemical neu- should be integrated into pest manage-

54 Vol 49 No. 1 January 2018 Thai Botanical Extracts against Blattella germanica 0 0 0 100 200 5.0±0.6 2.5±0.5 2.5±0.5 73.0±1.0 92.5±0.5 85.0±0.6 55.0±0.6 55.0±0.6 55.0±0.5 90.0±1.0 75.0±0.6 48 hours 0 0 0 0 40 0.2±0.5 5.5±0.0 0.2±0.5 2.0±0.8 200 2.5±0.5 73.0±1.0 2.0±0.8 85.0±0.6 4.0±0.8 80.0±0.8 2.5±0.6 45.0±0.6 1.2±0.5 45.0±0.6 1.2±0.5 45.0±0.5 1.2±0.5 88.0±1.0 2.2±0.5 63.0±1.0 acetone extract fractions from from fractions extract acetone 24 hours 0 2.5±0.5 0 2.5±0.5 0 0 0 0 40 5.0±0.0 100 1.7±0.5 200 2.5±0.5 73.0±1.0 1.2±0.5 73.0±1.0 3.3±1.0 73.0±1.0 2.2±0.5 35.0±1.0 0.5±0.6 35.0±0.6 0.5±0.6 35.0±0.5 0.5±0.5 43.0±1.0 73.0±1.0 1.7±0.5 Piper retrofractum Piper 6 hours 0 2.5±0.5 0 2.5±0.5 0 0 0 0 40 3.5±0.0 100 1.7±1.0 200 62.5±0.5 0.5±0.6 68.0±1.0 1.7±0.5 68.0±1.0 0.7±1.0 25.0±0.6 0.2±0.5 25.0±0.5 0.2±0.6 25.0±0.6 0.2±0.5 25.0±0.6 62.5±0.5 1.5±0.6 Table 6 Table 4 hours Percent mortality ± SD Percent 0 40 3.0±1.0 100 1.3±1.0 0.5±0.6 0.2±0.5 0.2±0.5 0.2±0.6 1.3±1.0 1.0±0.8 column chromatography. column

0 0 0 0 200 2.5±0.5 0 2.5±0.5 0 55.0±0.6 97.5±0.0 20.0±0.8 nymph stage following exposure to to exposure following stage nymph 2 hours 0 0 0 0 0 0 0 40 2.0±0.8 1.3±1.0 germanica

200 5.0±0.6 40.0±0.8 40.0±0.8 0.5±0.6 55.0±0.6 40.0±0.8 0.5±0.6 55.0±0.6 88.0±1.0 10.0±0.8 0.2±0.5 15.0±0.6 10.0±0.5 0.2±0.5 15.0±0.5 10.0±0.5 0.2±0.5 15.0±0.5 40.0±0.8 0.8±1.0 48.0±1.0

Blattella 1 hour

0 0 0 0 0 2.5±0.5 0 2.5±0.5 0 0 0 40 0.2±0.5 0.8±1.0 0.5±0.6 0.0±0.5 0.0±0.5 0.0±0.5 insect) /

g a µ 1% Tween-20 in distilled water. in distilled water. 1% Tween-20 Percent mortality of of mortality Percent F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 Vehicle a Fraction Concentration (

Vol 49 No. 1 January 2018 55 Southeast Asian J Trop Med Public Health ment programs against B. germinica. This Piper species collected in different areas.J is the first report of insecticidal activity Nat Med 2014; 68: 211-4. of P. retrofractum extracts against Ger- Bodiwala HS, Singh R, Dey CS, Sharma SS, man cockroach, B. germanica. However, Bhutani KK. Antileishmanial amides and insecticidal activity of the extracts against lignans from and Piper retro- B. germanica in field environment and fractum. J Nat Med 2007; 61: 418-2. appropriate formulations of this extracts Chang KS, Ahn YJ. Fumigant activity of (E)- need to be determined. anethole identified in Illicium verum fruit against Blattella germanica. Pest Manag Sci ACKNOWLEDGEMENTS 2002; 58: 161-6. Chansang U, Zahiri NS, Bansiddhi J, et al. The study was supported by the Ratch- Mosquito larvicidal activity of aqueous adapiseksompotch Fund (RA59/050), the extracts of long pepper (Piper retrofractum Faculty of Medicine, Chulalongkorn Vahl.) from Thailand. J Vector Ecol 2005; University, the National Science and Tech- 30: 195-200. nology Development Agency (Thailand) Chompoosri J, Thavara U, Tawatsin A, Sathan- Research Chair Grant, and the Integrated triphop S, Yi T. Cockroach surveys in the Innovation Academic Center (IIAC) northern region of Thailand and Guangxi Chulalongkorn University Centenary Province of China. Southeast Asian J Trop Academic Development Project. Med Public Health 2004; 35: 46-9. de Paula VF, Barbosa LCA, Demuner A, Piló- CONFLICTS OF INTEREST Veloso D, Picanço MC. Synthesis and insecticidal activity of new amide deriva- The authors declare no conflicts of tives of piperine. Pest Manag Sci 2000; 56: interest. 168-74. Deng Y, Shi D, Yin Z, et al. Acaricidal activity REFERENCES of petroleum ether extract of neem (Aza- Amerasan D, Murugan K, Kovendan K, et al. dirachta indica) oil and its four fractions Adulticidal and repellent properties of separated by column chromatography indigenous plant extracts against Culex against Sarcoptes scabiei var. cuniculi larvae quinquefasciatus and Aedes aegypti (Diptera: in vitro. Exp Parasitol 2012; 130: 475-7. Culicidae). Parasitol Res 2012; 111: 1953-64. Dev S, Koul O. Insecticides of natural origin. AhbiRami R, Zuharah WF, Thiagaletchumi Hardwood Academic Publishers, 1999; M, Subramaniam S, Sundarasekar J. Lar- 55: 756-7. vicidal efficacy of different plant parts of Dua YH, Jia RY, Yin ZQ, et al. Acaricidal activity railway creeper, Ipomoea cairica extract of four fractions and Octadecanoic acid- against dengue vector mosquitoes, Aedes tetrahydrofuran-3,4-diyl ester isolated albopictus (Diptera: Culicidae) and Aedes from chloroform extracts of Neem (Aza- aegypti (Diptera: Culicidae). J Insect Sci dirachta indica) oil against Sarcoptes scabiei 2014; 14: 1-6. var. cuniculi larvae in vitro. Vet Parasitol Banerji A, Sarkar M, Datta R, Sengupta P, Abra- 2009; 209: 175-8. ham K. Amides from Piper brachystachyum Elango G, Rahuman A, Zahir A, et al. Evalu- and Piper retrofractum. Phytochemistry 2002; ation of repellent properties of botanical 59: 897-901. extracts against Culex tritaeniorhynchus Bao N, Ochir S, Sun Z, Borjihan G, Yamagishi Giles (Diptera: Culicidae). Parasitolol Res T. Occurrence of piperidine alkaloids in 2010; 107: 577-84.

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