New Thai herbal shampoos as pediculicides for killing head louse, Pediculus humanus capitis De Geer (Phthiraptera)

Sirawut Sittichok, Orawan Wongnet, Mayura Soonwera

Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut's Institute of Technology Ladkrabang, Ladkrabang,Bangkok 10520, Thailand

Keywords:Pediculus humanus capitis Thai herbal shampoo Cratoxylum formosum + eucalyptus essential oil shampoo Ovicicide Nymphicide Adulticide

ABSTRACT Objective: To evaluate the efficacy of Thai herbal shampoos [Cartoxylum formosum (C.formosum) + eucalyptus essential oil (EO), C. formosum + citrus EO, Solanum trilobatum +eucalyptus EO, Solanum trilobatum + citrus EO, Moringa oleifera + eucalyptus EO and Moringa oleifera + citrus EO] for killing all stages of Pediculus humanus capitis (Phthiraptera). Methods:A filter paper contact method was applied with three concentrations (0.05, 0.10 and 0.20 mL/cm2)of each Thai herbal shampoo as well as permethrin pediculicide (positive control) and drinking water (negative control) against eggs, nymphs and adults of Pediculus humanus capitis. Mortality rates of the eggs were recorded after 7 days of incubation while those of nymphs and adults were recorded after 5 minutes of contact. Results: All herbal shampoos at the high concentration were highly effective against nymphs and adults, but not effective against the eggs. C. formosum+ eucalyptus EO and C. formosum + citrus EO shampoos at all concentrations exhibited the highest efficacy against nymphs and adults with 100% mortality rate at 5 min and LC50 values of 0.004 and 0.005 mL/cm2, respectively. All formulation of Solanum trilobatum and Moringa oleifera shampoos added with eucalyptus EO showed mortality rates against nymphs at 92.0%-100.0% and 76.0%-100.0% and against adults at 84.0%-100.0% and 20.0%-32.0%, respectively.Permethrin pediculicide was not effective against the eggs, but showed 68.0%-92.0% and 28.0%-60.0% mortality rates against nymphs and adults. Conclusions: These results indicate that C.formosum + eucalyptus EO shampoo can be used as an effective nymphicide and adulticide against Pediculus humanus capitis.

1. Introduction

Head lousePediculus humanus capitisDe Geer (Pediculidae:Phthiraptera) is a small and wingless insect with the body side of 2.5-4.5 mm. It is an ectoparasitic insect that feeds only on human blood more than three times per day. The most common symptoms of head lice infestation are red spots, irritation, pruritus of the scalp,and the secondary symptoms are sleep loss, blood loss, anemia and psychological distress[1-4]. Traditionally, pediculicides based on neurotoxic insecticides (lindane, malathion, carbaryl, permethrin and phenothrin) are the first option for head lice treatment. Unfortunately,head lice resistance to neurotoxic insecticides has occurred and been reported[1-4]. Moreover, adverse effects of chemical pediculicides such as irritation and burning sensation to the scalp, ataxia, tremor and seizure have been noted[4,5]. Presently, there are considerable concerns about chemical pediculicides related to human health, long chemical persistence in the environment, food supply, water, and air. Most importantly, head lice resistance to chemical pediculicides has already developed[6-8]. Thus, new pediculicides for head lice treatment are needed. More than ten products based on plant extracts from neem,clove, henna, Zingiberacera plants, Piperaceae plants and essential oil from Zingiberacera plants, Rutaceae plants, eucalyptus, lavender,coconut and yalang-yalang have been tested as pediculicide for head lice treatment[9-15]. Pediculicides from plants have been recommended as good and safe alternative pediculicides for head lice treatment because their toxicity to human’s nerve and respiratory systems is low[16,17]. Natural pediculicides from plants have been investigated for nymphicidal and adulticidal activities against head lice[11-14,15].Moreover, natural products are effective, easily biodegradable,inexpensive and environmental friendly, having no negative effects on non-target organisms[16-18]. The chemical compounds from plant extracts and plant essential oils (EOs) exhibited an inhibition effect on acetylcholinesterase on the nervous system of insects and caused its paralysis and death[19,20].

Several papers published in 2004, 2008, 2016, and 2017 report that EOs fromEucalyptus globulus(E. globulus) leaves,Cinnamomum aromaticumbark,Eugenia aromaticaflowering buds,Pimpinella anisumfruits,Sesamum indicumseeds, aerial parts ofMentha spicataandThymus vulgariswere active against eggs and female adults of head lice[21-25]. Shampoos of 10% crude aqueous extracts ofCitrus aurantifoliaandCitrus hystrixfruits exhibited highly effective pediculicidal activity against head lice[10]. Mehlhornet al[26] and Al-Quraishyet al[27] found that neem shampoo was highly toxic against eggs and adults of head lice. However, published papers related to ovicidal, nymphicidal and adulticidal acitivities ofCratoxylum formosum(C. formosum),Solanum trilobatum(S. trilobatum) andMoringa oleifera(M. oleifera) extracts against head lice are limited and many papers that reported about the Thai herbs presented in this study inform only of their therapeutic properties and health benefits but not of their pediculicidal activity against head lice[28-34].All Thai herbs investigated in this study are also used in traditional Thai medicine for treatment of food poisoning, diarrhea, internal bleeding, cough, and pyrexia and as expectorants. The major chemical constituents ofC. formosum, S. trilobatumandM. oleiferaextracts,EOs ofE. globulusandCitrus sinensis(C. sinensis)were phenolic compounds and monoterpenes[35-39]. Many papers have pointed out that monoterpenes cause mortality of insects by inhibiting the activity of acetylcholinesterase enzyme in the nervous system of insects[16-20].Therefore, the objective of this study was to determine the efficacies of new Thai herbal shampoos fromC. formosum,S. trilobatumandM. oleiferaadded with either eucalyptus essential oil (eucalyptus EO)or citrus essential oil (citrus EO) as well as the efficacies of permethrin pediculicide (Scully Anti-Lice Shampoo®, 0.5% w/w permethrin) and drinking water (Kaesad®) against all stages of head lice: eggs, nymphs and adults.

2. Material and methods

2.1. Six Thai herbal shampoos

Freshly picked leaves of 5-year-oldC. formosumtrees, fresh fruits of one-year-oldS. trilobatumtrees and fresh fruits of five-year-oldM. oleiferatrees were collected from Nakhon-Ratchasima province,Thailand during the summer season, April-May, of 2016. All plant specimens were positively identified by a botanical taxonomist at the Faculty of Agricultural Technology, King Mongkut’s Institute Technology Ladkrabang (KMITL). Leaves and fruits of plants were cleaned, cut into small pieces, and put in a 5 L flask. One thousand grams of each plant was soaked in 2 000 mL of 95% ethyl alcohol at room temperature for 5 d. Crude extract was separated by suction filtered. Ethyl alcohol in the filtrate was evaporated with a rotary evaporator at 70 ℃ until a crude extract was achieved. Plant EOs of fresh leaves of five-year-oldE. globulustrees (for eucalyptus EO) and fresh fruit peels of five-year-oldC. sinensistrees (for citrus EO) were extracted by water distillation in a modified Clevenger type apparatus for 6-8 h. Water was removed from the extracts by using anhydrous sodium sulphate. All of the plant extracts and essential oils were used to prepare 6 Thai herbal shampoos at 10% concentration by a medical plant scientist at KMITL [1.C. formosum+ eucalyptus EO shampoo(10% ethanolic extacts ofC. formosumleaves + 10%E. globulusEO+ water + emulsifier), 2.C. formosum+ citrus EO shampoo (10%ethanolic extacts ofC. formosumleaves + 10%C. sinensisEO +water + emulsifier), 3.M. oleifera+ eucalyptus EO shampoo (10%ethanolic extacts ofM. oleiferaseeds + 10%E. globulusEO + water +emulsifier), 4.M. oleifera+ citrus EO shampoo (10% ethanolic extacts ofM. oleiferaseeds + 10%C. sinensisEO + water + emulsifier), 5.S. trilobatum+ eucalyptus EO shampoo (10% ethanolic extacts ofS. trilobatumseeds + 10%E. globulusEO + water + emulsifier),6.S. trilobatum+ citrus EO shampoo (10% ethanolic extacts ofS.trilobatumseeds + 10%C. sinensisEO + water + emulsifier)]. All plant shampoo were stored in the laboratory (25 ℃ and 70% RH).Permethrin pediculicide was used as positive control and drinking water as negative control. Permethrin shampoo (Scully Anti-Lice Shampoo®, 0.5% w/w permethrin) was manufactured by Sherwood Chemicals Manufacturing Co. Ltd, Chachoengsao province, Thailand,and drinking water (Kaesad®) was manufactured by the Faculty of Agroindustry King Mongkut’s Institute of Technology Ladkrabang,Bangkok, Thailand. (www.kmitl.ac.th/agrind).

2.2. Approval of protocol for collection of eggs, nymphs and adults of head lice from human beings

The protocol for collection of all stages of head lice from human beings was approved by the KMITL Ethic Committee, Ladkrabang,Bangkok, Thailand with a registration number of 2560-01-04-003 in May 2016. All stages of head lice were collected from the heads of 50 severely infested subjects who were students and parents of some students at several primary schools in Ladkrabang district, Bangkok,Thailand. Their hair was cut with scissors and put in small insect boxes (4 cm×7 cm×3 cm) for egg collection. Nymphs and adults were collected by using head lice combs. Each stage of head lice was separated under a stereomicroscope within 15-20 min after the collection.

2.3. Ovicidal, nymphicidal and adulticidal activity experiments

A filter paper contact method was used to evaluate the ovicidal,nymphicidal and adulticidal activities of six Thai herbal shampoos.Permethrin pediculicide and drinking water were used as positive and negative controls, respectively. Each shampoo at concentrations of 0.05, 0.10 and 0.20 mL/cm2was dropped onto a 4.5-cm diameter filter paper. Ten fertile eggs/nymphs/adults of head lice were put in contact with it for 7 d (for eggs) and 5 min (for nymphs and adults). The eggs,nymphs and adults were incubated under laboratory conditions (25 ℃and 70% RH). The control treatments with permethrin pediculicide(Scully shampoo®, 0.5% w/v permethrin) and drinking water were arranged concurrently with the treatments of Thai herbal shampoos.Each treatment was replicated five times. The mortality of eggs was recorded at 7 d and those of nymphs and adults were recorded at 5 min. The criterion for mortality of eggs was that either their operculum did not open or opened but the nymph inside was dead. The criterion for mortality of nymphs and adults was that all of their internal and external parts did not move [21,22,26].

2.4. Statistical analysis

A completely randomized design was used in this experiment.Mortality of eggs, nymphs and adults were analyzed by analysis of variance (ANOVA) and Duncan’s multiple comparison with an SPSS program for windows version 22. Significant differences between treatments were determined atP＜0.05. The percentage mortality rate of eggs, nymphs and adults were calculated by the following formula:Mortality rate (%) = (NT/NC)×100

Where NT is the total number of dead eggs/nymphs/adults and NC is the total number of treated eggs/nymphs/adults.

3. Results

Table 1 and 2 showed the ovicidal, nymphicidal and adulticidal activities of Thai herbal shampoos at three concentrations (0.05, 0.10 and 0.20 mL/cm2) with eucalyptus and citrus EOs supplements in terms of mortality rate and LC50values against eggs at 7-day exposure and against nymphs and adults at 5-min exposure. All Thai herbal shampoos exhibited strong activity against nymphs and adults of head lice but weak or no activity against eggs. All formulations of Thai herbal shampoo added with 10% eucalyptus EO showed more nymphicidal and adulticidal activities than those added with 10% citrus EO. All Thai herbal shampoos at high concentration(0.20 mL/cm2) effected high mortality rates. The 0.20 mL/cm2of shampoos fromC. formosumadded with either 10% eucalyptus EO or 10% citrus EO effected mortality rates of 24.0%, 100% and 100.0% and 12.0%, 100% and 96.0% against eggs, nymphs and adults of head lice, respectively, while the 0.20 mL/cm2of shampoo fromS. trilobatumadded with eucalyptus EO effected mortality rates of 12.0%, 100% and 100% against eggs, nymphs and adults of head lice,respectively.M. oleiferashampoo at all concentrations added either with eucalyptus EO or citrus EO effected mortality rates of 76.0%-100.0% and 68.0%-96.0% against nymphs and 20.0%-32.0% and 20.0%-30.0% against adults of head lice, respectively. On the other hand,S. trilobatumandM. oleiferashampoos added with citrus EO,permethrin pediculicide and drinking water did not show any ovicidal activity at all. Permethrin pediculide effected mortality rates of 68.0%-92.0% and 28.0%-60.0% against nymphs and adults, respectively. As expected, drinking water did not show any ovicidal, nymphicidal and adulticidal activities at all.

All Thai herbal shampoos added with 10% eucalyptus EO were highly toxic to nymphs and adults with LC50values of 0.004-0.300 mL/cm2, while those added with 10% citrus EO showed LC50values of 0.005-0.400 mL/cm2. The LC50values ofC. formosum,S.trilobatumandM. oleiferashampoos added with 10% eucalyptus EO against eggs of head lice were 33.4, 44.8 and 44.6 mL/cm2,respectively, and the LC50value ofC. formosumshampoo added with 10% citrus EO was 43.8 mL/cm2. The LC50values of permethrin pediculicide against nymphs and adults were 0.020 and 0.040 mL/cm2, respectively. Drinking water did not show any LC50value. The relationships between mortality rate (%) and concentrations (0.05,0.10 and 0.20 mL/cm2) of six Thai herbal shampoos added with either 10% eucalyptus EO or 10% citrus EO against nymphs and adults of head lice showed that as concentration of Thai herbal shampoos increased from 0.05 to 0.20 mL/cm2, the mortality rate of nymphs and adults of head lice increased. The high concentration(0.2 mL/cm2) of all shampoo exhibited high mortality rate of all two stage of head lice. All Thai herbal shampoos added with eucalyptus EO were more effective against all head lice than those added with citrus EO.

Table 1 Mortality rates of eggs at 7 d and those of nymphs and adults of P. humanus capitis at 5 min after applied with six formulations of Thai herbal shampoos,permethrin pediculicide and drinking water (mean± SD).

Table 2 LC50 values of six formulations of Thai herbal shampoos, permethrin pediculicide and drinking water at 7 d against the eggs and at 5 min against nymphs and adults of P. humanus capitis (mL/cm2).

4. Discussion

The nymphicidal and adulticidal activities of all formulation ofC. formosumandS. trilobatumshampoos added with either 10%eucalyptus EO or 10% citrus EO at 0.20 mL/cm2concentration were significantly higher than permethrin pediculicide (positive control) and drinking water (negative control) (P＜0.05). All Thai herbal shampoos added with 10% eucalyptus EO at 0.05 mL/cm2concentration were more highly toxic to nymphs and adults of head lice than those added with 10% citrus EO. The maximum nymphicidal and adulticidal activities was 0.20 mL/cm2fromC.formosumshampoo added with 10% eucalyptus EO, which effected a mortality rate of 100% at 5 min against nymphs and adults and a mortality rate against head lice eggs of 24.0%. The results pointed that eucalyptus EO acted as a synergist agent in combination withC. formosumshampoo. Major chemical constituents of EO extracted fromE. globuluswere monoterpenoids, 1-8-cineole, α-terpinene, α- pinene, 2-β-pinene, β-myrcene, α-phellandrene,1-isopropenyl-3-methylbenzene, γ- terpinene, (E)-pinocarveol and 1-α-terpineol[40-42]. Many papers published in 2011, 2012 and 2013 reported that monoterpenoids from plant EOs caused mortality of insects by inhibiting the activity of acetylcholinesterase enzyme in the nervous system of the insects[20,35], and monoterpenoids showed the highest activity against eggs and adults of head lice[43].Similarly, two papers published in 2011 and 2015 reported that an anti-louse shampoo which contained neem seed extract penetrated readily into the cuticle of eggs and adults of head lice and blocked their oxygen uptake, causing mortality[26,27]. Our results showed thatC. formosumadded with eucalyptus EO shampoo effected 100%mortality rate at 5 min against nymphs and adults of head lice. These shampoo might penetrate into the cuticle of nymphs and adults and inhibited their tracheae system, causing paralysis and death. In contrast, these shampoo showed inactivity against eggs of head lice with an inhibition rate of only 24.0% at 7 days of incubation[26,27,43].The cuticle of the eggs is very different from those of nymphs and adults. The cuticle of the eggs is hydrophobic and impervious to water[26,27,43]. Since all Thai herbal shampoos tested in this study were aqueous formulation, they could not penetrate through the cuticle and showed low activity or inactivity against the eggs.

All formulations of Thai herbal shampoo added with citrus EO tested in this study showed lower nymphicidal and adulticidal activities than those added with eucalyptus EO andS. trilobatumandM. oleiferashampoos added with citrus EO did not show any ovicidal activity at all. Major chemical constituents ofC. sinensisEO are limonene, α-terpinene and α-pinene[44,45]. Similarly,two papers reported that limonene from plant EOs was low in activity against eggs and adults of head lice, and it was not very toxic to acetylcholinesterase enzyme in the nervous system of the insects[19,43]. On the other hand, α-pinene fromC. sinensisEO exhibited high toxicity againstCulex pipienslarvae[46].

Even though published papers about ovicidal, nymphicidal and adulticidal acitivities ofC. formosum,S. trilobatumandM. oleiferaextracts against head lice are limited, but two papers published in 2009 and 2012 reported that extracts ofM. oleiferaseeds showed larvicidal, oviposition deterrent and ovicidal activities against two mosquito species,Aedes aegyptiandCulex quinquefasciatus[47-49].Extracts ofS. trilobatumleaves exhibited larvicidal and pupicidal activities againstAedes aegypti,Culex quinquefasiatusandAnopheles stephensi[50] and an extract ofC. formosumshowed some therapeutic properties for humans such as antioxidant, anticancer, antimicrobial and anti-Alzheimer’s disease[51,52].

Our results showed that, all formulations ofC. formosumandS.trilobatumshampoos added with eucalyptus EO at 0.05 and 0.10 mL/cm2concentrations were more toxic to nymphs and adults of head lice than permethrin pediculicide (positive control; 68.0%-80.0% mortality rate for nymphs and 28.0%-36.0% mortality rate for adults). On the other hand, permethrin pediculicide was more toxic to adults of head lice than all formulations ofM. oleiferashampoos.All formulations ofC. formosumandS. trilobatumshampoos at high concentration (0.20 mL/cm2) added with eucalyptus EO showed 12.0%-24.0% mortality rate of the eggs while permethrin pediculicide and all formulations ofM. oleiferaandS. trilobatumshampoos added with citrus EO did not show any ovicidal activity at all. Permethrin pediculicide was one of the most common pediculicides in Thailand for treatment of human pediculosis, but head lice resistance to it has caused treatment failure[6-8]. Similarly, many papers reported that permethrin pediculicide showed 20%-30% mortality rate against nymphs and adults of head lice but did not kill eggs[6,7,53,54].Therefore, in order for a permethrin treatment to be effective, it needs to be applied repeatedly 2-3 times per week and treated for 7-10 d after the 1st application[7,54]. Unfortunately, permethrin pediculicide is persistent, harmful and highly toxic to human beings because it is neurotoxic and head lice resistance to it can develop.Common adverse effects after treatment with permethrin pediculicide are pruritus, burning and erythema[5-8,54]. For this reason, natural pediculicides derived from plant extracts are a more promising agent for head lice treatment. Our results showed thatC. formosum+ eucalyptus EO shampoo was highly effective pediculicide for killing two stage of head lice (nymphs and adults). TheC. formosum+ eucalyptus EO shampoo should not have any adverse effects to human beings sinceC. formosumhas been edible for native Thai people for a long time and commonly used as traditional Thai herbal medicine for diarrhea, food poisoning, wound healing, cough, fever,and stomach ache[36,37]. TheC. formosum+ eucalyptus EO shampoo has a high potential for developing into new pediculicide for head lice treatment especially for infested Thai children whose head lice infestation rate was over 20%[55].C. formosum+ eucalyptus EO shampoo is inexpensive and safe. However, it should be officially tested for acute and chronic toxicity before it is used as herbal pediculicide for head lice treatment.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by King Mongkut’s Institute of Technology Ladkrabang (KMITL), Bangkok, Thailand for academic year 2015/2016, Grant No. KMITL 2560-01-04-003, and the National Research Council of Thailand (NRCT) (Thesis Grant for Doctoral Degree Student Fly 2016). We wish to thank the botanical taxonomist and entomologist at KMITL for their identification of plants and insects. We also wish to express our gratitude to Mr.Pratana Kangsadal, the KMITL Proofreader, for reviewing and giving comments on the manuscript.