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Pediculocidal and scabicidal properties of Lippia multiflora essential oil
Journal of Ethnopharmacology 72 (2000) 305 – 311 www.elsevier.com/locate/jethpharm
Short communication
Pediculocidal and scabicidal properties of Lippia multiflora essential oil F.A. Oladimeji a, O.O. Orafidiya a,*, T.A.B. Ogunniyi b, T.A. Adewunmi b b
a Department of Pharmaceutics, Obafemi Awolowo Uni6esity, Ile-Ife, Nigeria Department of Medical Microbiology and Parasitology, Obafemi Awolowo Uni6ersity, Ile-Ife, Nigeria
Received 10 December 1999; received in revised form 15 March 2000; accepted 10 April 2000
Abstract The essential oil from the leaves of Lippia multiflora Moldenke (Verbenaceae) was tested for its pediculocidal and scabicidal activites against bodylice, headlice and scabies’ mites. The ‘knockdown’ times obtained for bodylice and headlice using lippia oil preparations were comparatively shorter than those obtained using benzyl benzoate and Delvap Super®, a brand of dichlorvos. The lethal effect of the lippia oil on headlice was increased when applied in an enclosed system that prevented volatilization of the oil while allowing maximum contact of the vapour with the headlice. A 20% v/v preparation of lippia oil applied to scabietic subjects for 5 consecutive days gave 100% cure compared with 87.5% cure obtained for benzyl benzoate preparation of the same concentration. The GC-MS analysis of oil revealed, among others, the presence of terpineol, a- and b-pinene which are known to be lethal to body and headlice. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lippia multiflora; Essential oil activity; Pediculosis; Scabies; GC-MS analysis
1. Introduction Pediculosis (lice infestation) and scabies have continued to be epidemic diseases in Nigerian prisons and among school children (Olasode and Onayemi, 1998). The treatment approach of lice infestation in some Nigerian prisons involves the use of pesticides like organophosphate (dichlorvos) and organochloride (DDT) either separately or in * Corresponding author.
combination with kerosene (a mixture of petroleum hydrocarbons). Among the school children and low income earners, kerosene remains the agent for the treatment of lice infestation. Incidentally, some of these agents are confirmed to be neurotoxic (Windholz, 1983; Maddin, 1991; Elgart, 1996), while, cases of lice resistance to some have been reported (Weston et al., 1997; Dawes et al., 1999). The risk involved in the use of these agents has, therefore, necessitated the search for more effective and safer drugs especially from natural sources for the treatment of these infestations.
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 2 2 9 - 4
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F.A. Oladimeji et al. / Journal of Ethnopharmacology 72 (2000) 305–311
Terpenoid compounds which are chemical constituents of essential oils have long been known to be insect repellent (Weston et al., 1997). Among plants containing terpenoids is Lippia multiflora Moldenke synonym Lippia adoensis Hochst (Verbenaceae), a shrub found growing in West Africa Savannah. The chemical composition of the essential oil of its leaves (Elakovich and Oguntimein, 1987; Valentin et al., 1995) and its biological activities (Benoit et al., 1996) have been studied. The essential oil of the plant has been documented as a promising fumigant in plant pest control (Olaifa et al., 1987). The present work describes the effectiveness of the essential oil of L. multiflora as an ectoparasitic agent against pediculosis and scabies.
2. Methodology
2.1. Plant material The leaves of L. multiflora were collected from wild plants growing in Ipetumodu, near Ile-Ife, South Western part of Nigeria and authenticated by Dr H.C. Illoh (Department of Botany, Obafemi Awolowo University, Ile-Ife, Nigeria) by comparison with voucher specimens (IFE 586, IFE 1850 and IFE 6238) deposited at the Herbarium of Obafemi Awolowo University Ile-Ife, Nigeria.
2.2. Extraction process The essential oil of the lippia leaves was obtained by hydrodistillation using the British Pharmacopoeia method (B.P. 1988). The extracted lippia oil was stored fully filled in brown bottles at 6°C until required for use.
2.3. Pediculocidal acti6ity 2.3.1. Biological material The bodylice (Pediculus humanus corporis) and headlice (Pediculus humanus capitis) were collected in petri-dishes from volunteers who were
inmates of the Nigerian Prison Services, Ilesa, Nigeria. The lice were separated into different size ranges using a handlens and a venier caliper. The lice were tested within 3h of collection.
2.3.2. Preparation of pediculocidal agents The following agents were prepared and used. (a) Delvap Super® (a brand of dichlorvos containing 1000 g dichlorvos per 1000 ml. The Candel, Victoria Island, Lagos, Nigeria). Based on the recommended dilution range and the method of usage at the prison cells, the following dilutions were prepared using distilled water as the dilution medium: 1 in 1000; 1 in 200; 1 in 100 and 1 in 100 using kerosene as the dilution medium (b) kerosene obtained locally (c) benzyl benzoate (100 and 25% v/v dilution in light liquid paraffin BP) (d) lippia oil (100%, 50, 25, 20, 15 and 10% v/v dilutions in light liquid paraffin BP) (e) light liquid paraffin BP as the placebo. 2.3.3. Direct contact method Each louse was carefully transferred into a glass dish and 0.02 ml of the agent was applied directly on the dorsal part of the louse using a 1 ml pipette. After 15 s of contact with the agent, the louse was transferred into a petri-dish lined with filter paper and observed using a handlens until dead or otherwise. The elapsed time was recorded for each test agent as the ‘knockdown’ time. The death of the louse was confirmed when there was cessation of motility or waggling of the appendages on touching with a needle. The agent was assumed to be ineffective when there was motility of the louse after 2 h of treatment. Between six and eight lice were used for each determination. 2.3.4. Direct contact in an enclosed system A glass dish of diameter 4.7 and 1.7 cm high was lined with filter paper, and five headlice were transferred into it. A total of 0.02 ml of the test agent was applied directly on the dorsal part of each louse and the dish was covered immediately with the lid. The lice were observed until death occurred and the elapsed time recorded as the ‘knockdown’ time.
F.A. Oladimeji et al. / Journal of Ethnopharmacology 72 (2000) 305–311
2.4. Scabicidal acti6ity 2.4.1. Study cases The study was carried out at the Nigerian Prison Services, Ilesa, Nigeria with the permission of the prison authority. Scabietic subjects were identified by the resident medical doctor of the prison, while diagnosis was confirmed microscopically by presence of the mites in the skin scrapings of the subjects (Cheesbrough, 1987). The scabietic lesions were localized on the hands, thighs, trunks, wrists and fingers of the subjects. When a case has been identified in a cell, all the subjects in such cell were screened. A total of 268 male inmate volunteers (subjects), whose ages ranged between 22 and 65 years and whose informed verbal consent was obtained in the presence of the prison’s resident medical doctor and officials were included in the study. The subjects were housed in ‘awaiting trial’, ‘convicted’ and ‘single’ cells within the prison. 2.4.2. Study design Single blind and group parallel design was adopted (Spilker, 1985), with blind imposed on the subjects and the staff of the prison’s clinic who applied the test agents. Subjects confirmed as having scabies were randomized into three treatment groups vis-a`-vis the test agent (lippia oil), the reference agent (benzyl benzoate) and the placebo (light liquid paraffin BP). Within the same treatment group, the subjects were subdivided into groups based on the concentration of the test agent applied and the treatment period. 2.4.3. Treatment schedule Using light liquid paraffin BP as the dilution medium, different concentrations of lippia oil and benzyl benzoate were prepared and applied between 3 and 5 days as follows: 3 consecutive days (treatment schedule A); 3 alternate days spanning 5 days (treatment schedule B); 5 consecutive days (treatment schedule C). The agents were applied to the affected part after morning bath (with non-medicated soap) using a cotton bud as the applicator. New cotton buds were used for every application and for each subject.
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2.4.4. E6aluation of efficacy of test agents Skin scraping was carried out 2 weeks after treatment and observed microscopically for the presence of scabies’ mites or larvae (Cheesbrough, 1987). Reinfestation was checked 4 weeks after treatment. The test agent activity was defined thus: (a) cure was absence of new lesion and the healing of old lesion, skin scrapings showed no infestation; (b) treatment failure was when skin scrapings microscopically confirmed the presence of mites or larvae 2 weeks after treatment; and (c) re-infestation was defined as complete cure at 2 weeks but with development of new lesions with positive microscopic findings at 4 weeks (Orkin and Maibach, 1996). 2.5. Analysis of lippia oil 2.5.1. Gas chromatography Lippia oil (2 mg) was dissolved in chloroform (1 ml) and 1 ml of the solution was injected by a splitless technique into a Hewlett-Packard 6890 gas chromatograph equipped with a FID and a HP-5 capillary column (cross linked 5% diPh, 95% dimethyl polysiloxane, 30 m× 0.32 mm i.d.× 0.25 mm film thickness). The column temperature was programmed at 50–210°C at 3°C/ min. The injector and detector temperatures used were 220 and 270°C, respectively. The carrier gas was nitrogen (2.3 ml/min). 2.5.2. Gas chromatography/mass spectrometry The oil sample (5 mg) was dissolved in chloroform (1 ml) and 0.6 ml of the solution was similarly injected by a splitless technique into a Fissions GC model 8000 gas chromatograph coupled to a mass spectrometer, MD 800 quadruple analysis operating at 70 eV. The capillary column was a HP50 + (30 m ×0.25 mm i.d. × 0.25 mm film thickness). The column temperature was programmed at 50–210°C at 3°C/min, and the injector temperature was 220°C. The carrier gas was helium (5 psi). The components of the oil were identified by their retention times and by comparison of their mass spectra with those from Wiley Library software.
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3. Results and discussion Table 1 shows the activity of lippia oil and other agents on bodylice. While lippia oil proved superior to benzyl benzoate and Delvap Super® (dichlorvos), it was not as effective as kerosene. Benzyl benzoate had no activity against bodylice, while Delvap Super® was not effective at prescribed concentration range (1 in 1000 – 1 in 200). Kerosene significantly improved the activity of Delvap Super® at 1 in 100 dilution. The lice treated with pure benzyl benzoate and Delvap Super® (1 in 200) looked quite dead initially, they later recovered at varying time intervals after the 2 h observation period. The smaller bodylice were more susceptible to lippia oil. This could be related to the chitinous shield of the louse which Table 1 Pediculocidal activities of lippia oil and other agents against bodylice (Pediculus humani corporis)a Agent/conc.
‘Knockdown’ time (min)b (Size range) 53.5mm
4.0–5.0 mm
]5.5 mm
Lippia oil (% 6/6) 100 3.49 90.97 50 4.94 9 0.77 25 10.69 90.84 20 13.87 9 2.46 15 15.63 9 1.59 10 23.38 9 2.93
5.299 0.59 9.009 1.00 19.449 1.81 22.94 9 1.80 24.30 93.27 33.03 94.57
6.55 90.41 11.64 9 2.13 23.559 1.60 ND 25.75 91.46 35.4492.88
Benzyl benzoate (% 6/6) 100 NE 25 NE
NE NE
NE NE
Del6ap Super ® Concentrate 1 in 100 1 in 200 1 in 1000
(dichlor6os) 20.97 94.25 63.67 95.03 NE NE
31.759 4.71 77.339 7.09 NE NE
35.09 9 3.80 92.259 14.62 NE NE
Del6ap Super ®+kerosene (1 in 100) 0.35 90.05 Kerosene 1.33 90.30
0.46 9011 1.94 9 0.77
0739 0.08 3.149 0.13
Light liquid paraffin BP
NE
NE
NE
Each value represents means 9S.D. from at least six bodylice. ND, not determined; NE, no effect. b Direct contact method. a
Table 2 Pediculocidal activities of lippia oil and other agents against headlice (Pediculus humani capitis)a Agent/conc.
‘Knockdown’ time (min) Direct contactb
Direct contact in enclosed systemc
Lippia oil (%v/v) 100 2.33 90.91 50 4.08 90.94 25 8.82 91.13 20 10.59 91.84 15 12.00 91.19 10 21.95 92.83
1.68 90.28 2.45 9 0.46 5.83 90.16 6.15 9 0.82 9.20 9 1.05 16.50 9 2.18
Benzyl benzoate (%6/6) 100 22.36 95.93 25 57.09 910.96
21.92 93.72 58.25 96.94
Del6ap Super ® (dichlor6os) Concentrate 20.33 94.51 1 in 100 50.33 92.31 1 in 200 85.60 9 11.44 1 in 1000 NE
19.87 92.53 51.2094.06 84.0599.92 NE
Del6ap Super ®+kerosene (1 in 100) 0.4190.08 Kerosene 1.25 90.17
0.38 9 0.10 1.10 9 0.24
Light liquid paraffin BP
NE
NE
Each value represents means 9S.D. n =6. c n =5; NE; no effect. a
b
becomes harder with age (James and Hardwood, 1969), thus reducing its permeability to the agent. Hence, the increase in the knockdown time observed for the older lice. The headlice were more susceptible to the agents than bodylice (Table 2). The sizes of headlice used in the study varied between 1.0 and 2.5 mm. At 25% v/v concentration, lippia oil was found to be more effective than benzyl benzoate of the same concentration. However, kerosene was more effective than lippia oil, benzyl benzoate and Delvap Super® concentrate. The apparent higher efficacy of kerosene, therefore, justifies its use by the local populace in treating body and headlice and its use in the prison cells as dilution medium for Delvap Super®. Lippia oil appears to be more effective in an enclosed system after
F.A. Oladimeji et al. / Journal of Ethnopharmacology 72 (2000) 305–311
direct contact with headlice (Table 2). Such method of application has no significant effect on the knockdown time obtained for benzyl benzoate, Delvap Super® and kerosene (P = 0.05), which are relatively non-volatile. Most pediculocidal agents act directly on the louse by entering their bodies through the external chitinous skeleton (Scabbie, 1945), however, the enhanced efficacy of lippia oil using an enclosed system indicated that the oil vapour, consisting of the highly volatile constituents of the oil have lethal effect on the headlice. The anti-scabies properties of lippia oil and that of benzyl benzoate are indicated in Table 3. Since the benzyl benzoate was applied at maximum concentration of 25%v/v (P.Cx., 1979), comparison between the lippia oil and benzyl benzoate was limited to concentrations between 10 and 25% v/v. The percentage of subjects cured with lippia oil preparations was found to be significantly different (P= 0.05) from those of benzyl benzoate under treatment schedule A. In contrast, a comparison of percentages cured obtained with lippia oil and benzyl benzoate preparation under treatment schedule C indicated no significant differ-
309
ence in their efficacy. The t-test value calculated for the two agents at concentrations between 10 and 25% v/v was 2.611, while the t-test value at 3 degrees of freedom was 3.182 (P= 0.05). However, the percentages cured using 20% v/v concentration of lippia oil preparation under treatment schedules A and C were 75 and 100%, respectively, compared with 62.5 and 87.55% obtained for benzyl benzoate preparation at the same concentration (Table 3). It could, therefore, be inferred from the result that while a 20%v/v lippia oil preparation would possess enough scabicidal power to eradicate the mite infestation, a 25% v/v preparation of benzyl benzoate will be required to give similar effect. Statistical analysis of treatment schedules A, B and C for lippia oil preparations using analysis of Variance (ANOVA) shows significant difference between them (P= 0.05). It is obvious from Table 3 that the lippia oil preparations may have to be applied for 3–5 consecutive days before it can effectively eradicate the mites infestation. A further comparison of treatment schedules A and C for lippia oil preparations using Student’s t-test showed significant difference at P= 0.05, indicat-
Table 3 Scabicidal activities of lippia oil and benzyl benzoate preparationsa Agent/conc.b
Number curedc Treatment schedule A
Treatment schedule B
Treatment schedule C
Lippia oil (%6/6) 10 15 20 25 30 40 50
1 4 6 6 7 8 7
(12.5) (50) (75) (75) (87.5) (100) (87.5)
0 (0) 2 (25) 3 (37.5) 5 (62.5) 6 (75) 5 (62.5) ND
3 6 8 8 8 7 8
(37.5) (75) (100) (100) (100) (87.5) (100)
Benzyl benzoate (%6/6) 10 15 20 25
0 2 5 4
(0) (25) (62.5) (50)
0 1 3 3
1 4 7 8
(12.5) (50) (87.5) (100)
Light liquid paraffin BP
ND
a
(0) (12.5) (37.5) (37.5)
ND
0 (0)
Treatment schedule A = 3 consecutive days; treatment schedule B = 3 alternate days; treatment schedule C = 5 consecutive days. Eight subjects were used for each concentration of test agent/treatment schedule. c Figures in parenthesis are percentages cured; ND, not determined. b
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Table 4 Composition (%) of the essential oil of Lippia multiflora Compound
% Composition
a-Pinene b-Pinene b-Cymene Limonene Linalool Terpineol Geraniol Thymol Citronellyl acetate
1.0 4.0 8.4 15.4 26.7 2.9 20.4 4.2 1.8
ing that the 5 consecutive days treatment was more effective. Therefore, the notion that patients with uncomplicated scabies may not require more than two appropriate applications of anti-scabies agent (Commens, 1994) may not hold for lippia oil preparations. A 20% v/v lippia oil preparation which was found effective using 3 or 5 consecutive days treatment schedule gave a low percentage cured when the treatment schedule was changed to 3 alternate days (Table 3). In an earlier trial study, the lippia oil and benzyl benzoate at 25% v/v concentration were applied on six scabietic subjects as single application. Treatment failure was recorded with both agents. Such failure could be due to the fact that the incubation period of the mite’s ova is 3–4 days (Mackenzie, 1941). Therefore, if the ova remain viable within the thickened skin, larvae might appear after the single application. Again, the extent of the lesions and presence of secondary infections could account for why the single application or short duration of treatment causes treatment failure (Charles and Charles, 1992). In a cell, all known cases of scabies were included in the study, however, the ‘contact’ (who may be acting as a reservoir of the infections) was not treated. These ‘contacts’ could be the source of reinfestation observed. The percentages of subjects reinfested in the ‘awaiting trial’, ‘convicted’ and ‘single’ cells were 23.7, 22.2, and 61.1% for lippia oil, and 33.3, 16.7 and 66.7% for benzyl benzoate, respectively. While the magnitude of the reinfestation varied with the type of cell in which
the subjects were housed, there was no significant difference (P= 0.05) between those subjects treated with lippia oil and benzyl benzoate preparations. Each ‘awaiting trial’ cell or ‘convicted’ cell is a large open room housing up to 20 subjects but with reduced contact among them, while the ‘single’ cell with a floor space of 2.230 m2, originally designed for one subject housed up to six subjects. Such overcrowding resulting in closer contact could be responsible for high incidence of re-infestation observed in the ‘single’ cells. The identified components of lippia oil are listed in Table 4 in order of their elution by the GC. The identity of four major peaks (cymene, linalool, geraniol and thymol) were further confirmed by GC-MS. Terpineol and a-pinene which are present in small quantities have been found to kill bodylice, with terpineol being faster in action (Weston et al., 1997). a- and b-pinene are also known insecticides (Windholz, 1983). The scabicidal activity of lippia oil is likely to be due, at least in part, to these three compounds. Similarly, oils of juniper, eucalyptus, lavender, geranium, lemon, rosemary and cypress which contain terpineol, camphene and a-pinene have lethal effects on head and bodylice (Weston et al., 1997). From the above findings, the essential oil of L. multiflora may be considered a potential pediculocidal and scabicidal agent when compared with benzyl benzoate, which presently is the drug of choice especially for the management of scabies. A concentration of 20% v/v of the oil in a suitable formulation basis applied for 5 consecutive days may be sufficient for treating mite infestation. Application of an occlusive covering or a cap for treatment of headlice will enhance the activity of the oil. Further studies on the lethal effect of the oil on the lice nits will determine the treatment duration for complete eradication of headlice infestation.
Acknowledgements The authors express their gratitude to the following people. The staff and medical personel of the Nigerian Prison Services, Ilesa, Nigeria for providing an enabling environment for the study.
F.A. Oladimeji et al. / Journal of Ethnopharmacology 72 (2000) 305–311
Professor E. Dagne, Department of Chemistry, Addis Ababa University, Ethiopia for the GC-MS analysis of the lippia oil.
References Benoit, F., Valentin, A., Pelissier, Y., Diafouka, F., Marion, C., Kone-Bamba, D., Kone, M., Mallie, M., Yapo, A., Bastide, J.M., 1996. In vitro antimalarial activity of vegetal extracts used in West African traditional medicine. American Journal of Tropical Medicine and Hygiene 54, 67–71. British Pharmacopoiea, 1988. Her Majesty’s Stationery Office, London, pp. A137 – A138. Charles, V., Charles, S.X., 1992. The use and efficacy of Azadirachta indica ADR (‘Neem’) and Curcuma longa (‘Turmeric’) in Scabies — a pilot study. Tropical and Geographical Medicine 44, 178–181. Cheesbrough, M., 1987. Medical Laboratory Manual for Tropical Countries. University Press, Cambridge, p. 409. Commens, C.A., 1994. We can get rid of scabies: new treatment available soon. Medical Journal of Australia 160, 317 – 318. Dawes, M., Hicks, N.R., Fleminger, M., Goldman, D., Hamling, J., Hicks, L.J., 1999. Treatment of headlice. British Medical Journal 318, 385–386. Elakovich, S.D., Oguntimein, B.O., 1987. The essential oil of Lippia adoensis leaves and flowers. Journal of Natural Products 50, 503 – 506.
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Elgart, M.L., 1996. A risk-benefit assessment of agents used in the treatment of scabies. Drug Safety 14, 386 – 393. James, M.T., Hardwood, R.F., 1969. Herm’s Medical Entomology. Macmillian, New York, pp. 135 – 146. Mackenzie, I.F., 1941. Scabies treated by a benzyl benzoate emulsion. British Medical Journal 2, 403 – 405. Maddin, S., 1991. Current Dermatological Therapy. W.B. Saunders, Philadephia, pp. 149 – 151. Olaifa, J.I., Erhun, W.O., Akingbohungbe, A.E., 1987. Insecticidal activity of some Nigerian plants. Insect Science Application 8, 221 – 224. Olasode, O.A., Onayemi, O., 1998. Scabies: revisit in a depressed economy. Central African Journal of Medicine 44, 18 – 21. Orkin, M., Maibach, H.I., 1996. Scabies treatment: current considerations. Current Problems in Dermatology 24, 151 – 156. Scabbie, E.B.S., 1945. Substances used in treatment of Pediculosis capitis: their relative value. British Medical Journal 1, 409 – 412. Spilker, B., 1985. Guide to Clinical Studies and Developing Protocols. Raven Press, New York, pp. 14 – 33. The Pharmaceutical Codex, 1979. The Pharmaceutical Press, London, p. 91. Valentin, A., Pelissier, Y., Benoit, F., Marion, C., Kone, D., Mallie, M., Bastide, J.M., Bessiere, J.M., 1995. Composition and antimalaria activity in vitro of volatile components of Lippia multiflora. Phytochemistry 40, 1439 – 1442. Weston, S.E., Burgess, I., Williamson, E.M., 1997. Oils against headlice in ‘development in pharmacognosy’. The Pharmaceutical Journal 259 (September 20), 482. Windholz, M., 1983. The Merck Index. Merck, Rahway, USA, p. 761, 1073.
Short communication
Pediculocidal and scabicidal properties of Lippia multiflora essential oil F.A. Oladimeji a, O.O. Orafidiya a,*, T.A.B. Ogunniyi b, T.A. Adewunmi b b
a Department of Pharmaceutics, Obafemi Awolowo Uni6esity, Ile-Ife, Nigeria Department of Medical Microbiology and Parasitology, Obafemi Awolowo Uni6ersity, Ile-Ife, Nigeria
Received 10 December 1999; received in revised form 15 March 2000; accepted 10 April 2000
Abstract The essential oil from the leaves of Lippia multiflora Moldenke (Verbenaceae) was tested for its pediculocidal and scabicidal activites against bodylice, headlice and scabies’ mites. The ‘knockdown’ times obtained for bodylice and headlice using lippia oil preparations were comparatively shorter than those obtained using benzyl benzoate and Delvap Super®, a brand of dichlorvos. The lethal effect of the lippia oil on headlice was increased when applied in an enclosed system that prevented volatilization of the oil while allowing maximum contact of the vapour with the headlice. A 20% v/v preparation of lippia oil applied to scabietic subjects for 5 consecutive days gave 100% cure compared with 87.5% cure obtained for benzyl benzoate preparation of the same concentration. The GC-MS analysis of oil revealed, among others, the presence of terpineol, a- and b-pinene which are known to be lethal to body and headlice. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lippia multiflora; Essential oil activity; Pediculosis; Scabies; GC-MS analysis
1. Introduction Pediculosis (lice infestation) and scabies have continued to be epidemic diseases in Nigerian prisons and among school children (Olasode and Onayemi, 1998). The treatment approach of lice infestation in some Nigerian prisons involves the use of pesticides like organophosphate (dichlorvos) and organochloride (DDT) either separately or in * Corresponding author.
combination with kerosene (a mixture of petroleum hydrocarbons). Among the school children and low income earners, kerosene remains the agent for the treatment of lice infestation. Incidentally, some of these agents are confirmed to be neurotoxic (Windholz, 1983; Maddin, 1991; Elgart, 1996), while, cases of lice resistance to some have been reported (Weston et al., 1997; Dawes et al., 1999). The risk involved in the use of these agents has, therefore, necessitated the search for more effective and safer drugs especially from natural sources for the treatment of these infestations.
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 2 2 9 - 4
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F.A. Oladimeji et al. / Journal of Ethnopharmacology 72 (2000) 305–311
Terpenoid compounds which are chemical constituents of essential oils have long been known to be insect repellent (Weston et al., 1997). Among plants containing terpenoids is Lippia multiflora Moldenke synonym Lippia adoensis Hochst (Verbenaceae), a shrub found growing in West Africa Savannah. The chemical composition of the essential oil of its leaves (Elakovich and Oguntimein, 1987; Valentin et al., 1995) and its biological activities (Benoit et al., 1996) have been studied. The essential oil of the plant has been documented as a promising fumigant in plant pest control (Olaifa et al., 1987). The present work describes the effectiveness of the essential oil of L. multiflora as an ectoparasitic agent against pediculosis and scabies.
2. Methodology
2.1. Plant material The leaves of L. multiflora were collected from wild plants growing in Ipetumodu, near Ile-Ife, South Western part of Nigeria and authenticated by Dr H.C. Illoh (Department of Botany, Obafemi Awolowo University, Ile-Ife, Nigeria) by comparison with voucher specimens (IFE 586, IFE 1850 and IFE 6238) deposited at the Herbarium of Obafemi Awolowo University Ile-Ife, Nigeria.
2.2. Extraction process The essential oil of the lippia leaves was obtained by hydrodistillation using the British Pharmacopoeia method (B.P. 1988). The extracted lippia oil was stored fully filled in brown bottles at 6°C until required for use.
2.3. Pediculocidal acti6ity 2.3.1. Biological material The bodylice (Pediculus humanus corporis) and headlice (Pediculus humanus capitis) were collected in petri-dishes from volunteers who were
inmates of the Nigerian Prison Services, Ilesa, Nigeria. The lice were separated into different size ranges using a handlens and a venier caliper. The lice were tested within 3h of collection.
2.3.2. Preparation of pediculocidal agents The following agents were prepared and used. (a) Delvap Super® (a brand of dichlorvos containing 1000 g dichlorvos per 1000 ml. The Candel, Victoria Island, Lagos, Nigeria). Based on the recommended dilution range and the method of usage at the prison cells, the following dilutions were prepared using distilled water as the dilution medium: 1 in 1000; 1 in 200; 1 in 100 and 1 in 100 using kerosene as the dilution medium (b) kerosene obtained locally (c) benzyl benzoate (100 and 25% v/v dilution in light liquid paraffin BP) (d) lippia oil (100%, 50, 25, 20, 15 and 10% v/v dilutions in light liquid paraffin BP) (e) light liquid paraffin BP as the placebo. 2.3.3. Direct contact method Each louse was carefully transferred into a glass dish and 0.02 ml of the agent was applied directly on the dorsal part of the louse using a 1 ml pipette. After 15 s of contact with the agent, the louse was transferred into a petri-dish lined with filter paper and observed using a handlens until dead or otherwise. The elapsed time was recorded for each test agent as the ‘knockdown’ time. The death of the louse was confirmed when there was cessation of motility or waggling of the appendages on touching with a needle. The agent was assumed to be ineffective when there was motility of the louse after 2 h of treatment. Between six and eight lice were used for each determination. 2.3.4. Direct contact in an enclosed system A glass dish of diameter 4.7 and 1.7 cm high was lined with filter paper, and five headlice were transferred into it. A total of 0.02 ml of the test agent was applied directly on the dorsal part of each louse and the dish was covered immediately with the lid. The lice were observed until death occurred and the elapsed time recorded as the ‘knockdown’ time.
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2.4. Scabicidal acti6ity 2.4.1. Study cases The study was carried out at the Nigerian Prison Services, Ilesa, Nigeria with the permission of the prison authority. Scabietic subjects were identified by the resident medical doctor of the prison, while diagnosis was confirmed microscopically by presence of the mites in the skin scrapings of the subjects (Cheesbrough, 1987). The scabietic lesions were localized on the hands, thighs, trunks, wrists and fingers of the subjects. When a case has been identified in a cell, all the subjects in such cell were screened. A total of 268 male inmate volunteers (subjects), whose ages ranged between 22 and 65 years and whose informed verbal consent was obtained in the presence of the prison’s resident medical doctor and officials were included in the study. The subjects were housed in ‘awaiting trial’, ‘convicted’ and ‘single’ cells within the prison. 2.4.2. Study design Single blind and group parallel design was adopted (Spilker, 1985), with blind imposed on the subjects and the staff of the prison’s clinic who applied the test agents. Subjects confirmed as having scabies were randomized into three treatment groups vis-a`-vis the test agent (lippia oil), the reference agent (benzyl benzoate) and the placebo (light liquid paraffin BP). Within the same treatment group, the subjects were subdivided into groups based on the concentration of the test agent applied and the treatment period. 2.4.3. Treatment schedule Using light liquid paraffin BP as the dilution medium, different concentrations of lippia oil and benzyl benzoate were prepared and applied between 3 and 5 days as follows: 3 consecutive days (treatment schedule A); 3 alternate days spanning 5 days (treatment schedule B); 5 consecutive days (treatment schedule C). The agents were applied to the affected part after morning bath (with non-medicated soap) using a cotton bud as the applicator. New cotton buds were used for every application and for each subject.
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2.4.4. E6aluation of efficacy of test agents Skin scraping was carried out 2 weeks after treatment and observed microscopically for the presence of scabies’ mites or larvae (Cheesbrough, 1987). Reinfestation was checked 4 weeks after treatment. The test agent activity was defined thus: (a) cure was absence of new lesion and the healing of old lesion, skin scrapings showed no infestation; (b) treatment failure was when skin scrapings microscopically confirmed the presence of mites or larvae 2 weeks after treatment; and (c) re-infestation was defined as complete cure at 2 weeks but with development of new lesions with positive microscopic findings at 4 weeks (Orkin and Maibach, 1996). 2.5. Analysis of lippia oil 2.5.1. Gas chromatography Lippia oil (2 mg) was dissolved in chloroform (1 ml) and 1 ml of the solution was injected by a splitless technique into a Hewlett-Packard 6890 gas chromatograph equipped with a FID and a HP-5 capillary column (cross linked 5% diPh, 95% dimethyl polysiloxane, 30 m× 0.32 mm i.d.× 0.25 mm film thickness). The column temperature was programmed at 50–210°C at 3°C/ min. The injector and detector temperatures used were 220 and 270°C, respectively. The carrier gas was nitrogen (2.3 ml/min). 2.5.2. Gas chromatography/mass spectrometry The oil sample (5 mg) was dissolved in chloroform (1 ml) and 0.6 ml of the solution was similarly injected by a splitless technique into a Fissions GC model 8000 gas chromatograph coupled to a mass spectrometer, MD 800 quadruple analysis operating at 70 eV. The capillary column was a HP50 + (30 m ×0.25 mm i.d. × 0.25 mm film thickness). The column temperature was programmed at 50–210°C at 3°C/min, and the injector temperature was 220°C. The carrier gas was helium (5 psi). The components of the oil were identified by their retention times and by comparison of their mass spectra with those from Wiley Library software.
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3. Results and discussion Table 1 shows the activity of lippia oil and other agents on bodylice. While lippia oil proved superior to benzyl benzoate and Delvap Super® (dichlorvos), it was not as effective as kerosene. Benzyl benzoate had no activity against bodylice, while Delvap Super® was not effective at prescribed concentration range (1 in 1000 – 1 in 200). Kerosene significantly improved the activity of Delvap Super® at 1 in 100 dilution. The lice treated with pure benzyl benzoate and Delvap Super® (1 in 200) looked quite dead initially, they later recovered at varying time intervals after the 2 h observation period. The smaller bodylice were more susceptible to lippia oil. This could be related to the chitinous shield of the louse which Table 1 Pediculocidal activities of lippia oil and other agents against bodylice (Pediculus humani corporis)a Agent/conc.
‘Knockdown’ time (min)b (Size range) 53.5mm
4.0–5.0 mm
]5.5 mm
Lippia oil (% 6/6) 100 3.49 90.97 50 4.94 9 0.77 25 10.69 90.84 20 13.87 9 2.46 15 15.63 9 1.59 10 23.38 9 2.93
5.299 0.59 9.009 1.00 19.449 1.81 22.94 9 1.80 24.30 93.27 33.03 94.57
6.55 90.41 11.64 9 2.13 23.559 1.60 ND 25.75 91.46 35.4492.88
Benzyl benzoate (% 6/6) 100 NE 25 NE
NE NE
NE NE
Del6ap Super ® Concentrate 1 in 100 1 in 200 1 in 1000
(dichlor6os) 20.97 94.25 63.67 95.03 NE NE
31.759 4.71 77.339 7.09 NE NE
35.09 9 3.80 92.259 14.62 NE NE
Del6ap Super ®+kerosene (1 in 100) 0.35 90.05 Kerosene 1.33 90.30
0.46 9011 1.94 9 0.77
0739 0.08 3.149 0.13
Light liquid paraffin BP
NE
NE
NE
Each value represents means 9S.D. from at least six bodylice. ND, not determined; NE, no effect. b Direct contact method. a
Table 2 Pediculocidal activities of lippia oil and other agents against headlice (Pediculus humani capitis)a Agent/conc.
‘Knockdown’ time (min) Direct contactb
Direct contact in enclosed systemc
Lippia oil (%v/v) 100 2.33 90.91 50 4.08 90.94 25 8.82 91.13 20 10.59 91.84 15 12.00 91.19 10 21.95 92.83
1.68 90.28 2.45 9 0.46 5.83 90.16 6.15 9 0.82 9.20 9 1.05 16.50 9 2.18
Benzyl benzoate (%6/6) 100 22.36 95.93 25 57.09 910.96
21.92 93.72 58.25 96.94
Del6ap Super ® (dichlor6os) Concentrate 20.33 94.51 1 in 100 50.33 92.31 1 in 200 85.60 9 11.44 1 in 1000 NE
19.87 92.53 51.2094.06 84.0599.92 NE
Del6ap Super ®+kerosene (1 in 100) 0.4190.08 Kerosene 1.25 90.17
0.38 9 0.10 1.10 9 0.24
Light liquid paraffin BP
NE
NE
Each value represents means 9S.D. n =6. c n =5; NE; no effect. a
b
becomes harder with age (James and Hardwood, 1969), thus reducing its permeability to the agent. Hence, the increase in the knockdown time observed for the older lice. The headlice were more susceptible to the agents than bodylice (Table 2). The sizes of headlice used in the study varied between 1.0 and 2.5 mm. At 25% v/v concentration, lippia oil was found to be more effective than benzyl benzoate of the same concentration. However, kerosene was more effective than lippia oil, benzyl benzoate and Delvap Super® concentrate. The apparent higher efficacy of kerosene, therefore, justifies its use by the local populace in treating body and headlice and its use in the prison cells as dilution medium for Delvap Super®. Lippia oil appears to be more effective in an enclosed system after
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direct contact with headlice (Table 2). Such method of application has no significant effect on the knockdown time obtained for benzyl benzoate, Delvap Super® and kerosene (P = 0.05), which are relatively non-volatile. Most pediculocidal agents act directly on the louse by entering their bodies through the external chitinous skeleton (Scabbie, 1945), however, the enhanced efficacy of lippia oil using an enclosed system indicated that the oil vapour, consisting of the highly volatile constituents of the oil have lethal effect on the headlice. The anti-scabies properties of lippia oil and that of benzyl benzoate are indicated in Table 3. Since the benzyl benzoate was applied at maximum concentration of 25%v/v (P.Cx., 1979), comparison between the lippia oil and benzyl benzoate was limited to concentrations between 10 and 25% v/v. The percentage of subjects cured with lippia oil preparations was found to be significantly different (P= 0.05) from those of benzyl benzoate under treatment schedule A. In contrast, a comparison of percentages cured obtained with lippia oil and benzyl benzoate preparation under treatment schedule C indicated no significant differ-
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ence in their efficacy. The t-test value calculated for the two agents at concentrations between 10 and 25% v/v was 2.611, while the t-test value at 3 degrees of freedom was 3.182 (P= 0.05). However, the percentages cured using 20% v/v concentration of lippia oil preparation under treatment schedules A and C were 75 and 100%, respectively, compared with 62.5 and 87.55% obtained for benzyl benzoate preparation at the same concentration (Table 3). It could, therefore, be inferred from the result that while a 20%v/v lippia oil preparation would possess enough scabicidal power to eradicate the mite infestation, a 25% v/v preparation of benzyl benzoate will be required to give similar effect. Statistical analysis of treatment schedules A, B and C for lippia oil preparations using analysis of Variance (ANOVA) shows significant difference between them (P= 0.05). It is obvious from Table 3 that the lippia oil preparations may have to be applied for 3–5 consecutive days before it can effectively eradicate the mites infestation. A further comparison of treatment schedules A and C for lippia oil preparations using Student’s t-test showed significant difference at P= 0.05, indicat-
Table 3 Scabicidal activities of lippia oil and benzyl benzoate preparationsa Agent/conc.b
Number curedc Treatment schedule A
Treatment schedule B
Treatment schedule C
Lippia oil (%6/6) 10 15 20 25 30 40 50
1 4 6 6 7 8 7
(12.5) (50) (75) (75) (87.5) (100) (87.5)
0 (0) 2 (25) 3 (37.5) 5 (62.5) 6 (75) 5 (62.5) ND
3 6 8 8 8 7 8
(37.5) (75) (100) (100) (100) (87.5) (100)
Benzyl benzoate (%6/6) 10 15 20 25
0 2 5 4
(0) (25) (62.5) (50)
0 1 3 3
1 4 7 8
(12.5) (50) (87.5) (100)
Light liquid paraffin BP
ND
a
(0) (12.5) (37.5) (37.5)
ND
0 (0)
Treatment schedule A = 3 consecutive days; treatment schedule B = 3 alternate days; treatment schedule C = 5 consecutive days. Eight subjects were used for each concentration of test agent/treatment schedule. c Figures in parenthesis are percentages cured; ND, not determined. b
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Table 4 Composition (%) of the essential oil of Lippia multiflora Compound
% Composition
a-Pinene b-Pinene b-Cymene Limonene Linalool Terpineol Geraniol Thymol Citronellyl acetate
1.0 4.0 8.4 15.4 26.7 2.9 20.4 4.2 1.8
ing that the 5 consecutive days treatment was more effective. Therefore, the notion that patients with uncomplicated scabies may not require more than two appropriate applications of anti-scabies agent (Commens, 1994) may not hold for lippia oil preparations. A 20% v/v lippia oil preparation which was found effective using 3 or 5 consecutive days treatment schedule gave a low percentage cured when the treatment schedule was changed to 3 alternate days (Table 3). In an earlier trial study, the lippia oil and benzyl benzoate at 25% v/v concentration were applied on six scabietic subjects as single application. Treatment failure was recorded with both agents. Such failure could be due to the fact that the incubation period of the mite’s ova is 3–4 days (Mackenzie, 1941). Therefore, if the ova remain viable within the thickened skin, larvae might appear after the single application. Again, the extent of the lesions and presence of secondary infections could account for why the single application or short duration of treatment causes treatment failure (Charles and Charles, 1992). In a cell, all known cases of scabies were included in the study, however, the ‘contact’ (who may be acting as a reservoir of the infections) was not treated. These ‘contacts’ could be the source of reinfestation observed. The percentages of subjects reinfested in the ‘awaiting trial’, ‘convicted’ and ‘single’ cells were 23.7, 22.2, and 61.1% for lippia oil, and 33.3, 16.7 and 66.7% for benzyl benzoate, respectively. While the magnitude of the reinfestation varied with the type of cell in which
the subjects were housed, there was no significant difference (P= 0.05) between those subjects treated with lippia oil and benzyl benzoate preparations. Each ‘awaiting trial’ cell or ‘convicted’ cell is a large open room housing up to 20 subjects but with reduced contact among them, while the ‘single’ cell with a floor space of 2.230 m2, originally designed for one subject housed up to six subjects. Such overcrowding resulting in closer contact could be responsible for high incidence of re-infestation observed in the ‘single’ cells. The identified components of lippia oil are listed in Table 4 in order of their elution by the GC. The identity of four major peaks (cymene, linalool, geraniol and thymol) were further confirmed by GC-MS. Terpineol and a-pinene which are present in small quantities have been found to kill bodylice, with terpineol being faster in action (Weston et al., 1997). a- and b-pinene are also known insecticides (Windholz, 1983). The scabicidal activity of lippia oil is likely to be due, at least in part, to these three compounds. Similarly, oils of juniper, eucalyptus, lavender, geranium, lemon, rosemary and cypress which contain terpineol, camphene and a-pinene have lethal effects on head and bodylice (Weston et al., 1997). From the above findings, the essential oil of L. multiflora may be considered a potential pediculocidal and scabicidal agent when compared with benzyl benzoate, which presently is the drug of choice especially for the management of scabies. A concentration of 20% v/v of the oil in a suitable formulation basis applied for 5 consecutive days may be sufficient for treating mite infestation. Application of an occlusive covering or a cap for treatment of headlice will enhance the activity of the oil. Further studies on the lethal effect of the oil on the lice nits will determine the treatment duration for complete eradication of headlice infestation.
Acknowledgements The authors express their gratitude to the following people. The staff and medical personel of the Nigerian Prison Services, Ilesa, Nigeria for providing an enabling environment for the study.
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Professor E. Dagne, Department of Chemistry, Addis Ababa University, Ethiopia for the GC-MS analysis of the lippia oil.
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