Formulation of an effective mosquito-repellent topical product from Lemongrass oil.
Ointment and cream formulations of lemongrass oil in different classes of base and the oil in liquid paraffin solution have been evaluated for mosquito repellency in a topical application. Mosquito repellency was tested by determining the bite-deterrence of product samples applied on an experimental bird's skin against a 2-day starved culture of Aedes aegypti L. mosquitoes. The 1% v/v solution and 15% v/w cream and ointment preparations of the oil exhibited [greater than or equal to]50% repellency lasting 2-3 h, which may be attributed to citral, a major oil constituent. This activity was comparable to that of a commercial mosquito repellent. Base properties of the lemongrass oil formulations influenced their effectiveness. The oil demonstrated efficacy from the different bases in the order of hydrophilic base > emulsion base > oleaginous base.
Key words: Cymbopogon citratus, essential oil, mosquito repellency, formulation.
Lemongrass oil is the essential oil obtained from the aerial parts of Cymbopogon citratus (DC) Stapf., family Poaceae. The plant has been widely recognized for its ethnobotanical and medicinal usefulness (Dalziel, 1937). The insecticidal (Arias et al., 1992), antimicrobial (Syed et al., 1995), and therapeutic properties (Akendengue, 1992) of its oil and extracts have been reported. Trado-medicinal preparations of the oil have been used both internally for alleviating colds and fever symptoms (Comerford, 1996) and externally to treat skin eruptions, wound and bruises (Spring, 1989). Plant essential oils in general have been recognized as an important natural resource of pesticides -- insecticides (Raguraman and Singh, 1997; Gbolade, 2001), larvicides (Adebayo et al., 1999), and repellents (Thorsell et al., 1998; Oyedele et al., 2000). The repellents are designed as topical preparations or combustible products that are able to protect the user or environment from harmful insects, such as mosquitoes, which trans mit diseases through their bite. The G.C. analysis of Indian Cymbopogon essential oil as well as its repellent effects against mosquito vectors of malaria (Anopheles stephensi), filariasis (Culex quinquefasciatus), and dengue fever (Aedes aegypti) have been described (Tyagi et al., 1998).
Personal protection by the use of repellents is of considerable importance within the integrated disease-vector control endeavour (Curtis et al., 1989). In this light, the present study was carried out to evaluate the protection affordable against Aedes aegypti L. by six formulations of lemongrass oil prepared from different classes of base.
* Materials and Methods
Extraction of lemongrass oil
Lemongrass oil was obtained by hydro distillation (B. P., 1980) from the fresh aerial parts of Cymbopogon citratus growing within Ile-Ife town. The plant was authenticated at the Department of Pharmacognosy (Obafemi Awolowo University, Ile-Ife, Nigeria) herbarium.
Preparation of lemongrass oil formulations
Graded concentrations of lemongrass oil (0.25-25% v/v) in liquid paraffin were prepared. Typical formulations of the oil were also made for repellency testing by incorporating the requisite quantity of oil into selected representative formulation bases to obtain stable products containing 15% v/w of the oil. This concentration of oil was selected based on some preliminary data (Table 1) and corroborated by an earlier study (Oyedele et al., 2000). The following bases were investigated: Oleaginous ointment bases namely, white petrolatum only, and simple ointment containing cetostearyl alcohol (5% w/w), hard paraffin (5% w/w), wool fat (5% w/w), and white petrolatum (85% w/w) (The Pharmaceutical Codex, 1979); surfactant-stabilized cream bases namely, a glyceryl monostearin cream base containing glyceryl monostearate (5% w/w), sodium lauryl sulphate (3% w/w), cetostearyl alcohol (2% w/w), liquid paraffin B.P. (25% w/w), and water (to 100%) (Oyedele et al., 2000), and a cetomacrogol cream base containing cetomacro gol emulsifying wax B.P. (9% w/w), liquid paraffin B .P. (6% w/w), white petrolatum (15% w/w), and water (70% w/w); as well as a hydrophilic ointment base containing macrogol 4000 (20% w/w) and macrogol 600 (80% w/w) (Orafidiya et al., 2001).
Breeding of mosquitoes
Aedes aegypti adult mosquitoes were raised in netted cages (37 x 30 x 28 [cm.sup.3]) under laboratory conditions (25-30[degrees]C, R.H. 60-70 %) from the larval colony already established in our laboratories (Adebayo et al., 1999). Adult female mosquitoes were fed regularly with blood from exposed skin of live chicken and the males with a 10% sugar solution.
When 80-100 adult mosquitoes had been raised, they were fed regularly for 4-5 days and then blood-starved (but water-provided) for the next 48 h before using them for repellency test. The test sample was applied thinly to the experimental bird's bare skin using camelhair brush. The bird was then placed against the netted cage. The rate of biting attack of the test mosquitoes was determined in triplicate experiments of 2-minute exposure of the treated bird's skin to the insects as previously described (Oyedele et al., 2000). The number of mosquitoes that bite within 2 min. was counted and compared to the control bite. (Principles of laboratory animal care, NIH publication No. 85-23, were complied with.) Repellency was calculated as percent bite deterrence ([+ or -] SEM) as previously reported (Oyedele et al., 2000; Gbolade et al., 2000). This was determined at hourly intervals until 5 h following application of the test sample. Results of the tests were compared with those of the bland formulation bases and th at of liquid paraffin (solvent for the oil) as negative controls, and against that of citral (Sigma), a major constituent in lemongrass oil (Ekundayo, 1985). A commercial topical insect-repellent preparation, Wipe(R), containing diethyl toluamide (20%) and dimethyl phthalate (10%) (SmithKline Beecham Pharmaceuticals Plc.) was used as the positive control.
Results and Discussion
Liquid paraffin solutions of lemongrass oil exhibited concentration-dependent repellency (Table 1). High concentrations (20%, 25%) provided complete (100%) protection lasting 1 h. Lower concentrations (1-15%) also exhibited total repellency that was short-lived immediately following application of the respective solutions. The 1% and 5% oil concentrations sustained more than 50% repellency for 3 h while the 10-25% concentrations gave higher protection (>90% repellency) over the same period (Table 1). Citral (15% v/v in liquid paraffin) exhibited repellent effects that were comparable to those of 5% lemongrass oil (Table 1), indicating that this constituent may play an important role in the repellent action of the oil. Tyagi et al (1998) have earlier suggested citronellol and geraniol as other constituents of the oil that may contribute greatly to its protective properties.
In earlier studies, essential oils from Hemizygia and Ocimum species have been found to produce similar concentration-dependent mosquito repellency. Liquid paraffin solutions of Ocimum gratissimum L. and O. basilicum L. oils (1% and higher concentrations) were shown to exhibit bite-protection ([greater than or equal to]70%) for 2-3 h and resulted in complete protection at the 20% and 35% concentrations respectively (Gbolade et al., 2000). Also Hemizygia welwitschii (Rolfe) M. Ashby oil at 5-15% solutions in liquid paraffin was reported to produce 85-100% antifeedant activity for 4 h (Oyedele et al., 2000). These solutions were shown to give total repellency at 12% and 15% concentrations, which lasted 2 and 4 h respectively, and this indicates a better activity compared with lemongrass oil in this present study. Furthermore, Dua et al (1996) have reported more than 50% protection from the bite of Aedes mosquitoes by methanolic extracts of Lantana camara L. flowers for 4 h, while the repellency produced by Euca lyptus oil lasted 5 h (Trigg and Hill, 1996).
Table 2 presents a comparison of the repellent effects of lemongrass oil (15%v/w) in different formulations. This concentration of the oil in each preparation produced an initial total protection, which the hydrophilic ointment sustained for 1 h. The hydrophilic base best supported the repellent action of the oil for 2 h. Throughout the test duration, this ointment demonstrated the highest repellency potential among the others, and was closest to [Wipe.sup.(R)] in activity (Table 2). However, the 15% lemongrass oil solution (Table 1) was superior to [Wipe.sup.(R)] in that it sustained higher repellency for longer duration. The surfactant-based creams were next in efficacy, providing 86-100% protection for 1 h (Table 2). The oleaginous ointments, which consisted mainly of hydrocarbon constituents, were the least effective. Their appreciable activity (>70% repellency) also lasted 1 h (Table 2). In general, the formulations in each particular class of base were similar in their repellent actions. The negative co ntrols exhibited no repellent activity.
Liquid paraffin solution of Hemizygia oil has been reported to be a more effective mosquito repellent than the emulsion formulations containing similar concentrations of the oil (Oyedele et al., 2000). Emulsification of an oil results in entrapment of the dispersed droplets, which hinders release (Rees and Collett, 1975). Another correlation of the properties of formulation base with activity of preparations of an essential oil showed that a hydrophilic ointment of lemongrass oil made from a 1:1 w/w blend of macrogol 4000 and macrogol 400 produced greater antibacterial effects than the same concentration of oil in emulsion products (Onawunmi, 1994). This was attributed to better release from the base.
The repellent effects of all the products dwindled with time, as might be expected. The vapour phase-mediated repellency of an essential oil fades with time obviously due to diffusion and continuous dilution of the vapour in the local convectional air currents, resulting in its gradual reduction and elimination from the vicinity. The observed influence of formulation base types on product repellency thus indicates that the rate of release as vapour of the repellent principle of the essential oil (studied under the same laboratory conditions) was higher from the hydrophilic ointment preparation than from the surf actant and hydrocarbon-based (cream and ointment) products, while the oil solutions most facilitated the release of the repellent. Volatile oils diffuse faster from liquid (solutions) than from the semisolid preparations (Oyedele et al., 2000). The slow release of the volatile repellent principle of lemongrass oil from the oleaginous formulations can thus be attributed to the lipophilic affinity of th e oil and its constituents for the bases, as well as the viscosity of the base.
Six different formulations of lemongrass oil have been evaluated in this study. The mosquito bite-deterrent effect of 15%v/w hydrophilic ointment formulation of the oil is very promising for topical use, and the preparation can be reapplied should a longer effect be required provided no untoward effect is produced.
Table 1 Mosquito repellency of lemongrass oil in liquid paraffin Time (h) after Bite-deterrence (1%) of lemongrass oil solutions application of sample Concentration (% v/v) of oil in liquid paraffin 0.25% 0.5% 1% 0 13 [+ or -] 4 66 [+ or -] 14 100 1 0 14 [+ or -] 7 76 [+ or -] 8 2 0 0 57 [+ or -] 4 3 0 0 51 [+ or -] 8 5 0 0 30 [+ or -] 6 Time (h) after Bite-deterrence (1%) of lemongrass oil solutions application of sample Concentration (% v/v) of oil in liquid paraffin 5% 10% 15% 0 100 100 100 1 80 [+ or -] 10 96 [+ or -] 4 96 [+ or -] 4 2 57 [+ or -] 4 92 [+ or -] 8 94 [+ or -] 6 3 53 [+ or -] 15 93 [+ or -] 5 94 [+ or -] 6 5 40 [+ or -] 35 40 [+ or -] 5 41 [+ or -] 11 Time (h) after Bite-deterrence (1%) of lemongrass oil solutions application of sample Concentration (% v/v) of oil in Positive control liquid paraffin 20% 25% 15% Citral 0 100 100 100 1 100 100 85 [+ or -] 5 2 96 [+ or -] 4 96 [+ or -] 4 65 [+ or -] 5 3 94 [+ or -] 6 94 [+ or -] 6 50 [+ or -] 10 5 41 [+ or -] 18 44 [+ or -] 4 35 [+ or -] 10 Table 2 Mosquito repellency of lemongrass oil formulations Time (h) after Bite-deterrence (%) of preparations containing 15% v/w oil application of sample Oleaginous ointments White Simple petrolatum ointment based based 0 100 100 1 72[+ or -]8 96[+ or -]4 2 28[+ or -]12 47[+ or -]6 3 0 9[+ or -]6 5 0 4[+ or -]4 Time (h) after Bite-deterrence (%) of preparations containing 15% v/w oil application of sample Surfactant-stabilized creams Monostearin Cetomacrogol cream cream 0 100 100 1 86[+ or -]8 87[+ or -]3 2 42[+ or -]8 51[+ or -]6 3 29[+ or -]8 35[+ or -]6 5 14[+ or -]8 13[+ or -]6 Time (h) after Bite-deterrence (%) of preparations containing 15% v/w oil application of sample Hydrophilic ointment Positive control Macrogol ointment Commercial Insect-repellent [Wipe.sup.(R)] 0 100 95[+ or -]4 1 100 84[+ or -]5 2 64[+ or -]4 74[+ or -]4 3 37[+ or -]4 55[+ or -]7 5 22[+ or -]8 43[+ or -]11
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A. O. Oyedele (1)
A. A. Gbolade (2)
M. B. Sosan (3)
F. B. Adewoyin (4)
O. L. Soyelu (3)
O. O. Orafidiya (1)
(1.) Department of Pharmaceutics
(2.) Department of Pharmacognosy
(3.) Department of Plant Science and the
(4.) Drug Research and Production Unit, Obafemi Awolowo University, Ile-Ife, Nigeria
Dr. A. O. Oyedele, Department of Pharmaceutics, Obafemi Awolowo University, Ile-Ife Nigeria. e-mail: firstname.lastname@example.org