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98 Current Research in Microbiology and Biotechnology Vol. 1, No. 3 (2013): 98-103 Research Article Open Access ISSN: 2320-2246 Study of Citronella leaf based herbal mosquito repellents using natural binders Nandini Rani, Aakanksha Wany, Ambarish Saran Vidyarthi and Dev Mani Pandey* Department of Biotechnology, Birla Institute of Technology, Mesra, Jharkhand- 835215, India * Corresponding author: Dev Mani Pandey, email: [email protected] ABSTRACT Citronella grass has been serving from years as a mosquito repellent in the field of ancient and modern medicine. Commercially available mosquito repellents are chemical based and disastrous to human health. An attempt has been made to prepare a 100% herbal product based on Citronella leaf remains which is left out and of no use after steam distillation. It is cheap, effective and environment friendly. It is a first and preliminary work based on formulating and evaluating herbal mosquito repellent cakes using natural binders such as neem powder, potato starch, corn starch, coconut shell powder, wood powder and cow dung. The efficacy of prepared citronella leaf cakes were evaluated on three different parameters such as flammability, burning time and mosquito repellency test. Also, the cakes were sprayed with different concentrations of Citronella oil. Based on the results obtained from these parameters, the residual percentage of each combination of cakes was calculated and it suggested that Neem powder cake has the most effective repellency activity when impregnated with 10% Citronella oil. Keywords: Citronella leaf cakes; flammability; mosquito repellent; natural binders; residual percentage INTRODUCTION Controlling mosquitoes is of utmost importance in the present day scenario with rising number of mosquito borne diseases. An alarming increase in the range of mosquitoes is mainly due to deforestation, industrialized farming and stagnant water. Thus, special products like mosquito repellents for combating mosquitoes are required. The products used for mosquito control have varying degrees of effectiveness. Carbon dioxide and lactic acid present in sweat in warm-blooded animals act as an attractive substance for mosquitoes. The perception of the odor is through chemoreceptors present in the antennae of mosquitoes. Insect repellents work by masking human scent. A number of natural and chemical mosquito repellents were studied in many research papers and review papers [3, 4, 7, 9, 10, 13, 12] that work to repel mosquitoes. Mosquito repellents based on chemicals has a remarkable safety profile, but they are toxic against the skin and nervous system like rashes, swelling, eye irritation, and worse problems, though unusual including brain swelling in children, anaphylactic shock, and low blood pressure [10, 11]. Hence, natural mosquito repellents were preferred over chemical mosquito repellents. All of the mosquitoes coils registered and sold contain solely synthetic pyrethroid, for example, d-allethrin, d- transallethrin and transfluthrin as active ingredients. These coils provide a high degree of reduction in numbers of host-seeking mosquitoes [16, 17, 5]. However, many people still dislike smell of the mosquito coils containing synthetic pyrethroid when they are burned; and these people also feel that the coils may be harmful for their health. Attempts have been made to find out new active ingredients, especially those derived from natural plants to replace the synthetic pyrethroid [4]. The nine potential plants namely Greater galangale (Alpiniaga langa), Fingerroot (Boesenbergia pandurata), Turmeric (Curcuma longa), Cardamom (Elettaria cardamomum), Neem (Azadirachta indica), Siamese cassia (Cassia siamea), Citronella grass (Cymbopogon nardus), Eucalyptus (Eucalyptus citriodora) and Siam weed (Eupatorium odoratum) that expressed high degree of repellency against mosquitoes are recommended as new active ingredients for inclusion in mosquito coil formulations. These were studied for their efficacy in reducing human-mosquito contact when used in mosquito coils in urban areas of Received: 15 April 2013 Accepted: 27 April 2013 Online: 04 May 2013 http://crmb.aizeonpublishers.net/content/2013/3/crmb98-103.pdf

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Current Research in Microbiology and Biotechnology Vol. 1, No. 3 (2013): 98-103 Research Article Open Access

IISSSSNN:: 22332200--22224466

Study of Citronella leaf based herbal mosquito repellents using natural binders Nandini Rani, Aakanksha Wany, Ambarish Saran Vidyarthi and Dev Mani Pandey* Department of Biotechnology, Birla Institute of Technology, Mesra, Jharkhand- 835215, India * Corresponding author: Dev Mani Pandey, email: [email protected]

ABSTRACT Citronella grass has been serving from years as a mosquito repellent in the field of ancient and modern medicine. Commercially available mosquito repellents are chemical based and disastrous to human health. An attempt has been made to prepare a 100% herbal product based on Citronella leaf remains which is left out and of no use after steam distillation. It is cheap, effective and environment friendly. It is a first and preliminary work based on formulating and evaluating herbal mosquito repellent cakes using natural binders such as neem powder, potato starch, corn starch, coconut shell powder, wood powder and cow dung. The efficacy of prepared citronella leaf cakes were evaluated on three different parameters such as flammability, burning time and mosquito repellency test. Also, the cakes were sprayed with different concentrations of Citronella oil. Based on the results obtained from these parameters, the residual percentage of each combination of cakes was calculated and it suggested that Neem powder cake has the most effective repellency activity when impregnated with 10% Citronella oil.

Keywords: Citronella leaf cakes; flammability; mosquito repellent; natural binders; residual percentage

INTRODUCTION Controlling mosquitoes is of utmost importance in the present day scenario with rising number of mosquito borne diseases. An alarming increase in the range of mosquitoes is mainly due to deforestation, industrialized farming and stagnant water. Thus, special products like mosquito repellents for combating mosquitoes are required. The products used for mosquito control have varying degrees of effectiveness. Carbon dioxide and lactic acid present in sweat in warm-blooded animals act as an attractive substance for mosquitoes. The perception of the odor is through chemoreceptors present in the antennae of mosquitoes. Insect repellents work by masking human scent. A number of natural and chemical mosquito repellents were studied in many research papers and review papers [3, 4, 7, 9, 10, 13, 12] that work to repel mosquitoes. Mosquito repellents based on chemicals has a remarkable safety profile, but they are toxic against the skin and nervous system like rashes, swelling, eye irritation, and worse problems, though unusual including brain swelling in children, anaphylactic shock, and low blood pressure [10, 11]. Hence, natural mosquito repellents were preferred over chemical mosquito repellents.

All of the mosquitoes coils registered and sold contain solely synthetic pyrethroid, for example, d-allethrin, d-transallethrin and transfluthrin as active ingredients. These coils provide a high degree of reduction in numbers of host-seeking mosquitoes [16, 17, 5]. However, many people still dislike smell of the mosquito coils containing synthetic pyrethroid when they are burned; and these people also feel that the coils may be harmful for their health. Attempts have been made to find out new active ingredients, especially those derived from natural plants to replace the synthetic pyrethroid [4]. The nine potential plants namely Greater galangale (Alpiniaga langa), Fingerroot (Boesenbergia pandurata), Turmeric (Curcuma longa), Cardamom (Elettaria cardamomum), Neem (Azadirachta indica), Siamese cassia (Cassia siamea), Citronella grass (Cymbopogon nardus), Eucalyptus (Eucalyptus citriodora) and Siam weed (Eupatorium odoratum) that expressed high degree of repellency against mosquitoes are recommended as new active ingredients for inclusion in mosquito coil formulations. These were studied for their efficacy in reducing human-mosquito contact when used in mosquito coils in urban areas of

Received: 15 April 2013 Accepted: 27 April 2013 Online: 04 May 2013

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Thailand. The potential of volatile oils extracted from turmeric, citronella grass and hairy basil as topical repellents against both day- and night-biting mosquitoes has been demonstrated [13]. In the case of Citronella species, for example, the components present in the oil are responsible for the desirable repellent characteristics of the plant against mosquitoes [2]. The repellent efficiency of 38 essential oils against mosquito bites was compared, including the species Aedes aegypti and found Citronella oil as the most effective and provided 2 hours of repellency [14, 15]. The chemical composition of citronella oil was studied and it was found that the crude essential oil consists of active ingredients that markedly suppressed the growth of several species of Aspergillus, Penicillium and Eurotium [6]. The most active compounds among the 16 volatiles examined in Citronella oil, consisting of 6 major constituents of the essential oil and 10 other related monoterpenes, were citronellal and linalool. Most of the essential oil-based repellents tend to give short-lasting protection for less than two hours. Citronella oil has demonstrated good efficacy against 44 mosquitoes in concentrations ranging from 0.05 % to 15 % (w/v) alone or in combination with other natural or commercial insect repellent products [1]. The characteristic of the oil is due to the presence of four main components, citronellal, eugenol, geraniol and limonene [8, 11]. A coil product preparation using sawdust, rice husk and corncob based fillers along with herbal oils and herbal powders indicating towards the improvisation of mosquito repellents by supplementing it with natural fillers and binders have been devised [10]. The present study was carried out to evaluate repellency of mosquito cakes derived from plants. An attempt has been made to develop a Citronella based herbal mosquito repellent cake which is more effective, cheap and keeps environment pleasant and health friendly using different binders such as sawdust, coconut shell powder, Neem powder etc. MATERIALS AND METHODS Collection of Citronella leaf remains left after steam distillation Citronella (Cymbopogon winterianus) leaf remains were collected from Pharmaceutical Medicinal Plant garden, BIT Mesra, Ranchi, India after steam distillation from steam distillation plant. Bundles of leaves were cut into small pieces with the help of sterile sharp scissors. Preparation of Citronella leaf cakes Cut leaf pieces were grounded into paste using electrical grinder by adding distilled water. 50-100g of Citronella leaf paste was taken and was plated accordingly (Fig 1). Wet weights of cakes were taken. For the determination of dry weight cakes were allowed to dry in sun for 24 hrs and dry weight was taken.

Figure 1. Preparation of Citronella leaf cake and drying

Formulation of cakes using different natural binders The different natural binders (500g each) were purchased commercially from local vendors. Different natural binders used were wood powder, potato starch, corn starch, coconut shell powder, neem powder and cow dung. The same procedure was followed for different combinations using different natural binders for the preparation of cakes. Supplementation of Citronella leaf paste with binders and impregnation with oil Each cake was prepared as 20% (w/w) binder (i.e. wood powder, coconut shell powder, Neem powder, cow dung, cornstarch, potato starch) in each cake formulation, whereas the citronella leaf paste (80%) in all cake formulations was same. As a result, a total of six cake formulations comprising different binders with Citronella leaf as active ingredients were prepared for testing. Citronella leaf cakes with no supplementation were used as reference cake. Wet weight and dry weight of each cake were taken after 24 hours of drying. Simultaneously, different concentrations of citronella oil such as 5%, 10%, 15% using methanol as a carrier alcohol was evenly sprayed on different combinations of cakes. Ten cakes (replicates) were prepared from each combination. Evaluation of mosquito repellent activity For investigating mosquito repellent activity the prepared cakes were checked for its flammability, burning efficiency with respect to burning time and eventually its effective repellent activity. Flammability test of these cakes were conducted to check its consistent combustibility. Further the time taken to burn the cake, smoke produced and its causal effect such as irritation, coughing, tears were observed and recorded. Ash produced by cakes were weighed and recorded. The cakes were burnt in selected mosquito prone areas in the evening and night period such as bushes, shrubs, laboratory corners, department premises and cafeteria. RESULTS AND DISCUSSION In the present study we aimed to formulate a natural, herbal mosquito repellent based on citronella leaves which are a natural source of essential oils. Among the plant families with promising essential oils used as mosquito repellents, Cymbopogon spp., Ocimum spp. and Eucalyptus spp. are the most cited [12]. Individual compounds present in these mixtures with high

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repellent activity include α-pinene, limonene, citronellol, citronellal, camphor and thymol. Still, synthetic chemicals are still more frequently used as repellents than essential oils, these natural products have the potential to provide efficient, and safer repellents for humans and the environment. Collection of plant material In pharmaceutical medicinal plant garden, BIT Mesra, steam distillation plant is operated for extraction of citronella oil from Cymbopogon winterianus (Java Citronella). After steam distillation the left out leaves were collected for this study and it was selected as the base for mosquito repellent citronella leaf cakes. Approximately, three kg of Citronella leaf remains were

collected from steam distillation plant and were finely grounded. Preparation of Citronella leaf cakes in combination with natural binders For the preparation of mosquito repellent coils, different fillers can be used, however, in this study natural binders were utilized [10, 4]. Cow dung based formulation along with optimized ingredients like Neem, Tulsi, rice; sawdust etc. has also been reported [4]. Citronella leaf cakes were prepared by plating 60g of leaf paste in a sterile petri dish. Wet weight of cakes and dry weights (after 24 hours) of prepared cakes were recorded (Table 1 and Fig 2). A significant amount of water loss is recorded i.e. 53.57g in all combinations.

Table 1. Average wet and dry weights of Citronella leaf cakes

S. No Name of the sample

Average wet

weight* (g)

Average dry weight*

(g)

Amount lost (g)

1 Citronella Leaf Paste 54.48 7.53 46.95 2 Leaf Paste + 20% Wood powder 65.03 12.64 52.39 3 Leaf Paste + 20% Neem powder 74.68 16.14 58.54 4 Leaf Paste + 20% Potato starch 70.63 18.82 51.81 5 Leaf Paste + 20% Corn starch 71.35 22.00 49.35 6 Leaf Paste + 20% Cow dung 76.69 14.88 61.81

7 Leaf paste + 20% Coconut shell powder 63.16 9.00 54.16

Average = 53.57

*The wet and dry weights are average of 10 replicates

Table 2. Parameters to check flammability of citronella leaf cakes

S. No. Name of the sample

Dry weight

(g)

Ash weight

(g)

Time taken to

burn (Minutes)

Residual (%) Irritation Remarks

1 Citronella leaf 7.43 3.23 12 43.47 Less Irritation Fully burnt

2 Citronella + 20% wood powder 13.59 2.9 21 21.34 Less Irritation Fully burnt

3 Citronella + 20% coconut shell powder 9.5 2.4 6 25.26 Less Irritation Fully burnt

4 Citronella + 20% Neem powder 21.29 5.27 22 24.75 Less Irritation Fully burnt

5 Citronella + 20% cowdung 13.48 5.22 19 38.72 High Irritation Fully burnt

6 Citronella + 20% corn starch 21.17 9.85 26 46.53 No Irritation Not burnt completely

7 Citronella + potato starch 21.21 10.25 36 48.33 Very less Irritation

Not burnt completely

These natural binders are easily available and can be purchased. It provides excellent binding to all the ingredients and holds it strongly together. Suitable binder is the one which gives slow and prolonged burning along with uniform binding ability. Neem powder cake and wood powder cake proved efficient candidates for mosquito repellent activity based on Citronella leaves when added in 20% concentration. Cow dung based cake produced more smoke and thus is a potential antioxidant which is in accordance with the result [4]. Evaluation of Citronella leaf cakes The efficacy of prepared citronella leaf cakes were evaluated on three different parameters such as

flammability, burning time and mosquito repellency test. a) Flammability test and burning time To observe the flammability of the cakes, the cakes were burnt using candles (Fig 3). The quantity of ash, irritation produced by different combination of cakes and the time taken to burn completely were recorded (Table 2). The ash weights of each combination and their burning time were recorded. Depending upon ash weight and dry weight, residual percentage was calculated. Residual percentage is calculated by: Residual (%) = Ash weight / Dry weight X 100

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For a good and consistently burning mosquito repellent cake, it is necessary that the cake should be burnt completely, producing low smoke and irritation and less residual percentage. A high residual percentage suggests incomplete burning of cakes and hence two combinations gave highest residual percentage i.e. corn flour and potato starch (46.53% and 48.33% respectively). Also, the burning time of cakes suggested that the cakes with starchy combinations took much time to burn (26 minutes – corn flour and 36 minutes- potato starch) and thus increased their residual percentage. Subsequently, wood powder, coconut shell powder and neem powder combinations took less time to burn giving less residual percentage. Thus, it can be suggested that the neem powder and wood powder combinations were good for making mosquito repellent cakes as the burning time and residual percentage and irritation caused were very less as compared to other combinations. On the other hand, coconut shell powder, irrespective of giving a lesser residual percentage burnt in a very short period of time i.e. 6 minutes which cannot be selected for formulation of mosquito repellent cakes. Hence, the flammability test and burning time gave two best combinations for formulating mosquito repellent cakes i.e. wood powder and neem powder, wood powder does not have any medicinal property but is easily available. However, neem powder cake is the best option, being medicinally active and its easy availability.

Figure 2. Different combination of Citronella leaf cake

Figure 3. Burning of Citronella leaf cake and its ash Table 3. Mosquito repellency test in different areas of Department of Biotechnology

S. No. Areas Reports given by people Remarks

1 Laboratory corners

Smoke caused irritation Mosquito repelled

2 Department premises Mosquitoes escaped Mosquito repelled

3 Supervisor's room

Mosquitoes moved outside the room Mosquito repelled

4 Cafeteria Mosquitoes escaped Mosquito repelled

Table 4. Parameters to check mosquito repellency of citronella leaf cakes with different concentrations of citronella oil

S. No. Name of the sample

Concentration of citronella

oil (%)

Dry weight

(g)

Ash weight

(g)

Time taken

to burn (mins)

Residual (%) Irritation Remarks

0 7.1 3.27 20 46.05 No No mosquitoes while burning 5 7.87 3.15 15 40.02 No No mosquitoes while burning

10 7.83 3.05 18 38.95 No No mosquitoes while burning 1

Citronella leaf

15 6.98 3.1 15 44.4 No No mosquitoes while burning 0 15.22 3.69 20 24.24 No No mosquitoes while burning 5 13.73 4.28 21 31.17 No No mosquitoes while burning

10 13.96 3.99 18 28.58 No No mosquitoes while burning 2

Citronella leaf + 20% Wood

powder 15 14.41 5.11 19 35.46 No No mosquitoes while burning 0 18.07 9.9 45 54.7 No Not burnt completely 5 19.39 10.25 50 52.8 No Not burnt completely

10 21.21 10.54 40 49.6 No Not burnt completely 3

Citronella leaf + 20% Potato

starch 15 20.26 10.78 45 53.2 No Not burnt completely 0 25.7 9.5 50 36.9 Less Not burnt completely 5 26.09 9.95 45 38.13 Less Not burnt completely

10 24.06 10.01 44 41.6 Less Not burnt completely 4

Citronella leaf + 20% Corn

starch 15 19.79 9.5 40 48.0 Less Not burnt completely 0 12.16 5.69 25 46.7 High No mosquitoes but irritating 5 16.85 7.02 20 41.6 High No mosquitoes but irritating

10 14.1 6.58 22 46.6 High No mosquitoes but irritating 5

Citronella leaf + 20% Cow

dung 15 14.48 6.57 21 45.37 High No mosquitoes but irritating 0 9.26 3.85 18 41.5 Less 2-3 mosquitoes were flying 5 9.39 3.29 15 35.03 Less 2-3 mosquitoes were flying

10 8.86 3.2 20 36.1 Less 2-3 mosquitoes were flying 6

Citronella leaf + 20% Coconut

shell powder 15 8.69 3.19 20 36.7 Less 2-3 mosquitoes were flying 0 16.2 3.91 30 24.1 Less No mosquitoes after 1 hour 5 16.35 5.49 25 33.5 Less No mosquitoes after 1 hour

10 15.72 5.16 20 32.8 Less No mosquitoes after 1 hour 7

Citronella leaf + 20% Neem

powder 15 16.54 4.13 25 24.96 Less No mosquitoes after 1 hour

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b) Mosquito repellency test Mosquito repellency test was done by simply selecting the mosquito prone areas in the evening and night period such as bushes, shrubs, laboratory corners, and cafeteria. The public remarks were noted down after allowing them to burn the cakes and checking if the mosquitoes are present or escaping away from the burning cakes (Table 3). Also, the mosquito repellency was checked by addition of citronella oil mixed with carrier alcohol (methanol) in different concentration i.e. 5%, 10%, 15% (Table 4). Kim et.al (2005) evaluated repellent efficacies of two medicinally active components and two natural aroma compounds citronella and citronellal, against mosquitoes, Culex pipiens pallens, was evaluated both in field and in vitro. The experiment conducted in vitro was carried out with hand bands impregnated with 30% citronella extract, 15% citronella extract and 30% citronellal extract, and with bands impregnated 30% citronella in field. They analyzed the data by the means of counting numbers bitten by mosquitoes per unit time, namely human bait method and percentage repellencies were calculated and statistically confirmed by t-test compared between control group and each experimental group. Based on the above study, different concentrations of Citronella oil (5%, 10% and 15%) mixed with methanol in all the different combinations were tried. 10% addition of Citronella oil in Neem powder mosquito cakes was found to be the best alternative for repellency activity. Citronella grass for their efficacy in reducing human-mosquito contact when used in mosquito coils has been studied [12]. They reported that mosquito coil containing leaves of citronella grass showed highest efficacy whereas that containing rhizomes of turmeric was least effective. Mosquitoes caught in their study included 12 species belonging to five genera (Aedes, Anopheles, Armigeres, Culex and Mansonia), but Cx. vishnui, Culex gelidus and Cx. quinquefasciatus were most predominant species. The best combination of citronella leaf cake was with neem powder showing less residual percentage of 24.1%, less irritating and average burning time of 21 minutes. Similarly, combination of citronella leaf cake with wood powder also showed low residual percentage of 24.2%, no irritation and an average burning time of 20 minutes. The effectiveness of both the combination was further increased by adding 10% citronella oil. Neem powder with citronella leaf as a base is the best option as it has antibacterial and other medicinal properties. Instead nascent citronella leaf cakes can also be used as a mosquito repellent. Thus, a good attempt was made in formulating citronella leaf based herbal mosquito repellent cake comprising Neem powder impregnated with 10% citronella oil. On the basis of public remarks and different areas tested for mosquito repellency, neem powder cake with 0%, and 15% citronella oil addition gave less residual

percentage of 4.1% and 24.96 % and took 30 minutes and 25 minutes for complete burning, respectively. It showed less irritation and was able to repel mosquitoes for more than one hour. Similarly, wood powder cake gave less residual percentage of 24.24% and 28.58% with 0% and 10% citronella oil addition and showed burning time of 20 and 18 minutes, respectively. On the other hand, cakes with starch as a components showed a high residual percentage (potato starch with 0% Citronella oil has a residual percentage of 54.7%, while that of corn starch having 15% oil concentration showed residual percentage of 48 % with an average burning time of 40 minutes). On the other hand, nascent Citronella leaf cake having high residual percentage of 46.05%, showing no irritation has a tendency to repel mosquitoes away from the place. Thus, it can be concluded from the mosquito repellency test that neem powder and wood powder cakes with 10% citronella oil addition showed lesser residual percentage with average burning time of 20 minutes and can be considered as good mosquito repellent cakes because of its consistent burning ability with less irritating smoke and low residual percentage and high mosquito repellent ability. CONCLUSION Currently, the use of synthetic chemicals to control insects and arthropods raises several concerns related to environment and human health. An alternative is to use natural products that possess good efficacy and are environmentally friendly. Among those chemicals, essential oils from plants belonging to several species have been extensively tested to assess their repellent properties as a valuable natural resource. Citronella (Cymbopogon winterianus) oil is the essential oil whose repellent activities have been demonstrated, as well as the importance of its leaf remains which are generally thrown as waste are the main focus of this study. Citronella leaf remains in the form of cakes is the first and preliminary report. In addition, the use of other natural products in the mixture, such as binders, could increase the protection time, potentiating the repellent effect of some essential oils. On the basis of results obtained it can be concluded that the citronella leaf cakes with neem powder as a binder and impregnated with 10% citronella oil having less residual percentage, less burning time and irritation is able to repel mosquitoes effectively is the most suitable mosquito repellent. This report is the preliminary work done using Citronella leaf cakes as herbal mosquito repellents using natural binders. However, further extensive study by collecting specific number of mosquitoes in a glass chamber covered with cloth sieve and exposing them to the smoke generated by the herbal product with the varying concentrations and recording of mortality time and comparing with chemical based formula in the artificial mosquito coils need to be performed.

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