DEVELOPMENT OF HERBAL MOSQUITO-REPELLENT FORMULATIONS AND THEIR COMPARATIVE EVALUATION

Objective: The present research work aimed to develop herbal mosquito repellents as gels, incense sticks, and liquids for plug-in devices and also to assess their performance characteristics. Methods: Herbal materials possessing mosquito-repellent activity were selected from the literature review based on percentage repellency and protection period. In this study, lavender, rosemary, lemongrass, and cedarwood oils were selected as actives. Span 20 and tween 80 were used to emulsify the volatile materials and then incorporated into Carbopol 934 base to form F1 and F2 gels which were evaluated for homogeneity, pH, spread ability, viscosity, and extrudability. Incense sticks were developed using charcoal and jigat as base materials. The prepared incense sticks F3 and F4 were evaluated for burning time, ash weight, and smoke. Solutions F5 and F6 were prepared using surfactants and cosolvents and were evaluated for phase separation. All formulations were screened for their mosquito repellency by screen cage method and their performance was compared to that of the marketed herbal repellents. Results: Reared mosquitoes were identified as Culex species by microscopical observations of the antenna and maxillary palps. Mosquito repellency in 4 h is 93%, 87%, 89%, 79%, 89%, and 85% for F1, F2, F3, F4, F5 and F6, respectively. Among all, gel formulation F1 expressed the highest mosquito- repellent activity with 93% and is a better candidate. Conclusions: Gel, incense sticks, and liquids prepared for use in plug-in devices exhibited reasonably good percentage repellency comparable to that of the standard. All the developed formulations are suitable for indoor protection from mosquito bites. However, safety, stability, and field studies need to be carried out to demonstrate suitability for regular use


INTRODUCTION
Vectors are the organisms that transmit a parasite or pathogens from one infected person to another causing serious infections. According to the WHO, every year deaths due to vector-borne diseases account for over 17% with more than one billion cases and over one million deaths. In the Indian scenario, malaria, dengue, chikungunya, filariasis, encephalitis, and kala-azar are of major concern [1]. National Vector-Borne Disease Control Program data of 2012 indicated that 50% of total malaria cases are due to Plasmodium falciparum [1,2]. Lancet's study claims that the malaria toll in India is 40 times the government count [2].
Malaria is caused by the genus Plasmodium, namely P. falciparum, P. vivax, P. ovale, and P. malariae. Vectors for the genus Plasmodium are various mosquito species Anopheles (A. arabiensis, A. gambias, A. funestus, and A. stephensi), Aedes (A. aegypti and A. albopictus), and Culex falciparum [3]. The disease is transmitted through mosquito bites. Both humans and mosquitoes are considered to be parasite's hosts. Anopheles and Culex species are most active during the dawn and dusk and also at night whereas, Aedes species are active during the day time. Breeding sites for Anopheles and Aedes species are fresh water bodies and Culex species are polluted stagnant water bodies [4].
A few interventions to reduce the disease burden are the elimination of breeding sites, sanitation hygiene, and regular waste removal. For indoor protection, spraying insecticides, fitting doors and windows with wire mesh, appropriate clothing, mosquito nets, and use of mosquito-repellent creams, plug-in devices, coils, incense sticks, etc., [2,5,6]. Long-lasting insecticide nets (LLINs) are advocated by the WHO as they offer both physical and chemical protection. The use of LLINs reduced malaria incidence by 50% in sub-Saharan Africa which accounts for more than 90% of global cases. The durability and cost of nets are reasons for concern [7].
Commercially available mosquito repellents contain synthetic chemicals which are toxic and can cause skin/eye irritation, cough, and asthma and their long-term use is unwarranted in children, the elderly, and pregnant women [8,9]. The literature review indicated various herbs and essential oils, namely lavender, mint, sage, lemongrass, citronella, rosemary, basil, camphor, cedar, clove, eucalyptus, lemon eucalyptus, geranium, chamomile, peppermint, neem, pyrethrum to possess mosquito-repellent activity [8][9][10][11][12]. Researchers were successful in developing herbal formulations and many are available in the market for regular use.
The present research work aimed to develop a herbal formulation by designing it as a gel, incense stick, and solution for plug-in device and to evaluate and compare the repellent activity of the formulations with the marketed products.

Preparation of incense sticks
Incense sticks were prepared following the procedure mentioned by Bahadur et al., 2020 [14]. The formulated incense sticks contain a total of 25% w/w of actives. All the base ingredients, neem, charcoal, jigat, and camphor, were finely powdered and passed through a sieve and mixed thoroughly. Oils were added and mixed to attain uniformity. Water Qs were used to attain a dough-like consistency. Small quantities were hand rolled and dried in shade for 24 h.

Preparation of liquid for plug-in device
The liquid for the plug-in device was prepared following the procedure mentioned by Sekar and Rahim., 2017 [15]. The formulated liquid contains a total of 25% w/w of active ingredients. Solution of oils in ethanol was added to the aqueous phase containing tween 80. Emulsification was achieved by magnetic stirring for 30 min.

Evaluation of emulgel formulation Physical evaluation
The formulated gel was visually evaluated for color and transparency. The smoothness of the gel was evaluated by simply rubbing the formulation between fingers to feel for smoothness, clumps, roughness, and homogeneity [16].

pH
The pH of the gel was evaluated with the digitally calibrated pH meter. 1 g of the formulated gel was dissolved in 25 mL of distilled water. The measurement of pH was performed in triplicate and the average reading was calculated [16].

Spreadability
Two gram of formulated gel was sandwiched between two similar glass slides. 200 gm weight was applied on the slides to get rid of the entrapped air to form a uniform film between the slides. The top slide was dragged and the time taken to separate the glass slides from one another was determined from the formula: where M = weight placed on the upper slide (200 g); L = length of a glass slide (6 cm); T = time taken (sec) to separate the glass slides from one another [16].

Viscosity
The viscosity of the formulated gel was determined using the digital Brookfield viscometer using spindle no. 64 at 10 rpm and temperature of 25±1°C. The corresponding dial reading was noted [16].

Preparation of emulgel
Emulgels were prepared following the procedure mentioned by Kumar et al., 2022 [13], and a flow chart of the same is indicated in Fig. 1. The formulated gel contains total of 25% w/w of active ingredients.

Screening of incense sticks for mosquito-repellent activity
100 mosquitoes were transferred carefully into the net cage (60 cm × 50 cm). The screen cage was placed in the center of the room and an incense stick was lit and placed at a distance adjacent to the cage. The duration of the study was 4 h. The behavior of the mosquitoes was compared with the positive control (black dragon). The experiment was performed in triplicate [14].

Screening of liquid for mosquito-repellent activity
One hundred mosquitoes were transferred carefully into the net cage (60 cm × 50 cm). The formulated liquid in plug-in device was placed at one corner of the net cage. The duration of the study was 4 h. The behavior of the mosquitoes was compared with the positive control (Good night). The experiment was performed in triplicate.

Evaluation of emulgels
Gel formulations F1 and F2 were evaluated for appearance, homogeneity, and washability and the results are presented in Table 4.
The data obtained for spreadability, pH, extrudability, and viscosity are indicated in Table 5. The pH of the gel formulations lies in the normal pH range of the skin. Spreadability studies demonstrated that they spread uniformly with minimum shear. The extrudability is satisfactory indicating good flow. Table 6 includes data for burning time, ash weight, odor, irritability, and smoke visibility. The odor was pleasant and the subject did not experience any irritation.

Evaluation of liquid for plug-in device
No phase separation was observed in 24 h.

Identification of mosquito species
The observations specified below indicated that the reared mosquitoes are Culex species.

Mosquito-repellent activity
The formulated emulgels F1 and F2 were evaluated for mosquitorepellent activity and compared with the positive control (odomos).
Mosquito repellency in 4 h was determined for F1 and F2 and is 93% and 87%, respectively. The standard product (odomos) demonstrated 98% repellency. F1 displayed better results than F2.

Mosquito-repellent activity
The formulated incense sticks were evaluated for mosquito-repellent activity and compared with the positive control (black dragon).
Mosquito repellency in 4 h was determined for F3 and F4 and is 89%, and 79%, respectively. The standard product (Black dragon) demonstrated 100% repellency. F3 showed better results than F4.

Mosquito-repellent activity
The formulated liquid for plug-in device was evaluated for mosquitorepellent activity and compared with the positive control (Good night).
between two glass slides. A weight of 500 gm was placed on them and simultaneously opened the cap. The length of the extruded ribbon in 10 min was noted. The test was carried out in triplicate [16].

Washability
The ease of wash ability of gel with water was observed visually after application [16].

Evaluation of incense sticks formulation [17]
Burning time and ash The burning time of incense sticks varies depending on thickness, length, and quality. The time taken for the incense sticks to burn down (in min) was recorded. The total ash obtained was weighed.

Odor and irritability
The incense sticks were observed for odor and irritation to the eyes or throat on burning.

Smoke visibility
The visibility of smoke was observed.

Phase separation
The stability of the formulation was evaluated by visually observing the phase separation of the liquid.

Collection and rearing of mosquitoes
Mosquito larvae were collected from the stagnant waters of Hussain Sagar Lake, Hyderabad. They were fed using fish food and the pupae were allowed to emerge into adults. Sucrose solution was used as mosquito feed. Adult mosquitoes were transferred into another net cage to test mosquito-repellent activity [18].

Identification of mosquito
Larvae and adult species were identified by morphological and microscopical examination [19]. Fig. 3 depicts the features of larvae and adults.
Screening of emulgels for mosquito-repellent activity 100 mosquitoes were transferred carefully into the net cage (60 cm × 50 cm). Initially, the volunteer's forearm was thoroughly washed with soap and dried. The right forearm acts as control and was placed in the net cage for 30 s to assess their activity. A minimum of 10 landings is an indication of the suitability of commencement. If the mosquitoes were more than 10, the study was commenced. The left forearm was treated with the mosquito-repellent gel formulation and the untreated skin was covered with a glove. The forearm was placed inside the net cage for 3 min and the number of mosquitoes that landed and probed the skin was recorded.    control 45  37  18  55  F3  48  43  9  52  F4  51  43  6  49  2  Positive control 28  37  35  72  F3  30  52  18  70  F4  34  53  13  66  3  Positive control 10  34  56  90  F3  22  52  27  78  F4  28  51  27  72  4  Positive control l0  29  71  100  F3  11  54  35  89  F4  21  52  27  79 Mosquito repellency in 4 h was determined for F5 and F6 to be 89%, and 85%, respectively, whereas the standard product (Good night) with 96% repellency. F5 showed better results than F6.

CONCLUSION
Selected volatile oils were formulated as gels, incense sticks, and liquids for in plug-in devices. The actives were selected based on protection time and % repellency. The formulations were studied for mosquito-repellent activity using net cages. Results obtained indicated formulations containing lemongrass oil possess better repellency. Those containing cedarwood are slightly inferior. Gel F1 exhibited 93% repellency in 4 h and is a better candidate. The performance of all formulations is comparable to the marketed formulations. All the developed formulations are suitable for indoor protection from mosquito bites.
Further studies to demonstrate safety, stability, and field experiences to demonstrate suitability for regular use need to be carried out.

ACKNOWLEDGMENT
• The authors are thankful to the principal and management of Sri Venkateshwara College of Pharmacy, Madhapur, Hyderabad, for providing the necessary facilities to carry out the research work. • We thank Dr. Bannoth Reddya Naik, M.Sc., Ph.D, Professor, Department of Zoology, Osmania University, Hyderabad, for offering infrastructure assistance.

AUTHORS CONTRIBUTION
• All authors have contributed in the designing and executing the research work and preparation of the manuscript.