INTRODUCTION:

The world is eventually turning to herbal formulations which are known to be effective against a large number of diseases and ailments¹. However, adding an appropriate recognition stating that plant remedies are efficacious without having adverse affects. Many of the herbs and shrubs are found to have medicinal as well as curative values along with mosquito larvicidal and mosquito repellent properties. Because the application of synthetic liniment has a poignant effect on the surroundings as well as non-target organisms; products of plant origin with insecticidal properties have been tried as an aboriginal method to control several insect pests and vectors in the recent past².

Before synthetic ointments came into use, plants and their derivatives were being used to kill the pests of agriculture, veterinary and public health importance. The use of plant-based embrocation is encouraged as they are comparatively less detrimental to the environment, biodegradable and have minimal or no use of a synthetic chemical which may result in causing a health hazard to the user³. Moreover, many potent plants having biologically active constituents that can lead to the discovery of new insecticidal preperations⁴. Therefore plant materials have attracted significant interest in mosquito control programs in recent times⁵.

Plant derivatives such as creams, essential oils, coils, incense, mist based products having plant-based derivatives have shown up in markets with high demand as compared to synthetic products. This has provided researchers with new scopes in the field to come up with more plants possessing mosquito repellent properties. Many polyherbal formulations having added qualities have also become the central element for recent research in this sector.

A mosquito repellent is a substance applied to skin, clothing or other surfaces that rejects insects (and arthropods in general) from infecting that surface. As most of the mosquito repellent products and devices are either of synthetic origin or have an evident effect on human health leading to several complications, the need for a repellent that is more effective and less harmful has arisen^6,7.

Substantial efforts have been made to expound and persuade the use of environmentally friendly insecticides that are not enduring, have low toxicity against the fauna and are moderately safe. Since time immemorial, insect repellent formulations have been found to possess spirits of camphor⁹, oil of tar⁹, oil of Pennyroyal and castor oil⁹ in their composition along with having effective results against the insects and arthropods.

From the Vedic era, various plants like Vitex negundo^10,11,12,13, Ocimum species, Azadirachta indica¹⁴ are used as mosquito repellents or are used as mosquitocidal. Ramasamy R 2001 et. al., performed the development of mosquito repellent fabrics using nanoparticles loaded with V. negundo leaf extract. The nanoparticles were loaded with V. negundo leaf extract using a traditional pad-dry method. The resultant fabrics showed 100% mosquito repellent effectiveness¹⁵. Ranasinghe M.S.N 2014 et. al., reported that the hexane extract of Azadirachta indica seeds, hexane extract of V. negundo leaves, essential oils from Ocimum sanctum leaves, are having promising mosquito repellent activities¹⁶.

Based on the present market scenario of mosque repellants and based on ethnomedicinal claims the present study was aimed to develop and evaluate an herbal mosquito repellent cream incorporating leaf extracts of Vitex negundo, Ocimum gratissimum and Azadirachta indica.

MATERIAL AND METHODS:

Collection of the plant material:

The Vitex negundo, Ocimum gratissimum and Azadirachta indica were collected from different village area of the Dhemaji District of Assam during the month of June-July, 2016. The fresh lives were identified by the Department of Botany, A.D.P. College, Nagaon.

Preparation of the plant extract:

The plant material of Vitex negundo, Ocimum gratissimum and Azadirachta indica were thoroughly washed with clean water and allowed for drying at 40^oC in a hot air oven for 3 days. The dried material was crushed in a mixture grinder to a fine powder.

Soxhlet extraction:

The leaf powder of Vitex negundo and Azadirachta indica were subjected to soxhlet extraction separately using solvent diethyl ether, hexane and ethanol according to their polarity. The extraction was carried out fora duration of 4 to 5 hours. This process was repeated third times and the obtained extracts were pooled for evaporation in a water bath at 60^oC for obtaining residue. The obtained residues were kept in a Petri dish and stored in the refrigerator at 40^oC. The leaf of Ocimum gratissimum was washed, shade dried and then chopped in to crush powder using a mixer. 40g of the powder was filled in the thimble and extracted successively with Water, Methanol and Chloroform using a Soxhlet extractor for 48hrs. All the extracts were concentrated using a rotary evaporator and stored at 4^oC in a refrigerator.

Preparation of optimized ingredients:

For the preparation of mosquito repellent cream, coal powder and cow dung¹⁷ has been selected as a base and Flour (Maida)¹⁷ have been selected as a binder. To impart porosity to the strongly bonded mixture and to ease continued burning, rice grain and sawdust have been used. The ingredients formula for the mosquito repellent was as expressed in Table 1.

Table1: The constituents in the mosquito repellent cream

Sl. No.	Ingredients	Weight Percentage
1	Azadirachta indica leaf extract	7.77%
2	Ocimum gratissimum leaf extract	3.0%
3	Vitex negundo leaf extract	3.0%
5	Rice dust	4.35%
6	Cow dung / Coal powder	40.4%
7	Flour (Maida)	6.35%
8	Peppermint oil	7.3%
9	Vegetable ghee	8.0%
10	Glycerin	10%

Preparation of Repellent testing Model:

A rectangular-shaped (43×15×18cm) and a Y shaped (21×10×10cm) mosquito repellent cages were prepared.

Preparation of the cream:

Table 1 summarizes the common heuristics of incorporation sequence and method used for the preparation of cream. As the majority of the heuristics are based on common practices, they can be derived from the basic information of the principal phenomenon of emulsion preparation. The extracts were mixed with cow dung and coal powder separately. The above-mentioned amounts of optimized ingredients are added where moida were used as a binder and peppermint oils were used as perfume. A small amount of Petroleum jelly was added to the formulation with continuous stirring at 70^oC. Then cream was estimated for various physical limitations¹⁷. Though the same plants were extracted with different solvents thus plant extracts were named accordingly:

· DEEAI+DEEVN+MEOG

· HEEAI+HEVN+MEOG

· EEAI+EEVN+MEOG

(*DEEAI= Diethyl ether extract of Azadirachta indica, DEEVN= Diethyl ether extract of Vitex negundo, HEEAI=Hexane extract of Azadirachta indica, HEVN= Hexane extract of Vitex negundo, MEOG=Methanolic extract of Ocimum gratissimum, EEAI= Ethanolic extract of Azadirachta indica, EEVN= Ethanolic extract of Vitex negundo.)

Six different formulations were prepared from the above extract which was based on cow dung and coal powder.

Table2: Types of Mosquito repellent formulations

Sl no	Types		Formulations
1	Type 1	Cow dung based	DEEAI+DEEVN+MEOG+ OI
2	Type2		HEEAI+HEVN+MEOG+ OI
3	Type3		EEAI+EAVN+MEOG+ OI
4	Type4	Coal powder based	DEEAI+DEEVN+MEOG+ OI
5	Type5		HEEAI+HEVN+MEOG+ OI
6	Type6		EEAI+EAVN+MEOG+ OI

Characterization and Optimization of Cream Formulation:

Different trial products of the cream formulation were characterized and optimized based on their aesthetic appearance, emulsification, pH, and consistency taking into account softness, greasiness, and stickiness. Sensorial observations including aesthetic appearance and consistency were assessed by ten observers^18,19,20.

Aesthetic Appearance:

The prepared cream must be aesthetically elegant in terms of its physical appearance, color, odor, and texture. Therefore, the cream formulations were subjected to sensorial observations²⁰.

Emulsification:

Indecent emulsification usually brings on phase separation/cracking and precipitation. Consequently, the batches were observed for fine emulsification, which leads to an elegant product²⁰.

pH Determination:

A distinct amount of cream (100 mg) was weighed, diluted in distilled water, and mixed well. The pH of the cream was recorded using Digital pH Meter (Mettler Toledo). pH evaluation was carried out for all experimental formulations. The measurement was carried out in triplicate²⁰.

Consistency:

Each batch of the cream was evaluated for its consistency by examining its softness, greasiness, and stickiness. The formulation should be of uniform consistency which could spread and soften easily when stress is applied. It must also be nongreasy and nonsticky.

Stability Studies:

In accordance with International Conference on Harmonization (ICH) guidelines, stability analyses of optimized formulations were performed. The optimized cream formulations were stored in tightly closed glass containers for 90 days at 25°C temperature and 60% relative humidity in a humidity chamber. At predestined intervals, 0, 30, 60, and 90 days, samples were collected and their physicochemical evaluation parameters such as color, consistency, phase separation, texture analysis, and pH were evaluated²⁰.

Spreadability:

Spreadability was determined by the apparatus which consists of a wooden block, which was provided by a pulley at one end²¹. By this method, spreadability was measured based on the slip and drag characteristics of cream. About 1 gm of cream was placed on the ground slide. The cream was then sandwiched between this slide and another glass slide having the dimension of the fixed ground slide and provided with the hook. The top plate was then subjected to a pull of 100gm. With the help of string attached to the hook and the time (in seconds) required by the top slide to cover a distance of 7.5cm be noted^22,23.

Spread ability was determined by using formula-

S = ML/T

Where, S = Spreadability,

M = Weight tied to upper slide,

L = Length of a glass slide and

T = Time taken to separate the slides from each other.

Evaluation of mosquito repellent activity:

Mosquito repellent activity was assessed by using the home-made test cage as described in the American Society for Testing and Materials (ASTM) standard E951-83 Laboratory testing of non-commercial mosquito repellent formulation on the skin. The formulations to be tested were applied to the skin.

The mosquitoes used in this experiment were caught using a net while biting humans between 7pm and 10pm. Mosquitoes were starved for 24hours and 20 mosquitoes were placed in the cage (43×15×18cm). Test timing was between 6pm and 9pm since the mosquitoes typically bite at night. The host-seeking behavior of the mosquitoes was tested before the experiment. This was done by introducing a pre-cleaned skin in the cage and counting the number of mosquitoes that allied within 10seconds. The skin which had been rubbed with 1 gm of the formulated herbal repellent was exposed to the cage and the number of mosquitoes that aligned or biting the arm was recorded each minute for 5minutes. Mosquitoes were given an over one-hour interval and the above procedure was followed for each of the other plant extracts and essential oils. Each test was replicated thrice to get a mean value of mosquito repellent action.

For a repellent to be successful, it must first have a high percentage of protection against mosquito bites. Second, it should be toxicologically safe at the rate of application for which it is intended. Third, it should be easy to apply and pleasant on skin²⁴.

Percentage the mosquito repellency²⁵

Percentage the mosquito repellency for the plant extract shown in Table 2 was calculated as below,

C - N

% of mosquito repellency= ------------------------×100

Where,

C = Number of Mosquitoes aligned/left and aligned/bit when the Standard was used.

N = Number of mosquitoes align/left and aligned/bit when the extract was used.

Statistical Analysis:

Data are presented as mean ± standard error mean (SEM). Data for different types were compared using one-way analysis of variance (ANOVA). All statistical analyses were performed using GraphPad Prism, Version 5.01 (GraphPad software. Inc., USA).

RESULTS:

Formulation Parameters:

Table 3: Formulation parameters of Cow dung based and Coal powder-based Herbal Mosquito Repellent (Type1 to Type6)

	Batch	Homogeneity	Skin irritation
Coal Powder Based	Type1	Good	Nil
	Type2	Good	Nil
	Type3	Good	Nil
Cow Dung Based	Type1	Good	Nil
	Type2	Good	Nil
	Type3	Good	Nil

Fig 1: pH and Spreadability of formulated Herbal Mosquito Repellent (Type1 to 6)

Analysis of Mosquito Repellent activity

Table 4: Mosquito Repellency (Percentage) of the formulated Mosquito Repellent cream (Rectangular Shaped Model)

Batch	Replicate1	Replicate2	Replicate3	The calculated mean value of aligned/left and aligned/bite	Percentage of Mosquito repellency
Batch	Total no of mosquitoes aligned/left and aligned/bite	Total no of mosquitoes aligned/left and aligned/bite	Total no of mosquitoes aligned/left and aligned/bite	The calculated mean value of aligned/left and aligned/bite	Percentage of Mosquito repellency
Type1	3	3	2	2.67	66.62%
Type2	1	0	1	0.67	91.62%
Type3	4	2	3	3	62.5%
Type4	2	3	2	2.34	70.75%
Type5	2	1	1	1.34	83.25%
Type6	4	3	3	3	62.5%

Table 5: Mosquito Repellency (Percentages) of the formulated Mosquito Repellent cream (Y-Shaped Model)

Batch	Replicate 1	Replicate2	Replicate3	The calculated mean value of aligned/left and aligned/bite	Percentage of Mosquito repellency
Batch	Total no of mosquitoes aligned/left and aligned/bite	Total no of mosquitoes aligned/left and aligned/bite	Total no of mosquitoes aligned/left and aligned/bite	The calculated mean value of aligned/left and aligned/bite	Percentage of Mosquito repellency
Type1	3	2	2	2.67	66.62%
Type2	1	2	0	1	87.5%
Type3	5	3	2	3.34	58.25%
Type4	2	1	2	2.5	68.75%
Type5	3	2	1	2.34	70.75%
Type6	4	2	3	3	62.5%

Table 6: Mosquito Repellent activity (Percentages) of the Mosquito Repellent cream (Rectangular-Shaped Model)

Batch	Number of Mosquito Tested		No any action		Mosquito migrate away		Mosquito paralyzed		Remark
Batch	Total	%	No.	%	No.	%	No.	%	Repellence
Type1	20	100%	08	40%	08	40%	04	20%	Low repellency
Type2	20	100%	02	10%	06	30%	12	60%	High Repellency
Type3	20	100%	08	40%	09	45%	03	15%	Low repellency
Type4	20	100%	05	25%	07	35%	08	40%	Low repellency
Type5	20	100%	05	25%	04	20%	11	55%	High Repellency
Type6	20	100%	08	40%	09	45%	03	15%	Low repellency

Fig2: Standard Curve for percentage of mosquito repellent activity in Rectangular Shaped Model

Table 7: Mosquito Repellent activity (Percentages) of the Mosquito Repellent cream (Y-Shaped Model)

Repellent Sample	Number of Mosquito Tested		No any action		Mosquito migrate away		Mosquito paralyzed		Remark
Repellent Sample	Total	%	No.	%	No.	%	No.	%	Repellency
Type1	20	100%	07	35%	08	40%	05	25%	Low Repellency
Type2	20	100%	04	20%	04	20%	12	60%	High repellency
Type3	20	100%	08	40%	08	40%	04	20%	Low Repellency
Type4	20	100%	05	25%	07	35%	08	40%	Low Repellency
Type5	20	100%	05	25%	04	20%	11	55%	High Repellency
Type6	20	100%	07	35%	09	45%	04	20%	Low Repellency

Fig3: Standard Curve for a percentage of mosquito repellent activity in Y Shaped Model

DISCUSSION:

Many researchers have found that plants Vitex negundo, Ocimum gratissimum and Azadirachta indica have better repellent efficacy rather than other plant materials^25,27. However, a single plant extract obtained from plants contributes to poor longevity as mosquito repellents. Therefore, three plant extracts of the Vitex negundo, Ocimum gratissimum and Azadirachta indica are suitable to be used as a polyherbal mosquito repellent.

Preliminary batches of formulated Mosquito repellent cream were characterized for their elegancy, emulsification, spreadability, stickiness and consistency based on sensorial assessment. The cream was off-white in color and opaque with a homogeneous appearance (Table 3).

All prepared formulations had nearly constant pH, homogeneous, emollient, non-greasy and were easily eliminated after the application. The pH values of all developed formulations were found to be in the range of 6.95 to 8 (Fig 1). Under Stability Studies Color, consistency, viscosity, texture profile, and pH of the prepared creams were found to be consistent, and no separation was observed throughout a 90-day study, which revealed the reproducibility of the physical and chemical parameters which ensures the consistent quality of the developed cream formulation.

In the present investigation, repellent activities of Type1 to Type 6 formulations of Azadirachta indica, Vitex negundo and Ocimum gratissimum extracts were determined by using the Arm-in-Cage Model. In the arm-in-cage method experiment, although it appears that at 5minute exposure times at hourly intervals, the number of aligning and biting mosquitoes was generally reduced, the reduction is probably due to the satisfaction of the mosquitoes being sufficiently fed as time and exposure go up.

In this study, most of the formulated types of mosquito repellent have been shown to possess satisfactory repellency effects against insects. The remarkable activity was observed in the case of Type2 formulation. The most effective repellent activity of the Type2 formulation was 91.62% in Rectangular-shaped Model and 87.5% in the Y shaped Model. The Type1, Type3, Type4, Type5 and Type6 formulations showed 66.62%, 62.5%, 70.75%, 83.25% and 62.5% repellent activity in Rectangular shaped model (Table 4) and 66.62, 58.25%, 68.75%, 70.75% and 62.5% repellent activity in Y shaped model (Table 5) respectively.

Ranasinghe MSN et. al., 2016, reported on the repellent activity of the different types of plant extracts and essential oils. They found that all the essential oil show more repellency than plant extracts. During our study, it was found that the Type2 formulation (cow dung based) have shown higher repellent activity than Type1 and Type3 formulation of cow dung based. Similarly, Type5 formulation (coal powder-based) has shown higher repellent activity than Type4 and Type6 formulation of coal powder-based (Table 6, Fig 2)¹⁶. Pandharbale AR 2007, evaluated the mosquito repellent activity of Azadirachta indica and Ocimum gratissimum. They found that Tulsi oil showed higher mosquito repellency than Neem leaf extract²⁴. Gaddaguti V et. al., 2016, reported about Potential mosquito repellent compounds of Ocimum species against 3N7H and 3Q8I of Anopheles gambiae. They have found that 12 out of 35 compounds of the two Ocimum species were found to be idyllic for the intend and development of possible mosquito repellents. And they have claimed that herbal ingredients of Ocimum species can be considered for formulation for mosquito repellents²⁷.

No skin irritations or rashes were detected on the arms of the test volunteers’ with extracts, essential oils and the prepared mosquito repellent formulations. All formulations were safe concerning skin irritation and allergic sensitization as the primary irritation index was found to be zero, and there was no report of any edema or redness. However, a hot sensation was observed on the arms of the test volunteers with Type 1. Further improvements for the mosquito repellent formulations were redesigned by reducing the concentration of Type 1 ingredients to the level where no hot sensation was observed. The mosquito repellent formulations from plants are exceptionally fruitful due to the prosperity of insecticidal ingredients found in plants as defenses adjacent to insects²⁸. Repellents have an important place in protecting man from the bites of insect pests. An effective repellent will be useful in reducing man vector contact and in the interruption of disease transmission^28,29. The Azadirachta indica, Vitex negundo and Ocimum gratissimum not only has good mosquito repellent/mosquitocidal activity but also it has good odour characteristic and the effect was due to presence of various active ingredients in these plants^30,31.

CONCLUSION:

During this study, mosquito repellent activity of ingredients of polyherbal showed that the product was very efficient and safe to use. Statistically, it showed that the formulated product is very good and safe for use. During this study, we found that Hexane extract showed higher repellent activity. No volunteers complain about allergic consequences. So, it is a safe product. The formulation was ecological, economical and pocket friendly. Further investigations are needed to elucidate the efficacy of the herbal mosquito repellent formulations against a wide range of mosquito species and also to identify active compounds responsible for mosquito repellent activity to utilize them if necessary, in preparing a commercial product to be used as a mosquito repellent.

ACKNOWLEDGMENTS:

The authors are thankful to the Department of Herbal Science and Technology, ADP College, Nagaon, Assam, India, for providing the platform to conduct this work.

FUNDING:

The work was financially supported by DBT-Star College Scheme.

CONFLICT OF INTEREST:

The authors declare that they have no conflict of interest. All the tables and figures are self-made and original.

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Received on 03.11.2020 Modified on 05.12.2020

Research J. Pharm. and Tech 2021; 14(12):6262-6268.

DOI: 10.52711/0974-360X.2021.01083