Formulation and Evaluation of Polyherbal Mosquito Repellent Cream

Mosquitoes such as Aedes, Anopheles, and Culex are a serious threat to the public health as they are known vectors for various protozoans, viruses, and bacteria which result in many life- threatening diseases like malaria, filariasis, yellow fever, Japanese encephalitis, chikungunya, and dengue. These vectors have been considered as a major obstacle to socioeconomic development of developing countries particularly in the tropical region. Despite considerable efforts in recent years to control vector-borne diseases, malaria alone produces 250 million cases per year and 800,000 deaths including 85% children under five years (WHO, 2010). Therefore, the prevention of mosquitoes could be better than the cure of vector-borne disease. Hence, use of the mosquito repellents on exposed skin area is strongly recommended. Insect repellents usually work by providing a vapor barrier deterring the arthropod from encountering the skin surface. Most of the commercial mosquito repellents are prepared using nonbiodegradable, synthetic chemicals like N, N-diethyl-3-methylbenzmide (DEET), dimethyl phthalate (DMP), and allethrin, which may lead to their higher exposure to the environment and, hence, the unacceptable health risks. With an increasing concern on public safety, a renewed interest on the use of natural products of plant origin is desired because natural products are effective, environment friendly, biodegradable, inexpensive, and readily available. Many plant extracts have been identified as having insect-repellent effects and there has been increasing research in the last decade proving plant-based mosquito repellents are just as, if not more effective than DEET like Citronella Oil, Neem Oil etc. Plant products are emerging as a potential source of mosquito control and among them essential oils have special interest due to their insecticidal or repellent properties. Essential oils are volatile, naturally occurring, complex compounds characterized by a strong odour and are formed by aromatic plants as secondary metabolites. They are liquid, volatile, rarely coloured, lipid soluble and soluble in organic solvents with a density generally lower than that of water. There are 17,500 aromatic plant species among higher plants and approximately 3,000 essential oils are known out of which 300 are commercially important for pharmaceuticals, cosmetics, and perfume industries apart from pesticide or repellent potential. Owing to the attraction for natural products like essential oils, despite their wide use and being familiar to us as fragrances, it is important to develop a better understanding of their mode of biological action for new applications in human health, agriculture, and the environment. Some of them constitute effective alternatives or complements to synthetic compounds of the chemical industry without showing the same secondary effects. In nature essential oils play an important role in the protection of the plants as antibacterial, antiviral, antifungal, and insecticides. Hence the present study aims to develop an ecofriendly mosquito repellent, a substitute for chemical repellent by using different essential oils and to produce the natural repellent which is more cost effective, cheap and keeps the environment pleasant and health friendly. The aim of the present study is to formulate and evaluate a polyherbal mosquito repellent cream. The main objective of the study is to formulate a polyherbal mosquito repellent cream consisting of various herbal constituents such as Citronella oil, Eucalyptus oil, Tulsi oil, clove oil, neem oil, Sweet orange extract, Turmeric extract, etc. which exhibit mosquito repelling character.

Drug Profile

Cymbopogon nardus (Citronella)

Figure 1: Cymbopogon nardus.

Kingdom: Plantae

Order: Poales

Family: Poaceae

Genus: Cymbopogon

Species: C. nardus

Cymbopogon nardus or Citronella grass (S. Pengiri) is a coarse and clump-forming tropical grass native to Sri Lanka. Citronella grass can grow up to 1.5-1.8 m tall and citronella stems are like canes. Citronella essential oil is extracted by steam-distillation of citronella leaves. Citronella essential oil is used as an insect repellent and it can be found in dozens of registered pesticide products such as sprays, lotions, and candles. Due to its antifungal properties, citronella oil is also used to treat insect bites. Additionally, citronella essential oil is one of the most common oils used in aromatherapy as it has an ability to treat and prevent fever and headache. Because of citronella oil’s antiseptic properties, it is used in soaps, household cleaners and detergents. The main components of citronella essential oil are monoterpene hydrocarbons and alcohols such as geraniol (18-20%), citronellal (5-15%), citronellol (6.4- 8.4%), limonene (9-11%) and geranyl acetate (2%). Additionally, methyl eugenol, camphene, nerol, borneol, citronellic acid and citral are also present in citronella essential oil.

Eucalyptus globulus

Figure 2: Eucalyptus globulus.

Kingdom: Plantae

Order: Myrtales

Family: Myrtaceae

Genus: Eucalyptus

Species: E. globulus

Eucalyptus globulus (S. Eucalyptus) is named as “Blue Gum” and it is a medium to very tall forest tree which may reach 70 m in ideal conditions. However, Eucalyptus globulus is more commonly 15-25 m in height and it has a rough, greyish bark which is shed on the upper trunk and branches in long ribbons. Eucalyptus globulus leaves are glossy, dark green, thick, and leathery. The white flowers occur from winter to early summer and they are followed by hard, woody-capsules containing greyish fruits which are named as “Gum Nuts.” Eucalyptus essential oil which is extracted by steam-distillation of eucalyptus leaves is taken orally for pain and inflammation of respiratory tract mucous membranes, coughs, asthma, and bronchitis. It is also used as an antiseptic, insect repellent and treatment option for wounds. Eucalyptus oil is also popularly used as a fragrance in perfumes and cosmetics and it is found in mouthwashes, toothpastes and cough drops as well. The main chemical constituent present in eucalyptus oil is 1,8-cineole (60-70%). In addition, α-pinene (9%), β-pinene (0.4%), limonene (0.04%), terpinen-4-ol (0.3%), aromadendrene (2.5%), epiglobulol (0.4%) and globulol (2.7%) are also present in eucalyptus oil.

Curcuma longa (Turmeric)

Figure 3: Curcuma longa

Kingdom: Plantae

Order: Zingiberales

Family: Zingiberaceae

Genus: Curcuma

Species: C. longa

Curcuma longa (Turmeric) is an upright, relatively short and stout plant that rarely grows more than about 1 m in height. Its leaves are elongated, dark green, and pointed, often curling slightly along the margins. Each leaf arises on an individual stalk directly from the fleshy rhizome at its base. The rhizome appears scaly due to the remaining rings of previous leaves. Its outer skin is brownish but its flesh is deep orange-yellow inside. Rhizomes grow to about 5-8 cm x 1.5- 2.5 cm the flower stalk will appear among the leaves, also directly rising from the rootstock. The yellow-reddish flowers are arranged spirally along the cylindrical spike which may be partially protected by a leaf sheath. Turmeric is mainly used in culinary preparations and for medicinal purposes. For chemical purposes, it is used as an indicator and can identify a base. Turmeric turns red when it comes in contact with a base. Monoterpene hydrocarbons (46.9%) constitute the bulk of the Curcuma longa rhizome essential oil which is obtained by hydro-distillation and has antiseptic and anti- carcinogenic properties. Major constituents of the essential oil are β-bisabolene (13.9%), transocimene (9.8%), myrcene (7.6%), 1,8-cineole (6.9%), α-thujene (6.7%) and thymol (6.4%).

Ocimum sanctum (Tulsi)

Figure 4: Ocimum sanctum

Kingdom: Plantae

Order: Lamiales

Family: Lamiaceae

Genus: Ocimum

Species: O. tenuiflorum

Ocimum sanctum or Tulsi is a branched sub-shrub which is 30-60 cm tall, with simple opposite green or purple leaves that are strongly scented. Tulsi leaves have petiole and are ovate, up to 5 cm long, usually slightly toothed. Flowers are purplish in elongate racemes in close whorls. Tulsi is native throughout the tropics and wide-spread as a cultivated plant. It is cultivated for religious and medicinal purposes and for its essential oil. Tulsi essential oil is used as a medicine for fever, headache, lung disorders, heart disorders and many other diseases. Tulsi essential oil has antibacterial, anti-viral and anti-fungal properties as well. Tulsi essential oil is also used in massage oils, perfumes, aromatherapy baths, soap making and candle making. Tulsi leaf essential oil contains methyl eugenol (46-68%), (E)- caryophyllene (17-27%) and β-elemene (16.3%) as the major constituents.

Syzygium aromaticum (Clove)

Figure 5: Syzygium aromaticum

Kingdom: Plantae

Order: Myrtales

Family: Myrtaceae

Genus: Syzygium

Species: S. aromaticum

Syzygium aromaticum (Clove) is a triangular-shaped evergreen tree growing about 12 m tall with a smooth grey bark and about 12 cm long, glossy opposite leaves that resemble bay leaves. The attractive red and white bell-shaped flowers have four tiny petals surrounded by a long, four- parted calyx and numerous stamens. The clove buds are pink but calyx changes from yellow to deep red after the stamens fall. The fruit, called mother of cloves, is an edible purple berry about 2.5 cm long. The entire Syzygium aromaticum plant is extremely aromatic. Cloves are used in cooking, either in whole or in ground form as a spice throughout Europe and Asia. Clove essential oil is widely used and well known for its medicinal properties. Traditional uses of clove oil include use in dental care as an antiseptic and analgesic. The oil is active against oral bacteria associated with dental caries and periodontal diseases. Additionally, clove oil has anti-carcinogenic, anti-allergic, antioxidant and insecticidal properties. The major constituent in clove bud oils is eugenol (72.08-82.36%) and eugenyl acetate is the second major component (8.6-21.3%). In addition, β-caryophyllene (2.76-8.64%) and α-humulene (0.34- 1.04%) also see present in clove bud oil.

Citrus sinensis (Sweet Orange)

Figure 6: Citrus sinensis

Kingdom: Plantae

Order: Sapindales

Family: Rutaceae

Genus: Citrus

Species: C. sinensis

Citrus sinensis or Sweet Orange is a small shallow-rooted evergreen tree which is about 6-13 m in height with enclosed conical top and spiny branches. Citrus sinensis leaves which are 5- 15 cm long and 2-8 cm wide are dark green in colour, glossy and oval shaped. Greenish-white Citrus sinensis flower is small and it has five petals. Citrus sinensis fruits are reddish-green to yellowish-green in colour and consist of a leathery and tightly adhered peel which is 6 mm thick. Citrus sinensis peel protects the juicy inner pulp of the Sweet Orange fruit. Sweet Orange peels, leaves and flowers contain fine essences of oils that are used in the manufacture of cosmetic products and medicines. The main chemical components of Sweet Orange peel essential oil are α-pinene, sabinene, myrcene, limonene, linalool, citronellal, neral and geranial.

 Azadirachta indica (Neem)

Figure 7: Azadirachta indica

Kingdom: Plantae

Order: Sapindales

Family: Meliaceae

Genus: Azadirachta

Species: A. indica

Azadirachta indica is an evergreen and fast-growing tree that can reach a height of 15-20 m. The Neem branches are spread widely. The fairly dense crown is roundish or oval. The white and fragrant flowers arise from the junction of the stem and petiole. An individual flower is 5- 6 mm long and 8-11 mm wide. Neem fruit is a smooth olivelike drupe. The fruit skin is thin and turns yellow when ripe. The bitter-sweet pulp is yellowish-white. The white, hard inner shell of the fruit encloses one, rarely two or three, elongated seed having a brown seed coat. All parts of the tree such as seeds, leaves, flowers and bark, are used in the preparing of various different medical preparations. Neem products have medicinal properties that prove to be anti- fungal, anti-diabetic, antibacterial, antiviral and anti-fertility. Limonoid compounds contained in Neem seed extract seem to have the insecticide and pesticide properties. The main limonoid in Neem seed extract is azadirachtin. Additionally, azadiradione, fraxinellone, nimbin, salannin, salannol, vepinin and vilasinin are also present in Neem seed extract. Bitter property of the Neem oil is due to tannins, flavonoids and sesquiterpene derivatives.

Vitex negundo (Nika)

Figure 8: Vitex negundo

Kingdom: Plantae

Order: Lamiales

Family: Verbenaceae

Genus: Vitex

Species: V. negundo

Vitex negundo or Nika is a branched, small and slender tree which grows up to 4.5-5.5 m in height, flourishing mainly in the Indian subcontinent. Vitex negundo leaf upper surface is green and the lower surface is silvery in colour. Flower is bluish purple and becomes black when ripe. All parts of the plant, from root to fruit, possess a multitude of phyto-chemical secondary metabolites which impart an unprecedented variety of medicinal uses to the plant. The hexane extract of Vitex negundo leaf contains viridiflorol (19.55%), β-caryophyllene (16.59%), sabinene (12.07%), 4- terpineol (9.65%), γ-terpinene (2.21%), caryophyllene (1.75%), 1- ocetene-3-ol (1.59%) and globulol (1.05%). Ethanol extract of Vitex negundo leaf contains flavonoids as the major constituents along with a few terpenoids.

MATERIAL AND METHODOLOGY

Collection

The ingredients; Citronella oil, Eucalyptus essential oil, Turmeric, Tulsi essential oil, clove oil, Sweet Orange peel essential oil, Neem oil, Nika extract used in the preparation of 50g polyherbal mosquito repellent cream were extracted using appropriate methods.

Ingredients and its Role

Table No.1: List of ingredients and its purpose.

Equipment Required

Table No.2: List of equipment

Preparation of Herbal Mosquito Repellent Cream

1. Preparation of Oil Phase

1. Beeswax, Paraffin oil, Cetyl alcohol, Stearic acid, and all the prepared essential oils are added in a beaker kept over the water bath which is set on the temperature of 70 degree Celsius.

2. Preparation of Water Phase

1. Aqueous ingredients such as glycerine and water-soluble ingredients such as Potassium hydroxide and Methyl paraben are mixed and heated simultaneously at the same temperature.

3. Mixing Sequence

1. Prepare aqueous phase and oil phase into separate containers before mixing.
2. To make W/O emulsion, keep the amount of oil phase higher than or equal to aqueous phases.
3. Add aqueous phase to oil phase through wall of the container to ensure little loss of two phases.
4. Stir emulsion at constant temperature (65 to 75 degree Celsius) for 45 – 60 min for proper emulsification.

EVALUATION PARAMETERS

The herbal mosquito repellent cream was evaluated for different parameters such as, appearance, viscosity, pH, irritancy test, thermal stability test, etc.

Organoleptic properties

1. Colour:

Off-White colour with slight shimmer

2. Odour:

The odour of the cream was observed to be characteristic

3. Presence of foreign particles:

A little amount of herbal cream was taken and spread on glass slide and which is then observed against light.

4. Dilution test:

1g of cream was taken in 2 test tubes; one is diluted with water and other with oily medium. Both the test tubes were shaken and then observed.

Stability studies

1. Phase separation:

Cream was kept in closed container away from sunlight. Then phase separation was observed every 24hrs for 30 days.

2. Moisture absorption studies:

50mg of cream was taken on watch glass. A beaker was filled with water and kept in a desiccator without adsorbent, allowed to get saturated. Then watch glass with cream was introduced into desiccators and left it for 24hrs.

Moisture absorbed (%) = Final wt. – Initial wt. × 100

                                Initial wt.