Field of the Invention
The present invention relates to a herbal mosquito repellent formulation and a process for the preparation thereof. More particularly, the present invention relates to herbal incense sticks with mosquito repellent properties, which are prepared from different plants found in the North-East region of India.
Background of the Invention
Mosquitoes are known to transmit many diseases such as malaria, dengue, yellow fever, filariasis and Japanese encephalitis. Approximately 40 million people in India alone suffer from mosquito born diseases annually. Therefore, it is important to develop suitable mosquito repellent products. Mosquito repellents currently used for domestic purposes are mainly synthetic products with high mammalian toxicity. They are mostly synthetic pyrethroids which are less harmful than general insecticides but still are not totally safe for prolonged use.
Several workers have been studying the mosquito repellent properties of plant or plant mixture in different parts of the world. However, formulations in the form of incense stick are quite limited in the market. A few such process with published information/patents are described below.

Herbal mosquito repellent agarbatti prepared by traditional plant materials like essential oil of Tagetes minuta is competitive over synthetic compound (pyrethroid) or products i. e. commercial mats, coils and sprays (Tyagi et al., 1997 International pest control 39 (6) 184-185) and powder of the pyrethrum flowers (Howard, 1911 U. S. Der. Agr. Farmers'Bull. 444 1-15, Nagasawawa et al, 1951, Botyu-Kagaku 16 176-181).
Iyer et al in PCT/IN2004/000117 disclose novel herbal mosquito repellent compositions comprising extracts of Blumea lacera. The solvent extracts and essential oils of Blumea lacera were standardized, evaluated and characterized using chromatographic techniques. Further, herbal mosquito repellent formulations in liquid, vapour and mat form were prepared from the said extracts. However, the drawbacks associated therewith are involvement of chemical extraction and characterization of the active constituents which makes the preparation costlier, time consuming and require scientific expertise.
Citronellol and citronellal from citronella oil have been found effective oil constituents against mosquito (Travis et al 1946 J. Econ. Ent 42 686). Saleh (Phytochemistry, 1984, 23 (11) 2497-2498) have found that the essential oil of Artemisia monosperma possesses insecticidal activity against housefly.
Kumar et al in IN194307 recite a formulation useful as herbal mosquito repellent containing the essential oil of Tagetes minuta, cedar, Chrysanthemum cinerariaefolium (Pyrethrum powder) and Cyperus rotundus powder together with binders and fillers in the ratio of about

0.20-3.0, 0.5-15, 2.0-3.0, 2.0-3.0, 0.1-1 and 8-10 respectively. However, the drawbacks associated therewith are limited availability of plant materials and huge infra-structural requirement for extraction of essential oils making the preparation costly.
US186120 discloses a process for the preparation of a formulation useful as mosquito and insect repellent which comprises mixing the components such as pyrethrum extract, neem extract, oil from plant source and alcoholic base by known method in a ratio ranging 0.5:7.0:57.5:35.0 to 0.25:13.33:45.66:41.0. However, the drawbacks associated therewith are huge infra-structural requirement for extraction of oils making the preparation costly.
Keeping in view the hitherto know prior art, it may be summarized that the mosquito repellents available in the market till date are associated with the following disadvantages:
> the mats available in the market contain allethrin, a synthetic pyrethroid;
> most sprays contain synthetic compounds causing health hazards, limited activity and are costly;
> coils which are available contain allethrin and are harmful to health.
In contrast, indigenous plant materials having mosquito repellent properties would prove to be eco-friendly and safe to human health. Due to abundant availability of raw materials, these indigenous plant materials are suitable for any industry for making herbal mosquito repellent products. Therefore, the inventors of the present invention realized that there

exists a need to develop a safe mosquito repellent formulation. The formulation of the present invention has been prepared in the form of incense sticks.
Objects of the Invention
The main object of the present invention is thus to provide a herbal mosquito repellent formulation, the components of which are synergistic in action.
Another object of the present invention is to provide a formulation comprising an effective amount of plant materials together with binding material and wood dust for smooth burning.
Still another object of the present invention is to provide a formulation which contains Ocimum gratissimum, Anphineuron spp. and Polygonum hydropiper as insect repellents.
Yet another object of the present invention is to provide a formulation which can suitably substitute the synthetic preparations currently available in the market, which are known to cause some side effects on prolonged application.
A further object of the present invention is to provide a formulation which can be made by a simple procedure which is most cost effective and economic in comparison to synthetic preparations which require complex chemical synthetic procedures.

Another object of the present invention is to provide a formulation which is effective at a very low concentration, for which only a small amount of each of the material would be essential for making a preparation.
Summary of the Invention
The present invention provides a herbal formulation, which is useful against mosquitoes as repellent. The present invention also provides a mosquito repellent herbal incense stick and the formulation is prepared by mixing ground plant parts from plant species of North East India such as Ocimum gratissimum (leaf), Anphineuron spp (leaf) and Polygonum hydropiper (whole above ground part) together with binders (Litsea glutinosa) and fillers (wood dust). The formulation is effective to repel the mosquitoes significantly.
Accordingly, the present invention provides a herbal mosquito repellent formulation comprising leaf of Ocimum gratissimum, leaf of Anphineuron spp and above ground parts of Polygonum hydropiper optionally along with binders and filler materials
Detailed Description of the Invention
Ocimum gratissimum, Anphineuron spp and Polygonum hydropiper are found abundantly in Northeastern region at almost all places upto 1000 msl. These species are indigenous to this region. Usually, Anphineuron spp and Polygonum hydropiper are considered as weed. The description of the plant species is given below:-

1. Polygonum hydropiper Linn. - It is glabrous with erect and branched stem, leaves sub-
sessile or petioled lanceolate or oblong-lenceolate, stipules glabrous or sparsely
strigose which are very shortly ciliate. The racemes is filiform decurved interrupted,
bracts are glabrous grandular, perianth very glandular pink, nut usually trigonous
opaque granulate.
Distribution: Plains and hill of India, in wet patches Assam, Chittagong & Bengal ascending the Khasi Hills to 5000 feet, Sibsagar, Sylhet
2. Amphineuron sp. - Vern. Name - 'Bihlangani'- It is a terrestrial fern with creeping
rhizome. Stipes short hairy in grooves; scales linear- lanceolate, brown. Lamina large
pinnate, pinnae 20-27 pairs, sessile slightly oblique and falcate, obtuse or rounded.
Sori are mostly confined to lobes of pinnae, sporangia stalled.
Distribution: Common plant in Assam. (Pobitara sanctuary)
3. Ocimum gratissinum linn. - The plant is shrubby, glabrescent, leaves ovate acute
crenate or coarsely toothed, bracts sessile lanceolate owned from a rounded base.
Lateral triangular broader than lower, corolla are - inch! in length.
Distribution: Bengal, Chittagong, Assam
In India, most people have the habit of using incense sticks in the evening which is the time when mosquitoes start biting and causing problem in the houses. Therefore, if they can be

driven out in that time one can escape the trouble. Therefore, an incense stick was prepared using easily available plant products, which were evaluated as protectants against mosquito bites. It was found that the stick could provide considerable protection up to one hour of its duration of burning.
In the formulation of the present invention, the basic resources i.e., the plant species utilized are easily and abundantly available and can be easily raised for large scale production of plant materials.
The present invention provides a formulation having 'Ram Tulsi' (Ocimum gratissimum ), 'Bih Dhekia' (Anphineuron spp.), 'Bih longoni' (Polygonum hydropiper), gum (binding material) and wood dust (filler), which are mixed thoroughly and from which the incense sticks are prepared manually with the help of bamboo splints. The plants i.e. Ocimum gratissimum (leaf), Anphineuron spp. (leaf) and Polygonum hydropiper (whole above ground part) were collected fresh from the field and shade dried for 3 to 4 days till they became brittle. They were then powdered separately in the mixer grinder to 60 mesh size. They were then passed through the mesh and the powders passing through were collected separately and kept for use to prepare incense stick to be kept inside tightly closed plastic bags to prevent moisture entry.
The binder (gum) was obtained from the ground bark of Litsea glutinosa.

Fillers (wood dust) were collected from the saw mill. The same was sieved with 60 mesh size sieve.
The ground plant powders of Ocimum gratissimum: Anphineuron sp.: Polygonum hydropiper were mixed in the ratio in the range of 5 to 9 : 1 to 3 : 3.2 to 5. The mixture was then added to wood dust and binder gum in the proportion of Ocimum gratissimum : Anphineuron sp. : Polygonum hydropiper : Gum : Wood dust:: 5 to 9 : 1 to 3 : 3.2 to 5 : 2.9 to 5 : 1.1 to 2.5 respectively.
The formulation prepared from the aforesaid composition was used to prepare the herbal mosquito repellent incense stick.
The mixture was made in to a paste with the addition of little water. Care was taken to make the paste of uniform quality; where all the ingredients are distributed uniformly and no lump formation occurred. Thereafter, the paste was applied to the bamboo sticks of good burning quality in uniform manner by hand rolling. They were prepared manually, leaving about 1.5 inch gap from the bottom. Care was taken that the pastes remain in uniform condition covering the entire sticks. Another finishing layer of binder was optionally applied to the prepared incense stick for rusty colour appearance just by rolling the completed incense stick in to gum powder. After these, sticks were kept in shade for 3 days to dry completely and bonded adequately. At last, the sticks may be optionally dipped for

fragrance for 2 to 3 seconds uniformly in any scented essential oil solution and packed tightly inside the plastic bags. The formulation is found very effective to repel mosquitoes.
Novelty of the present invention resides in providing a mosquito repellent formulation with synergistic combination of indigenous plant products, which could repel the mosquitoes effectively. The activity of the present formulation for mosquito repellent herbal incense stick is highly significant as shown by the examples herein below and exhaustive field trials.
Different combinations of the ratios of plant parts were used to observe the efficacy as illustrated by the following examples. Combination in absence of a plant part was also evaluated for observing the success of the formulation as mosquito repellent. However, it was observed that the presence of all the plant parts together in above mentioned ratios was found effective for repelling mosquitoes significantly (table 1), depicting that the claimed formulation is synergistic.
The prepared mosquito repellent sticks were evaluated in 10' x 15' room space in terms of mosquito bites per unit minutej in comparison to another room of equal size with no application of sticks. Volunteers were used to evaluate the efficacy of sticks. The volunteers were allowed to sit in chair by keeping their legs perpendicular to floor surface. They were asked to remove cloths from feet to knee for exposing the area to mosquitoes for bite. Before burning the sticks, volunteers were allowed to sit and the number of mosquito bites per minute were counted. Afterwards, one formulated herbal incense stick was burnt. Again

after 10 minutes of burning the stick, the volunteer counted the no of mosquito bites per minute. Likewise, at every 10 minutes interval, 5 randomly selected minute data on mosquito bite was taken. Similar data was also recorded in control condition i.e. same room area without incense stick.
EXAMPLES
The following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention.
Example 1
This example depicts a method of preparation of a formulation useful to repel mosquitoes, which involves mixing of ground plant parts of the following plants along with conventional binder and filler in presence of water:
1) Bih longoni (Polygonum hydropiper) 20 g
2) Bih Dhekia (Anphineuron sp.) 20 g
3) Gum 15 g
4) Wood dust 10 g
The grounded parts of all the components mentioned above were mixed and a paste was prepared by adding 70 ml water. Incense sticks were made manually in bamboo splints. Incense sticks prepared with this combination showed less repellent activity to mosquito and burnt up to 1 hour (refer table 1).

Example 2
This example depicts a method of preparation of a formulation useful to repel mosquitoes, which involves mixing of ground plant parts of the following plants along with conventional binder and filler in presence of water:
1) Ram Tulsi (Ocimum gratissimum ) 20 g
2) Bih Dhekia [Anphineuron sps.) 25 g
3) Gum 15 g
4) Wood dust 10 g
The grounded parts of all the components mentioned above were mixed and a paste was prepared by adding 80 ml water. Incense sticks were made manually in bamboo splints. Incense sticks prepared with this combination exhibited poor performance as mosquito repellent and burnt up to 1 hour (refer table 1).
Example 3
This example depicts a method of preparation of a formulation useful to repel mosquitoes, which involves mixing of ground plant parts of the following plants along with conventional binder and filler in presence of water:
1) Ram Tulsi (Ocimum gratissimum ) 20 g
2) Bih longoni (Polygonum hydropiper) 22 g
3) Gum 20 g

4) Wood dust 15 g
The grounded parts of all the components mentioned above were mixed and a paste was prepared by adding 85 ml water. Incense sticks were made manually in bamboo splints. Incense sticks prepared with this combination exhibited poor performance as mosquito repellent and burnt up to less than 1 hour (refer table 1).
Example 4
This example depicts a method of preparation of a formulation useful to repel mosquitoes, which involves mixing of ground plant parts of the following plants along with conventional binder and filler in presence of water:
1) Ram Tulsi (Ocimum gratissimum ) 50 g
2) Bih Dhekia (Anphineuron sps.) 15 g
3) Bih longoni (Polygonum hydropiper) 70 g
4) Gum 60 g
5) Wood dust 30 g
The grounded parts of all the components mentioned above were mixed and a paste was prepared by adding 110 ml water. Incense sticks were made manually in bamboo splints. Incense sticks prepared with this combination exhibited good performance as mosquito repellent and burnt up to 1.5 hours (refer table 1).
Example 5
1) Ram Tulsi {Ocimum gratissimum ) 35 g

2) Bih Dhekia (Anphineuron sps.) 07 g
3) Bih longoni (Polygonum hydropiper) 22 g
4) Gum 20 g
5) Wood dust 08 g
The grounded parts of all the components mentioned above were mixed and a paste was prepared by adding 90 ml water. Incense sticks were made manually in bamboo splints. Incense stick manufactured from this combinations produced excellent result and burnt up to 1.30 hours.
The results illustrated in table 1 depict that the incense stick prepared from the combination given in example 5 recorded a mean protection time of 90 minutes (Table-1).
Table -1: Mean number of Mosquito bites recorded in time interval inside a room with different treatments

(Table Removed)

* All data are mean of 10 replications
Field trials were conducted in 90 houses in and around Jorhat during evening, when mosquito populations remain at peak. Families were selected with different socio-economic background and 70% of the selected families were identified from village where mosquito populations were quite high and varied. Responses so generated revealed that 20% people reported to feel disagreeable fragrance during burning of the stick; of course, they reported to have high repellent activity of the incense sticks. However, disagreeable fragrance may be overcome by using essential oil sprays in sticks. Rest 80% people did not feel any fragrance,

but mentioned to have significant mosquito repellent activity. Among the population 70% people reported that mosquito did not come to their vicinity about 1-2 hours after burning of the sticks.
Advantages of the invention:
> The formulation is safe, eco-friendly and provides significant mosquito repellent activity.
> The formulation is easy to prepare and can be prepared with hand rolling.
> Ingredients required for preparing the formulation are easily and abundantly available.
> The cost of production is marginal.
> Field trials indicate no/any human toxicity.
> The formulation i.e. mosquito repellent herbal stick can be used extensively by village people as it is easy to use and does not require even electricity like mat.
> The manufacturing of mosquito repellent herbal stick needs little investment for which the technology opens up huge rural employment generation in production and its marketing.

We claim:
1. A herbal mosquito repellent formulation comprising leaf of Ocimum gratissimum, leaf of Anphineuron sp. and above ground parts of Polygonum hydropiper optionally along with binders and filler materials.
2. A formulation as claimed in claim 1, wherein Ocimum gratissimum : Anphineuron spp.: Polygonum hydropiper : binder: filler are mixed in the ratio ranging between 5 to 9 : 1 to 3 : 3.2 to 5 : 2.9 to 5 :1.1 to 2.5 respectively.
3. A formulation as claimed in claim 1, wherein Ocimum gratissimum : Anphineuron spp.: Polygonum hydropiper : binder : filler are preferably mixed in the ratio of 5 : 1: 3.2 : 2.9 :1.1 respectively.
4. A formulation as claimed in claim 1, wherein the binder used is preferably Litsea glutinosa.
5. A formulation as claimed in claim 1, wherein the filler used is preferably wood dust.
6. A formulation as claimed in claim 1, wherein the plant parts of Ocimum gratissimum, Anphineuron sp. and Polygonum hydropiper are shade dried and grounded.
7. A formulation as claimed in claim 1, wherein the formulation is preferably in the form of an incense stick and is effective to repel mosquitoes while burning.
8. A herbal mosquito repellent formulation substantially as herein described with reference to the foregoing examples.