FIELD OF THE INVENTION
This invention relates to a mosquito repellent composition. This invention also relates to a process for the isolation of oil extract from leaves and stems of Chloroxylon swietenia DC. Further the oil is evaluated for knockdown and mortality rate of mosquitoes. It also relates to the process of preparation of extracts of C. swietenia and their characterization by chromatographic techniques like GC-MS and HPTLC.
BACKGROUND OF THE INVENTION
Insects have long been known to be a nuisance to human beings. In particular, significant attention has been focused on mosquitoes which are carriers and spreaders of diseases like malaria, filariasis, encephalitis, dengue fever, yellow fever etc. Many countries through out the world are attempting to control the mosquitoes and to eradicate the scourge of malaria and other diseases.
Thousands of plants have been screened for potential sources of insect repellents. It is known in the art that essential oils can repel insects preferably biting insects such as mosquitoes and are advantageous compared to those of synthetic compounds in terms of presenting lower toxicity to humans.
The natural oils known to repel mosquitoes or insects include Rotundial (from the leaves of Vitex rotundifolia Watanabe k et al., 1995) Biotech Biochem. 59(10), 1979-1980); citronella oil (U.S. Pat. No. 5,346,922); Eucalyptus oil (Watanabe et al., (1993). J. Agri. Food Chem. 41: 2164-2166) and oil (Sharma V. P et al., (1993). J. American mosquito control Association 9(3), 359-360) and oil of Hedeomapulgoides, oil of anisum and oil of Chrysanthemum (U.S. Pat. No. 5,208,209), peppermint oil, clove oil, cedar oil, lavender, geranium oil, lemongrass oil etc.
The natural products or herbal solutions which are reported as effective mosquito repellents require more frequent application (at least every 2hrs) and higher concentrations than DEBT. Studies on citronella revealed that it provides shorter complete protection time than the products based on DEBT.
Neem is known to have long been used in traditional South Indian Communities as an insect and mosquito repellent.
U.S Pat. No. 5,885,600 discloses an insect repellent composition and an antioxidant particularly from a combination of neem, citronella and cedarwood oils. Reference may be made to U.S. Pat. No. 5,776,477, which discloses a variable mixture of natural ingredients of Pennyroyal herb, calendula, citronella, pyrethrin and aloe vera. U.S. Pat. No. 6,291,745 reports limonene and other metabolites of geranyl pyrophosphate for insect control in plants.
In urban India almost every household uses mosquito repellents in the form of liquids, mats and coils. Various mosquito repellents which are currently available commercially consists of chemicals of synthetic origin like DEBT, allethrin, prallethrin, cyclothrin etc. The safety of these compounds is doubtful and repeated exposure to these compounds may have long term side effects.
Therefore there remains a need for a natural effective and safe substance that can repel mosquitoes and which can be formulated into a variety of compositions and delivery systems. With this objective, we have evaluated the mosquito repellent properties of Chloroxylon swietenia extracts. Chloroxylon swietenia (C.swietenia) belongs to the family Rutaceae is a tropical aromatic tree popularly known as East Indian Satin Wood. The potential use of the tree in mosquito control has never been investigated. Just the leaves as such show great mosquito repellent activity and thus the tribals of Andhra Pradesh hang leaf garlands in the houses to avoid mosquitoes. This is a common phenomenon observed in most of the tribal communities of Andhra Pradesh and India.
OBJECTIVES OF THE INVENTION
The main object of the present invention is to provide an effective insect repellent, which is safe to use.
Yet another object of the invention is to provide a natural mosquito repellent formulation in liquid form comprising an oil extract from leaves and stems of Chloroxylon swietenia DC.
Yet another object of the invention is to provide an insect repellent which effectively repels mosquitoes.
Still another object is to provide a process for the isolation of oil extract from leaves and stems of Chloroxylon swietenia DC.
In the present invention Knockdown and mortality rate effects of the essential oil extracts on mosquitoes is evaluated and compared with those of commercially available formulations. The composition is established as equivalent to the commercially marketed compounds. The toxicity of the oil is also evaluated on wistar rats to establish the acceptable levels of active ingredients to optimize knockdown (KD) and mortality rates.
SUMMARY OF THE INVENTION
Accordingly the present invention provides a mosquito repellent composition comprising :
essential oil extract 1.0 -3.0 gm (w/w)
butylated hydroxytoluene 0.1-0.2 gm (w/w)
deodourized kerosene 97.0-99.0 gm (w/w)
In an embodiment of the invention the essential oil extract used is isolated from the leaves or stems of Chloroxylon swietenia D C.
In another embodiment of the invention the oil extract isolated from leaves of Chloroxylon swietenia D C used has the following components: alphapinene, myrcene , limonene , cis-P -ocimene, trans-p-ocimene , linalool, geijerene, methyl chavicol, pregeijerene geranyl acetate, p-bourbonene , p-elemene, methyl eugenol , p-caryophyllene, bicyclogermacrene ,d-cadinene, T-cadinol and farnesol.
In yet another embodiment of the invention the oil extract isolated from the stems cf Chloroxylon swietenia D C used has the following components alphapinene, myrcene , limonene , cis- P -ocimene, trans-p-ocimene , linalool, geijerene; pregeijerene geranyl acetate, methyl eugenol , p-caryophyllene, bicyclogermacrene ,d-cadinene, T-cadinol, farnesol.
In yet another embodiment the mosquito repellent composition exhibits mosquito knock down KD50 value in the range of 10.48 to 30 minutes.
In yet another embodiment the mosquito repellent composition exhibits mosquito knock down KD90 value in the range of 18 to 23 minutes.
In yet another embodiment the mosquito repellent composition shows a mosquito mortality rate of 100% at the end of 2 hours.
In yet another embodiment the mosquito repellent composition shows a mosquito knockdown rate of 100% at the end of 20 minutes.
In yet another embodiment the mosquito repellent composition with 3% extract oil exhibits knock down KD50 value of 10.48 min. and KD90 value of 18.2 min.
In yet another embodiment the mosquito repellent composition with 2.5% extract oil exhibits KD50 value of 10.5 min. and KD90 value of 18.2 min.
In yet another embodiment the mosquito repellent composition with 2% extract oil exhibits KD50 value of 11.0 min. and KD90 value of 19.2 min.
In yet another embodiment the mosquito repellent composition with 1.6% extract oil exhibits KD50 value of 11.5 min. and KD90 value of 20.5 min.
In still another embodiment the mosquito repellent composition with 1% extract oil exhibits KD50 value of 12.0 min. and KD90 value of 22.7 min.
The present invention further provides a process for the isolation of oil extract from the leaves and stems of Chloroxylon swietenia D C , which comprises hydrodistilling the leaves or stems of Chloroxylon swietenia D C, for a period of about 5 hours followed by extraction with water in the form of steam to obtain the desired distil oil and drying the above said oil by known methods to obtain the desired extract.
In an embodiment of the invention the oil extract obtained from the leaves of Chloroxylon swietenia D C has the following components alphapinene, myrcene , limonene , cis-P -ocimene, trans-p-ocimene , linalool, geijerene, methyl chavicol , pregeijerene geranyl acetate, p-bourbonene , p-elemene, methyl eugenol , p-caryophyllene, bicyclogermacrene ,d-cadinene, t-cadinol and farnesol.
In yet another embodiment of the invention the oil extract obtained from the stem of Chloroxylon swietenia D C has the following components alphapinene, myrcene , limonene , cis-P -ocimene, trans-p-ocimene , linalool, geijerene, pregeijerene geranyl acetate, methyl eugenol , P-caryophyllene, bicyclogermacrene ,d-cadinene, T-cadinol, farnesol.
DETAILED DESCRIPTION OF THE INVENTION
The present investigations were undertaken to evaluate the mosquito repellent activity of essential oil extracts of C. swietenia. The oil was extracted by hydrodistillation and
analyzed by GC-MS and HPTLC. Knockdown and mortality rate of mosquitoes of the essential oil was evaluated.
HPTLC ANALYSIS
TLC was carried out on 10 cm x 10 cm HPTLC plates precoated with silica gel 60F254 which were further impregnated with 8% AgNOa. The oil samples were applied to the plates and were developed in a twin-trough chamber with Dichloromethane-acetone 9.8:0.2 (v/v) as mobile phase. The plates were scanned at X,=254nm by means of a TLC scanner 3 (Camag) in absorbance/reflection mode. The results were tabulated in Table-A.
Table-A
(Table Removed)
Evaluation of knockdown and mortality rate of mosquitoes
The cage was made of glass with internal dimensions of length=65 cm, breadth=54 cm, height=54 cm and volume^ 189540 cm3. A plug point is provided inside the cage for fitting the vaporizing machine with an external power on/off switch to control the time for which the machine can be switched on and off during the experiment. The sides of the cage are sealed properly to prevent the leakage of vapors from the cage.
The vaporizing machine with the test material has been fitted to the plug point inside the cage. Known number of mosquitoes of mixed population were introduced into the cage and the mosquitoes were allowed 10 min. time for acclimatization in the cage. After 10 min. the power switch was on and observations are made on the behaviour of mosquitoes and for knockdown and mortality rates.
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 The effect of closed cage environment without any test substance on the mosquitoes
To determine the effect of closed cage environment without any test substance, the mosquitoes were introduced into the cage. Mosquitoes were flying at a random pace in the beginning. After 10 min of acclimatization to the new environment, some of the mosquitoes had settled down. All the observations were made for a period of 5 hrs. Around 60% had settled down and 15% knocked down after 2hr. At the end of 4 hrs 40% knock down and 20% mortality was observed.
Example 2 Evaluation of the effect of deodorized kerosene on the behaviour of mosquitoes
To evaluate the effect of deodorized kerosene on the behaviour pattern of mosquitoes, deodorized kerosene was filled in a bottle and fitted to the vapourizing machine. All the observations were made for a period of 3hrs. Around 50% had settled down and 35% were knocked down at the end of Ihr. At the end of 2 hrs, 72% knockdown and 30% mortality was observed.
Example 3 Preparation of composition of essential oil extracts
Composition of essential oil extracts (1%) were preparation as follows
Essential oil extract 1.0 gm (w/w)
Butylated hydroxytoluene 0.1 gm (w/w)
Deodourized kerosene 98.9 gm (w/w)
Example 4 The effect of essential oil on knockdown and mortality rate of mosquitoes
To determine if C. swietenia oil was an effective mosquito repellent, 1% (w/w), 1.6% (w/w), 2% (w/w), 2.5% (w/w) and 3% (w/w) solutions of essential oil were prepared in deodorized kerosene (96.9% - 98.9% w/w) and butylated hydroxytoluene (0.1% w/w) and the liquid was transferred to a bottle and fitted to a vapourizing machine. All the observations were made for a period of 2 hrs and the knockdown and mortality rates were recorded. The knockdown rate was almost 100% at the end of 20 min for the samples containing 2.5% and 3% essential oil. The mortality rate was 98% at the end of 2 hrs of experimentation. The sample containing 1% oil showed around 92% mortality and sample
with 1.6% essential oil showed around 93% mortality. The mortality rate was around 96% with the sample containing 2% oil. The knocked down mosquitoes showed circular movements in the upside down position. All the dead mosquitoes had their wings folded inwards. Appendage shedding was also observed from 20 min of experimentation.
Example 5
The effect of fresh leaves of C. swietenia on mosquitoes knockdown and mortality rate
To determine the effect of fresh leaves of C. swietenia on the behaviour of mosquitoes, known amount of leaves (25 gm) of leaves taken in a wide mouth container covered with a piece of mosquito net placed inside the cage and all the observations were made for a period of 3 hrs. The knockdown rate was 96% and 92% mortality was observed at the end of 2 hrs.
Example 6
The effect of positive control sample which is commercially available in the liquid form
To determine the effect of positive control sample on mosquito knockdown and mortality, one product in the form of liquid containing 1.6% prallethrin was selected. The liquid was placed in position with the vaporizing machine inside the cage. All the observations were made for a period of 3hr. The knockdown rate was almost 100% at the end of 20 min. The mortality rate was around 98% at the end of 2 hrs. The knocked down mosquitoes showed circular movements in the upside down position. All the dead mosquitoes had their wings folded inwards.
Example 7 Evaluation of KD50 and KD90 of closed cage environment without any test substance
The KD50 and KD90 of closed cage environment without any test substance was evaluated with the said procedure, and the observations were made for each minute up to SOOminutes. The KD50 and KD90 were found to be 252.65 min and 425.40 min. respectively.
Example 8 Evaluation of KD50 and KD90 of the solvent deodorized kerosene
To evaluate the KD50 and KD90 values of the solvent deodorized kerosene, with the same procedure mentioned above and the knockdown rate was recorded for each minute up to 180 minutes. The KD50 was found to be 82.24 min and KD90 was found to be 136.92 min.
Example 9
Evaluation of KD50 and KD90 of positive control sample available commercially in the market in liquid form
To evaluate the KD50 and KD90 values of positive control samples available commercially containing 1.6% prallethrin, with the same procedure and the knockdown was recorded for each minute up to 60 minutes. The KD50 value was found to 10.34 min and KD90 was found to be 18.0 min.
Example 10
Evaluation of KD50 and KD90 of essential oil of C. swietenia in liquid form at
varying concentrations
The KD50 and KD90 of essential oil was evaluated with the above mentioned procedure, and the observations were made for each minute up to 60 minutes.
The KD50 value was found to be 10.48 min. and KD90 was found to be 18.2 min. for the sample containing 3% (w/w) of essential oil, 96.9% (w/w) deodourized kerosene and 0.1% (w/w) of butylated hydroxytoluene.
The KD50 was found to be 10.5 min and KD90 was found to be 18.4 min for the sample containing 2.5% (w/w) of essential oil, 97.4% (w/w) deodourized kerosene and 0.1% (w/w) of butylated hydroxytoluene.
The KD50 was found to be 11.0 min and KD90 was found to be 19.2 min for sample containing 2% (w/w) of essential oil, 97.9% (w/w) deodourized kerosene and 0.1% (w/w) of butylated hydroxytoluene.
The KD50 was found to be 11.5 min and KD90 was found to be 20.5 min for sample containing 1.6% (w/w) of essential oil, 98.3% (w/w) deodourized kerosene and 0.1% (w/w) of butylated hydroxytoluene.
The KD50 was found to be 12.6 min and KD90 was found to be 22.7 min for sample containing 1% (w/w) of essential oil, 98.9% (w/w) deodourized kerosene and 0.1% (w/w) of butylated hydroxytoluene.
Acute Inhalations toxicity of the test material in Wistar rats
Acute inhalation toxicity of the test material was determined by exposure of wistar rats of both sex to aerosol of 50%w/v concentration of test substance in dimethyl sulfoxide and 50%w/v concentration of test substance in deodorized kerosene generated by a glass atomizer at 0.4 ml/min injection rate. The average gravimetric concentration achieved was 1.94 ± 0.05 mg of the test substance per litre of chamber air (maximum attainable concentration). The rats were housed in special restainers and continuously exposed to aerosol (head and nose exposure) for 4 hrs. The animals were observed for 7 days after the day of exposure and then necropsied and subjected to pathological examination. It is interesting to note that there are no toxic signs, pre-terminal deaths and other abnormalities detected at necroscopy.
Advantages of the invention
1. The extracts are potent mosquito repellents.
2. It is envirofriendly
3. It is absolutely safe for human beings.
4. It is economically viable process.
Thus these results demonstrate that the essential oil extracts of C. swietenia is an effective and safe mosquito repellent.

We claim
1 A mosquito repellent composition comprising :
essential oil extract 1.0 -3.0 gm (w/w)
butylated hydroxytoluene 0.1 -0.2 gm (w/w)
deodourized kerosene 97.0-98.9 gm (w/w)
2. A composition as claimed in claim 1, wherein the essential oil extract used is
isolated from the leaves or stems of Chloroxylon swietenia D C .
3. A composition as claimed in claim 1, wherein the oil extract isolated from leaves of
Chloroxylon swietenia D C used has the following components: alphapinene, myrcene
, limonene , cis- P -ocimene, trans-p-ocimene , linalool, geijerene, methyl chavicol ,
pregeijerene geranyl acetate, p-bourbonene , p-elemene, methyl eugenol , P-
caryophyllene, bicyclogermacrene ,d-cadinene, T-cadinol and farnesol.
4. A composition as claimed in claim 1, wherein the oil extract isolated from the stems of
Chloroxylon swietenia D C used has the following components alphapinene, myrcene ,
limonene , cis- P -ocimene, trans-p-ocimene , linalool, geijerene, pregeijerene geranyl
acetate, methyl eugenol , p-caryophyllene, bicyclogermacrene ,d-cadinene, T-cadinol,
farnesol.
5. A composition as claimed in claim 1, exhibits mosquito knock down KD50 value in the
range of 10.48 to 30 minutes.
6. A composition as claimed in claim 1, exhibits mosquito knock down KD90 value in the
range of 18 to 23 minutes.
7. A composition as claimed in claim 1, shows a mosquito mortality rate of 100% at the end
of 2 hours.
8. A composition as claimed in claim 1, shows a mosquito knockdown rate of 100% at the
end of 20 minutes.
9. A composition as claimed in claim 1, with 3% extract oil exhibit knock down KD50 value
of 10.48 min. and KD90 value of 18.2 min.
10. A composition as claimed in claim 1, with 2.5% extract oil exhibit KD50 value of 10.5
min. and KD90 value of 18.2 min.
11. A composition as claimed in claim 1, with 2% extract oil exhibit KD50 value of 11.0
min. and KD90 value of 19.2 min.
12. A composition as claimed in claim 1, with 1.6% extract oil exhibit KD50 value of 11.5
min. and KD90 value of 20.5 min.
13. A composition as claimed in claim 1, with 1% extract oil exhibit KD50 value of 12.0
min. and KD90 value of 22.7 min.
14. A process for the isolation of oil extract from the leaves and stems of Chloroxylon
swietenia D C , which comprises hydrodistilling the leaves or stems of Chloroxylon
swietenia D C, for a period of about 5 hours followed by extraction with water in the
form of steam to obtain the desired distil oil and drying the above said oil by known
methods to obtain the desired extract.
15. A process as claimed in claim 14, wherein the oil extract obtained from the leaves of
Chloroxylon swietenia D C has the following components alphapinene, myrcene ,
limonene , cis- P -ocimene, trans-p-ocimene , linalool, geijerene, methyl chavicol ,
pregeijerene geranyl acetate, p-bourbonene , p-elemene, methyl eugenol , P-
caryophyllene, bicyclogermacrene ,d-cadinene, i-cadinol and farnesol.
16. A process as claimed in claim 14, wherein the oil extract obtained from the stem of
Chloroxylon swietenia D C has the following components alphapinene, myrcene ,
limonene , cis- P -ocimene, trans-p-ocimene , linalool, geijerene, pregeijerene geranyl
acetate, methyl eugenol , p-caryophyllene, bicyclogermacrene ,d-cadinene, i-cadinol,
farnesol.
17. A mosquito repellent composition and a process for the preparation thereof,
substantially as herein described with reference to the examples accompanying this
specification.