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(43) International Publication Date WO 2017/168449 Al5 October 2017 (05.10.2017) P O P C T

(51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,A01N 65/00 (2009.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM,

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(72) Inventors; and TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU,(71) Applicants : BARDE, Laxmikant Niranjan [IN/IN]; S/O TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE,

Mr. Niranjan Krushnrao Barde, Z.P. Krushi Colony, Ram- DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU,nagar, Balaji Society, Yavatmal - 445001, Maharashtra LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK,(IN). MAHAJAN, Nilesh Manoharrao [IN/IN]; S/O SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,Manoharrao Deoraoji Mahajan, 'Vrundavan', 09, Karde GW, KM, ML, MR, NE, SN, TD, TG).Nagar, Kathora Road, VMV Amravati - 444604, Maharashtra (IN). Declarations under Rule 4.17 :

— of inventorship (Rule 4.17(iv))(74) Agent: SAVANGIKAR, Vasant Anantrao; Krishna &

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kind of national protection available): AE, AG, AL, AM,

(54) Title: HERBAL BASED MOSQUITO REPELLENT

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(57) Abstract: This invention comprises a herbal composition for repellency of at least Anopheles, Aedes and Culex mosquitoes forat least eight hours when applied to the body. In one embodiment of this invention, the herbal composition comprises three or morethan three essential oils. The herbal composition of of this invention has been illustrated 5 with use of a cream containing three es -sential oils: Citronella oil, Catnip oil and Kaffir lime oil. However, the invention also includes within its scope any other composi -tion that comprises more than three or other combination of three essential oils.

HERBAL BASED MOSQUITO REPELLENT

FIELD OF INVENTION

Invention relates to mosquito repellants based on herbal active ingredient/s.

BACKGROUND OF THE INVENTION

Mosquito control and personal protection from mosquito bites are currently the most

important measures to control mosquito born diseases. The use of mosquito repellents

on exposed skin is, therefore, strongly recommended. As a result, it has already been

accepted as part of an overall integrated mosquito-borne disease control program. In

fact, in many circumstances, applying mosquito repellents to the skin may be the only

feasible way to protect against mosquito bites. Mosquitoes are well known vectors of

several disease causing pathogens. As per WHO, 500 million people in the world suffer

from one or the other tropical diseases that include malaria, lymphatic filariasis,

schistosomiasis, dengue, haemorrhagic fever (DHF), chikungunya, trypanosomiasis

and leishmaniasis. As per WHO report that about 1700 death per day were reported

from malaria especially in case of children.

Mosquito-borne diseases, such as malaria, filariasis, dengue hemorrhagic fever (DHF)

and chikunguiniya are still imposing to be major public health problems in the

Southeast Asian countries because of the tropical or subtropical climate. Mosquitoes

are responsible for spread of many diseases than any other group of arthropods. Aedes

aegypti Lin. is known to carry dengue and yellow fever; malaria is transmitted by

Anopheles species; and filarial disease by Culex quinquefasciatus Say. Dengue fever is

endemic over large areas of tropics and subtropics. The etiological agent is an arbovirus

and the major vector is the Aedes aegypti mosquito, which is found in 3600 Brazilian

municipalities. Global prevalence of lymphatic filariasis is 120 million and population

at risk is 1.3 to 1.5 billion every.

Anopheles stephensi L is a major vector in India which is responsible for about 40-50%

of the annual malarial incidence. Globally malaria kills 3 million people each year,

including 1 child every 30 seconds. WHO has declared the mosquito as "Public enemy

number one". It is estimated that every year at least 500 million people in the world

suffer from one or the other tropical diseases that include malaria, lymphatic filariasis,

schistosomiasis, dengue, chikungunya, trypanosomiasis and leishmaniasis.

Mosquito control and personal protection from mosquito bites are currently the most

important measures to control this disease. The use of mosquito repellents on exposed

skin is, therefore, strongly recommended. It has widely accepted that, applying

mosquito repellents to the skin may be the only feasible way to protect against

mosquito bites.

The use of mosquito repellents on exposed skin is, therefore, strongly recommended to

avoid these diseases.

There are a number of effective mosquito repellents containing synthetic chemicals

such as allethrin, N-N-diethyl-m- toluamide (DEET), N-N-diethyl phenylacetamide

(DEPA), N-N-diethyl benzamide (DEBA), dimethyl phthalate (DMP) and N, N-diethyl

mendelic acid amide (DEM) and picaridin. DEET & DEBA are currently the most

effective mosquito repellent and is available in various commercial compositions

containing as high as up to 95% DEET content. Although, repellent containing DEET

and DEBA are effective in discouraging mosquitoes from biting, it has been known to

cause headache, eye and skin irritation, shortness of breath, ataxia, dizziness, cancer

and defect in child birth. The adverse effects also include contact urticaria, skin

eruption or toxic encephalopathy in children. In addition, synthetic chemicals used for

control of vectors are causing irreversible damage to the ecosystem, as some of them

are non-degradable in nature. These problems highlight the need for the development

of effective as well as safer non-DEET alternatives.

Thus, plant essential oils, commonly used as fragrances and flavoring agents, are

preferred as mosquito repellents. Essential oils can be applied to humans in a similar

way to other conventional mosquito repellents and they tend to be selective and have

little or no harmful effects. Since botanicals are less likely to cause ecological damage,

a large number of plants have been screened for their insecticidal activities against

mosquitoes and some of these have been found to possess promising effects.

Protection against mosquito bites was reported for the genera:

1. Azadirachta indica A.Juss. (Synonyms:- Neem,Nimtree and Indi an Lilac,

Azadirachta indica var. / rValeton, Azadirachta indica var. siamensis Valeton,

Azadirachta indica siibsp. vartakii Kothari, Londhe & N.P.Singh, Melia

azadirachta L . Melia indica (A. Juss.) Brandis), Cymbopogan nardus L(Synonyms:

2 . Cymbopogon afronardus Stapf. Common names:- False citronella (Zaire), citronella

grass (Taiwan), blue citronella grass (Kenya), Naid grass (India).

3. Mentha piperita L. (Synonyms:- White peppermint, Peppermint, Mint plant, ,

4 . Eucalyptus metadata Hook.f (Common name:- Spotted gum Synonyms:- Corymhia

maculate (Hook. ) K.D. Hill & L.A. S.Johnson.

5 . Eucalyptus amygda Una (Labill).

6 . Eucalyptus risdnonii (Hook. )

7 . Lantana camara L (Common names:- big-sage (Malaysia ), wild-sage, red-

sage, white-sage (Caribbean ) and tickberry (South Africa) Synonyms:- Lantana

aculeata L.; Camara vulgaris L . .

Especially citronella oil, the essential oil from Cympobogan spp., is popular in

mosquito- repellent compositions. It has been studied as a possible alternative as a

mosquito repellant and demonstrated good efficiency against Aedes spp., Culex spp.,

and Anopheles spp. It is plant based insect repellent obtained by steam distillation of

fresh or dried leaves reported to be effective for not more than 3 hours.

Sritabutra (2013) (Sritabutra D.et. al. Asian Pac J Trop Dis 2013; 3(4): 271-

276). reported mixed Citronella oil with olive oil and coconut oil and tested its efficacy

on Ae. aeg p ti and Or. Quinquefasciatu with an insect repelling lotion containing

DEET 25.63% as a positive control. They reported that Citronella oil when combined

with olive oil gives repellency of 54 minutes against Ae. Aegypti and 165 minutes

against Cx e]uinc{uefasciatus. In combination with coconut oil it shows the repellency

of 82 mins against Ae. Aeg p ti and 105 minutess against Cx. c[uinc[uefasciatus .

Tawatsin et al (2001) (Apiwat Tawatsin et.al, Vector Ecology, Vol. 26, No. 1: 76-82,

2001) tested Citronella oil against An. dints for repellency by using mosquito cage

method. Findings indicated that pure oil was only effective for less than four hours.

Catnip oil obtained from Nepeta cataria belonging to family Lamiaceae (mint). It is

perennial herb and though it grows wild throughout the world. It is 10 times more

effective than DEET as illustrated in following experiment: 20 mosquitoes were placed

in a two-foot glass tube, half of which was treated with nepetalactone. After 10

minutes, only an average of 20 percent — about four mosquitoes — remained on the

side of the tube treated with a high dose (1.0 percent) of the oil. In the low-dose test

(0. 1 percent) with nepetalactone, an average of 25 percent — five mosquitoes — stayed

on the treated side. The same tests with DEET (diethyl-m-toluamide) resulted in

approximately 40 percent to 45 percent — eight-nine mosquitoes — remaining on the

treated side. (C. Peterson, J . Coats, (2001), Catnip Repels Mosquitoes More Effectively

Than DEET, American Chemical Society, Science Daily.) (Zhu, J.J. et. al,

"Nepetalactones from essential oil of Nepeta cataria represent a stable fly feeding and

oviposition repellent" (20\2). Faculty Publications: Department of Entomology . 340.).

However, in practice, the oil is to be applied not in closed environment but in an

environment open to the atmosphere on the body of the user, from which it evaporated

fast and does not give protection for desired period of 8 hours.

Kaffir lime oil obtained from Citrus hystrix belonging to family Rutaceae was reported

as an effective repellent with respect to repellency hour providing around 100%

protection for 8 hours when combined 5% vanillin applied topically against An.

stephensi , 100% protection against Ae. aeg p ti for 3 hours 100% protection against

Cx. quinquefasciatus for 1.5 hours and 100% protection against An. dims for 2.5 hours

laboratory study. Duration of repellency in case of Ae. Aegypti and Cx.

quinquefasciatus were found to be very less considering the practically rational

repellency.

Hill (2007) (Hill N, Lenglet A, Arnez AM, Cainero I : Randomised, double-blind

control trial of p-menthane diol repellent against malaria in Bolivia. BMJ 2007, 55)

evaluated repellency of the volatile oils by laboratory cage method using the human-

bait technique (Schreck and McGovern 1989, WHO 1996). The testing period lasted up

to eight hours, depending on the efficacy. The timing of the tests depended on whether

the target mosquitoes were day-or night-biters; Ae. aegypti was tested from 0800 h to

1600 h while An. di s and Cx. quinquefasciatus were tested between 1800 h and 0200

h . They found that when p-menthane diol repellent was combined with 5% vanillin and

applied topically repellency period for providing around 100% protection was for 8

hours against An.stephensi, 3 hours against Ae. Aegypti, for 1.5 hours against Cx.

quinquefasciatus and 2.5 hours against An. dims.

Pavitha 2014 (Pavitha P., S.Poornima, "Repellent Potential of Tagetes erecta L. and

Callistemon brachyandrus Lindl. Against Mosquito Larvae for Formulation of Herbal

Repellent Compounds", IJIRSET, Vol. 3, Issue 5, May 2014) studied to avoid harmful

effects of commercially available mosquito repellent creams in market, an attempt has

been made to prepare herbal formulations from natural products such as flowers of

Tagetes erecta L . and leaves of Callistemon brachyandrus Lindl. To evaluate the

repellent potential, phytochemical studies, larvicidal activity, smoke toxicity and

repellent activity tests were performed against Anopheles stephensi, Culex infulus and

Aedes aegypti. The result was that repellent activity test for cream formulation showed

89.87%, 87.5% and 90% protection and smoke toxicity test for incense log showed

66.25%, 70% and 67.5% protection against Anopheles stephensi. Culex infulus and

Aedes aegypti respectively for the observation period of 6 hrs. However, % repellency

for respective hour i.e. hour-wise repellency shall vary, data on which has not been

provided. Hence, it is not known for how many hours the protection remains effective.

Maia 2011 (Marta Ferreira Maia, Sarah J Moore, "Plant-based insect repellents: a

review of their efficacy, development and testing" Malaria Journal 10 (Suppl 1): S11)

studied the use of plant-based repellents for generations in traditional practice as a

personal protection measure against host-seeking mosquitoes. Recently, commercial

repellent products containing plant-based ingredients have gained increasing popularity

among consumers, as these are the composition in which active ingredients i.e.

essential oils are used are far below the level which is considered toxic to the skin i.e.

toxic level (LD 0 of all three oils) being considered as more than 4000 mg/kg body

weight commonly perceived as "safe" in comparison to long-established synthetic

repellents.

Mya 2015 (MM Mya, YY Aye, AW Oo, RK Saxena, 2015"Effect of Citrus hystrix DC

Leaves Ethanol Extract on Larvae of Aedes aegyptf Journal of Biological Engineering

Research and Review,; 2(2): 01-06) study was done on Vector control program- an

important public health mission for the eradication of vector-borne disease. Aedes

aegypti mosquito is a main vector for transmitting the viruses of dengue. Eradication of

Aedes aegypti at larval habitat from the domestic environment is the only way to

prevent dengue transmission. In this study Citrus hystrix (Kaffir lime) leaves ethanol

extract was used for the elimination of larvae of Aedes aegypti. Larvicidal effects of

Citrus extract in different dilutions on larvae of Aedes aegypti were monitored

according to WHO Guidelines for Laboratory Testing of Mosquito. Results of study

found that at 2.4%, 2.1%, 1.8%, 1.5% and 1.2% concentrations of Citrus hystrix leaves

ethanol extract caused 99.5%, 85.5%, 62.5%, 26.5% and 2% mortality of Aedes larvae

in 24 hrs. respectively. However 1.2% concentration showed almost negligible

larvicidal effect. Result of study suggests that high concentrations of Citrus hystrix

leaves ethanol extract can be used for the eradication of Aedes aegypti.

Nararak J . et al 2016 (Jirod Nararak, Sunaiyana Sathantriphop, Monthathip Kongmee,

Michael J. Bangs and Theeraphap Chareonviriyaphap "Excito-Repellency of Citrus

hystrix DC Leaf and Peel Essential Oils Against Aedes aegypti and Anopheles minimus

(Diptera: Culicidae), Vectors of Human Pathogens" Journal of Medical Entomology,

2016; 1-9) reported work done on the essential oils of Kaffir lime (Citrus hystrix DC.)

at four different concentrations (0.5, 1.0, 2.5, and 5.0% v/v) for their repellency,

excitation, and knockdown properties against laboratory strains of Ae. aegypti (L.) and

An. minimus Theobald using an excito-repellency test system. Both contact and

noncontact escape responses to leaf- and peel-derived Kaffir lime oils were observed.

Comparing unadjusted escape responses for An. minimus, leaf oil had strong combined

irritant and repellent activity responses at 1-5% concentrations (90.0-96.4% escape)

and the strongest spatial repellent activity at 1% and 2% (85.9%> and 87.2% escape,

respectively). The peel oil exhibited good excitation with repellency at concentrations

of 2 .5% (89 .8% escape) and 5% (96.28% escape), while concentrations 1-5% showed

more moderate repellent activity against An. minimus. For Ae. aegypti, 2.5% leaf oil

produced the greatest response for both contact (56.1% escape) and noncontact (63.3%

escape) trials, while 2.5% produced the strongest response among all concentrations of

peel oil, with 46.5% escape. However, after adjusting the contact trial escape (a

measure of combined excitation and repellency), the estimated escape due to contact

alone was a much weaker response than spatial repellency for both species.

Knockdown responses above 50% were only observed in Ae. aegypti exposed to 5%

leaf oil. Kaffir lime oils were more active against An. minimus than Ae. aegypti

mosquitoes. There were statistically significant differences between leaf (more active)

and peel oils at each concentration against An. minimus in contact and noncontact trials,

except at the highest (5%) concentration.

Peterson (2001) (Peterson, Christopher John, "Insect repellents of natural origin: Catnip

and Osage orange " (2001). Retrospective Theses and Dissertations. Paper 1101.). The

studies presented here to identify naturally occurring insect repellents. Two plants are

reported here, Catnip and the Osage orange. The first study confirmed repellency of

Osage orange extracts to the maize weevil, but found that repellency to two

components, Osajin and Pomiferin, was not significant. A second study evaluated the

effects of Catnip essential oil on German cockroaches. Extracts of the plant and

isolated compounds were more repellent than diethyl-m-toluamide (DEET). One

isomer of nepetalactone was more active than the other was. Peterson determined that

the chemoreceptors responsible for the repellent response were located on the antennae.

A third study reexamined some of the activity we saw in Osage orange extracts in the

first study. They examined the essential oils of the fruit, and identified several

sesquiterpene compounds. We tested the repellency of essential oil components to

German cockroaches, and found several of them to be repellent. In the last study we

examined the repellency of Catnip and Osage orange extracts to the yellow fever

mosquito. We determined that catnip essential oil significantly reduced the number of

insects near the treated area.

Sritabutra (2013) (Duangkamon Sritabutra, Mayura Soonwera 'Repellent activity of

herbal essential oils against Aedes aegypti (Linn.) and Culex quinquefasciatus (Say.)'

Asian Pac J Trop Dis 2013; 3(4): 271-276 Repellent activity of herbal essential oils

against Aedes aegypti (Linn.) and Culex quinquefasciatus (Say.)" Asian Pac J Trop Dis

2013; 3(4): 271-276) studied the mosquito repellent activity of herbal essential oils

against female Aedes aegypti and Culex quinquifasciatus . The method was carried on a

volunteer's forearm by using 0.1ml of each essential oil was applied to 3cmx l0cm of

exposed skin and their protection time was recorded for 3 min after every 30min. The

result was that essential oil from clove oil in olive oil and coconut oil gave the longest

lasting period of 76.50 min and 96.00 min against Aedes aegypti and the citronella

grass oil in olive oil, coconut oil and lemongrass oil in coconut oil exhibited protection

against Culex quinquifasciatus at 165.00, 105.00 and 112.50 min. It was concluded that

clove, citronella and lemongrass oil were the most promising for repellence against

mosquito species. These oils could be used to develop a new formulation to control

mosquitoes.

Patel et al (2012) (E. K . Patel, A . Gupta and RJ. Oswal A review on: mosquito

repellent methods" LJPCBS 2012, 2(3), 3 10-3 17 studied insect repellents by masking

human scent; a number of natural and chemical mosquito repellents were studied to

repel mosquitoes. The result was that chemical mosquito repellents has a remarkable

safety profile, but they are toxic against the skin and nervous system and result in

rashes, swelling, eye irritation and worse problems, and through some unusual side

effects including brain swelling in children, anaphylactic shock and low blood pressure.

It was concluded that natural mosquito repellents were preferred over chemical

mosquito repellents.

Gabi Baba at al (2012) (Gabi Baba, A.O. Lawal and Hauwa B . Shariff "Mosquito

repellent activity and phytochemical characterization of essential oils from Striga

hermonthica, Hyptis spicigera and Ocimum basilicum leaf extracts", British Journal of

Pharmacology and Toxicology 3(2): 43-48, 2012) screened the phytochemical s and

compared the mosquito repellent activities of essential oils from Hyptis spicigera L.,

Striga hermonthica D. and Ocimum basilicum L. (Basil) against Anopheles gambiae

and Culex quinquifasciatus under laboratory conditions by using alkaloids, saponins,

steroids, tannins and terpenoids were present in all 3 oils and cardiac glycosides were

detected in both Striga hermonthica and Hyptis spicigera, while anthraquinone and

phlobatin were present in S. hermonthica and H. spicigera on FTIR . In FTIR spectrum

revealed the presence of C=0,C-I, C-O-C, OH, C-N-S=0 and N0 2 these correlated

with the functional groups in the identified phytochemical. The result was that 50%

concentration, Ocimum basilicum and Hyptis spicigera oil exhibited higher repellent

potential on Anopheles gambiae with protection time of 183 and 120 min while Hyptis.

spicigera and Stroga hermonthica had protection time of 180 and 175min against

Anopheles gambiae and at 100% concentrations, Ocimum basilicum oil exhibited the

highest protection time against the two species of mosquito tested. It was concluded

that the essential oils of Striga hermonthica, Ocsimum basilicum and Hyptis spicigera

leaf extracts have been confirmed to have potentials as mosquito repellent agents

against Anopheles gambiae and Culex quinquifasciatus.

Thus, there is a long standing unmet need of safer mosquito repellants that do not

contain DEET, however, could possibly match the efficacy with respect to mosquito

repellency and duration of repellency of DEET.

Further, there is evidence of limitations of DEET. Stanczyk et al (2013) [Nina M .

Stanczyk, John F. Y. Brookfield, Linda M . Field, James G . Logan; Aedes aegypti

Mosquitoes Exhibit Decreased Repellency by DEET following Previous Exposure;

PLOS ONE Iwww.plosone.org February 2013 | Volume 8 | Issue 2 | e54438; pp 1-6]

have reported that Although DEET has been shown to be extremely effective, recent

studies have revealed that certain individual insects are unaffected by its presence. A

genetic basis for this has been shown in Aedes aegypti mosquitoes and the fruit fly

Drosophila melanogaster, but, for the Triatomine bug, Rhodnius prolixus, a decrease in

response to DEET occurred shortly after previous exposure, indicating that non-genetic

factors may also be involved in DEET "in sensitivity". In this study, we examined

host-seeking behavior and electrophysiological responses of A. aegypti after pre

exposure to DEET. We found that three hours after pre-exposure the mosquitoes

showed behavioral insensitivity, and electroantennography revealed this correlated with

the olfactory receptor neurons responding less to DEET. The change in behavior as a

result of pre-exposure to DEET has implications for the use of repellents and the ability

of mosquitoes to overcome them.

This finding makes it furthermore necessary to identify viable alternatives for DEET

for mosquito repellency.

The main objective of research work was to check the effectiveness and suitability of

one or more herbal bioactive ingredients as mosquito repellants for efficacy in the skin

application and making compositions from them suitable and effective for skin

application and safer than DEET, DEBA and without the use of these synthetic

ingredients.

SUMMARY

This invention comprises a herbal composition for repellency of at least Anopheles,

Aedes and Culex mosquitoes for at least eight hours when applied to the body. In one

embodiment of this invention, the herbal composition mentioned above comprises three

or more than three essential oils. The herbal composition of this invention has been

illustrated with use of three essential oils: Citronella oil, Catnip oil and Kaffir lime oil.

However, the invention also includes within its scope any other composition that

comprises more than three or other combination of three essential oils. In one

embodiment, the herbal composition of this invention as mentioned above comprises a

cream. The illustrated herbal composition comprises 5% w/v Citronella oil, 2% w/v

Catnip oil and 2.4% w/v Kaffir lime oil and excipients.

BRIEF DESCRIPTION OF FIGURES AND LEGENDS

Figure 1: Skin irritation study. 1: Photograph of normal Control rat with part of skin,

exposed by removing hair, on which nothing is applied; shows normal appearance of

the skin. 2 : Photograph of rat with part of skin exposed on which base composition was

applied in which no oil is added; shows normal appearance of the skin. 3 Photograph of

rat with part of skin exposed on which cream composition of this invention was

applied; this also shows normal appearance of the skin showing absence of any

irritation.

Figure 2 : Hourly % Repellency of cream composition of this invention

Figure 3: Comparative study with marketed products. F4 - Cream comprising

Citronella oil + Catnip oil+ Kaffir lime oil combination; MP1-; a prior art composition

available in market for applying on body surface comprising 12% DEBA and citronella

oil (strength not mentioned) used as fragrance. MP2- "Fabric Roll On" a prior art

composition available in market for applying to fabric and garments comprising 0.25ml

of Citronella oil & Eucalyptus oil, each. MP3- a prior art composition available in

market for applying to fabric and garments comprising Citronella oil alone.

DETAILED DESCRIPTION OF THE NVENTION

It was surprisingly found that although, when applied directly to the skin for the

purpose of mosquito repellency, individual essential oils do not retain mosquito

repellency for more than 2-3 hours against all the three mosquito species critical for

working as vector for most of the diseases i.e. Anopheles (An), Aedes (Ae) & Culex

(Cx), when two or more essential oils are mixed, in absence of DEET or any other

synthetic mosquito repellant as an ingredient in the mosquito repellant composition, the

spray and gel compositions based on these mixtures provided a mosquito repellency of

at least 5 hours; which is a practically significant improvement over prior art herbal

mosquito actives. The spray and gel compositions of this invention also remained

stable and retained the mosquito repellency also after storage for three months period

up to 91%, which is a practically significant level of stability.

However, the period of 5 hours was shorter than desired period of at least 8 hours;

hence an improvement in the compositions containing two or more essential oils was

needed. No herbal composition is disclosed in prior art that has repellency of at least

Anopheles, Aedes and Culex mosquitoes for at least eight hours when applied to the

body

It was surprising observation that when used in cream form, the efficacy was retained

on the skin surface for a longer period of time as compared to the spray & gel, thereby

enhancing the duration of repellency remarkably up to 8 hrs.

Hence, although the illustration provides a composition containing three essential oils,

particularly, Citronella oil, Catnip Oil and Kaffir lime oil, it provides an evidence that

it is possible to get a herbal composition to achieve repellency of at least Anopheles,

Aedes and Culex mosquitoes for at least eight hours when applied to the body. Hence,

this invention covers within its scope any herbal composition that shows repellency of

at least Anopheles, Aedes and Culex mosquitoes for at least eight hours when applied to

the body.

Three important considerations which were given attention to in this investigation for

evaluating mosquito repellency are as follows:

1 . Effectiveness: % repellency on hourly basis and average repellency of

individual pure oil when not used in combination

2 . Duration of repellency: This is important from the period of protection which

is considered as practically relevant for protection after the mosquito repellant

composition is applied on the body.

3 . Spectrum of repellency: Means repellency activity shown by an oil against

which types and range of mosquito species. If we use single oil, it gives a

variable effect in terms of mosquito species. It is evident from the prior art

literature that when single oil is used it doesn't show effect against all the

species. Even if it shows the repellency it does show practically relevant

duration of repellency.

To ensure maximum utility of a mosquito repellant composition for applying to the

skin/body, it is considered necessary that it should provide acceptable level of

repellency for at least 8 hours against species of Anopheles, Culex and Aedes

mosquitoes which are prevalent as major vectors for major diseases of public health

importance. A mosquito repellant composition would be ideal if it is effective against

all the prevalent species of Anopheles, Culex and Aedes.

In our study we tried same composition containing combination of two oils eg.

Citronella + Catnip; Citronella + Kaffir lime oil; Catnip + Kaffir lime oil. These all

combinations were studied for the mosquito repellency against all three species. It was

found that repellency effectiveness was different in different species and duration was

also less when two oils were used in combination as compared to the cream containing

the combination of all three oils. Further, it was also observed that cream was better

than the spray or emulgel with respect to retention on skin surface.

In one embodiment of this invention, the mosquito repellant composition for

application to the skin comprises Citronella oil, an oil obtained by steam distillation of

fresh or dried leaves of Cymbopogon nardus belonging to family Poaceae, Catnip oil

and Kaffir lime oil, . Catnip oil is obtained by steam distillation of dried leaves & peels

and flowering tops of Nepeta catariaL. (Mint) belonging to family Lamiaceae. Mint is

perennial herb and grows wild throughout the world. It is 10 times more effective than

DEET. However, duration of repellency was far lower than the same for 8 hours for

DEET. Kaffir lime oil obtained from steam distillation of leaves and peels of fruits of

Citrus hystrix belonging to family Rutaceae was reported as an effective repellent

with respect to repellency hour.

In a further embodiment of this invention, the composition of this invention comprises

spray, gel or cream mixture of essential oil comprising Citronella Oil, Catnip Oil and

Kaffir lime oil along with the excipients to make the composition stable

In a further embodiment of this invention, the herbal mosquito repellents are made in a

gel and spray forms.

The prepared compositions were tested for pH, viscosity, drug content, skin irritation

test, mosquito repellency activity and stability testing. Both gel and spray compositions

were found to be effective, safe and stable suggesting the suitability for the human use.

Comparative study of cream base composition was done with compositions already

available in the market that comprised essential oils as one of the ingredients; which

included a cream comprising DEBA 12% w/w in cream base and citronella oil as a

fragrance only, as % strength or cone is not mentioned; a liquid product comprising

Citronella oil 0.25ml & Eucalyptus oil 0.25ml per ml of the liquid meant to be applied

to a fabric and not for application directly on the skin leaving a probability that skin is

still left vulnerable for a mosquito bite; and - a product comprising Citronellal (strength

not mentioned).

In one aspect, this invention is an emulgel type preparation. Emulgel is a gel

preparation contains oils emulsified in aqueous gel base.

There are obvious advantages of emulgel over other topical preparations as: 1 .

Hydrophobic drugs can be easily incorporated into gels using oils or other solubilizer

like propylene glycol; 2 . It has better stability and loading capacity; 3 . Good production

feasibility as it is very easy to prepare.

The emulgelwas evaluated of characterized by following criteria: 1 . Physical

appearance; 2 . Rheology - includes viscosity; 3 . In- vitro permeation study also called

as drug diffusion study; 4 . Drug content analysis (assay); 5 . Spredability- ease of

spreading; 6 . Extrudability - ease of coming out of container; 7 . Stability testing; 8 .

Skin irritation study.

In a further aspect, the emulgel type preparation of this invention comprises a gelling

agent. In illustrative composition, acrylic acid polymer (carbopol®) is used as gelling

agent. However, any gelling agent other than carbopol® may be used that does the same

or similar function. In a still further aspect, the emulgel type preparation of this

invention comprises Citronella oil and Catnip oil as herbal mosquito repellent active

ingredients. Other herbal mosquito repellant active ingredients may also be used that

show same efficacy.

EXAMPLES

Table 1: Ingredients used for illustrative composition of polyherbal mosquito

repellant emulgel

The emulgel is targeted to have following quality attributes :-

1 Appearance: - White smooth aqueous based emulgel.

2 pH 7.2-7.4

3 Viscosity:- 1,80,000 cps, lrpm, spindle no. 64

4 Spredability:- Excellent

5 . Extrudability:- Good

6 . Skin Irritancy Test:- No irritation

7 . Mosquito repellency: - 100% for 1st & 2nd hr., 94% for 3rd hr., 89% for 4th hr.,

82% for 5 hr. and 75% for 6m hr.

8 Stability: - Stable (As per ICH guidelines)

EXAMPLE 1

POLYHERBAL BODY SPRAY MADE FROM CITRONELLA OIL AND

CATNIP OIL

Spray containing 5% w/v citronella oil and 2% w/v catnip oil were prepared according

to formula mentioned in table no. 2 . Compositions of three type of spray were

formulated: -

1 . Temporary emulsion spray (Fl)

2 . Alcohol based spray (F2)

3 . Aqueous based spray (F3)

Table 2 : Composition of Polyherbal body spray (100 ml)

EXAMPLE 2 :

POLYHERBAL EMULSIONS

TEMPORARY EMULSION (Fl)

Phase A : Butylated hydroxy toluene, Methyl paraben and Propyl paraben were

dissolved in propylene glycol. Citronella oil and Catnip oil were added in this mixture.

Phase B : Isopropyl myristate and Tween 80 were dissolved in the mixture of ethanol

and isopropyl alcohol then add sufficient amount of water. Phase A was mixed with

Phase B with moderate stirring by using overhead stirrer.

ALCOHOL BASED EMULSIONS (F2):

Phase A : Butylated hydroxy toluene, methyl paraben and propyl paraben were

dissolved in propylene glycol. Citronella oil and catnip oil were added in this mixture.

Phase B : Isopropyl myristate and Tween 80 were dissolved in the mixture of ethanol

and isopropyl alcohol. Phase A were mixed with Phase B with moderate stirring by

using overhead stirrer.

AQUEOUS BASED EMULSIONS (F3):

Phase A : Butylated hydroxy toluene, methyl paraben, propyl paraben were dissolved

in propylene glycol. Citronella oil and catnip oil were added in this mixture.

Phase B : Accurately weighed Tween was mixed in sufficient amount of water. Then

Phase A were added slowly in these phase B mixture to make emulsion with

continuous Stirring under overhead stirrer.

EXAMPLE 3

POLYHERBAL BODY CREAM MADE FROM CITRONELLA OIL, CATNIP

OIL AND KAFFIR LIME OIL

Table 3 : Ingredients used for illustrative composition of polyherbal mosquito repellent

cream preparation.

The cream is targeted to have following quality attributes:-

1. Appearance: - White smooth cream.

2 . pH 6.0-6.5

3 . Viscosity:- 80,000 cps, lrpm, spindle no. 63

4 . Spredability:- Excellent

5 . Extrudability:- Good

6 . Skin Irritancy Test:- No irritation

7 . Mosquito repellency: - 100% for l sthr- 4th hr., 94% for 5thhr., 90% for 6th

hr., 80% for 7thhr. and 75% for 8th hr.

8 . Stability: - Stable (As per ICH guidelines)

The cream type preparation of this embodiment invention comprises Citronella oil,

Catnip oil and Kaffir lime oil as herbal mosquito repellent active ingredients.

Cream containing 5% w/w citronella oil, 2% w/w catnip oil and 2.4% w/w Kaffir lime

oil was prepared as follows.

Table 4: Composition of Mosquito repellent cream

Sr. Ingredients Quantity

No. Taken(50gm)

1 Cetostearyl alcohol 10

2 Emulsifier 2.5

3 Liquid paraffin 5

4 Propyl paraben 0.01

5 Citronella oil 2.44

6 Catnip oil 0.97

7 Kafir lime oil 1.2

8 Methyl paraben 0.04

9 Propylene glycol 3.72

10 Purified water Qs up to 50gm

Method of preparation:-

Preparation of oil phase: Oil phase ingredients such as cetostearyl alcohol,

emulsifier, liquid paraffin, propyl paraben. Melt them using gentle heating according to

their melting points up to 75°C. Add essential oils (Citronella, Catnip and Kafir lime

oils) at 55°C into oil phase just before mixing the two phases.

Preparation of aqueous phase:- Aqueous phase ingredients such as purified water,

propylene glycol and methyl paraben. Heat it in water bath at 75°C.

Mixing sequence: Prepare aqueous and oil phase into separate containers before mixing.

Add aqueous phase to oil phase through wall of container. Stir emulsion at constant

temperature for proper emulsification. A thick cream base was prepared. Keep the stirring

speed of mixer or high-shear homogenizer below 2,000 rpm to avoid breaking down the

carbon chains, which leads to foaming of emulsion and causes instability.

Determination of pH:

The pH of spray was checked by using a digital pH meter (GLOBAL-pH DPH-507) at

room temperature. Initially, the pH meter was calibrated using standard buffer tablet of

pH 7 . Three types of spray compositions were taken in three beakers and pH was noted.

Drug content analysis:

Drug content was determined in each of the composition. About 0.1ml of sample was

transferred in 10ml of volumetric flask and make up the volume with distilled ethanol,

prepared the stock solution. 0.1ml was pipette out from the stock solution and diluted

with distilled ethanol. The content of Citronella oil and Catnip oil was determined by

using Shimadzu 1800 UV-visible spectrophotometer at λ 288 nm and 241 nm

respectively. The analysis was carried out for three times.

Skin Irritation Test:

The back hairs of albino mice were shaved using hair removal skin cream to expose a

skin area of about 5cm. The exposed area of the albino mice were treated with three

types of compositions i.e., Temporary emulsion, alcohol based and aqueous based and

covered with a gauge pad (bandage). The mice were labeled Fl, F2 and F3 respectively

for the different compositions. After 24 hrs., the tape and gauge were removed and

treated area evaluated for irritation, redness of skin

Table no. 5: Evaluation of mosquito repellant spray

Mosquito Repellency Testing:

Repellency of Citronella oil and Catnip oil based composition of herbal body spray was

compared using a test on human subjects by exposing hands treated with spray to caged

mosquitoes. Hands treated with different spray compositions were exposed to cage

containing 32 adult male and female mosquitoes of Culex quinquifasciatus, Aedes

aegypti and Anopheles stephensi for 5 hours study. This repellency testing was carried

out in aseptic cabinet having length 65cm, height 45cm and % repellency was

calculated by following formula:

% Repellency = [(Number of bites received on control arm - Number of bites

received on treated arm) / Number of bites received on control arm] x 100

Table 6: Mosquito repellency of polyherbal spray for 5hrs. testing

Stability studies of the compositions:

These studies were carried out on three types of spray compositions (Fl, F2 and F3) by

using REMI SC-6 plus stability chamber according to ICH guideline. The stability

conditions of 40±2°C/70±5%RH for 90 days. The physical stability of spray

compositions were observed periodically. All three types of compositions were

evaluated after one month for the pH, drug content and mosquito repellency test.

Skin Irritation Test: The back hair of 3 albino mice were shaved using hair removal skin

cream to expose a skin area of about 5cm . The exposed area of the 2 albino mice were

treated with primary base, cream composition and 1 albino mice was kept for control study

and covered with a gauge pad (bandage). The mice were labeled F l (control), F2(cream

base) and F3(cream composition) respectively. After 24 hrs. the tape and gauge were

removed and treated area evaluated for irritation; irritation is indicated by redness of skin.

Observation was done for 7 days.

Mosquito Repellent Activity: Mosquito repellent activity was assessed by using the test

cage as described in the American Society for Testing and Materials (ASTM) standard

E951-83 Laboratory testing of non-commercial mosquito repellent composition on the

skin. According to the method female mosquitoes of different species like Aedes aegypti,

Culex quinquifasciatus and Anopheles stephensi were transferred into the mosquito proof

enclosed cage with the help of aspirator tube. The compositions to be tested were applied

to the forearm (25cm ) of the volunteers. The applied hand was introduced into the cage

and the number of bites was recorded. After every 30 minutes the volunteer hands were

subjected to the cage for 5 minutes and the numbers of mosquito bites were noted. Same

procedure was implemented for control. Control study is done by placing the forearm in

cage without application of cream. According to this procedure different compositions

were tested by applying on the forearm with fresh mosquitoes every time of testing. The

Percentage protection provided by repellent cream can be expressed by the formula

% Repellency = [(No. of bites on Control arm - No. of bites on treated arm) / No.

of bites on Control arm] * 100

Stability studies of cream compositions: These studies were carried out on cream

composition by using REMI SC-6 plus stability chamber according to ICH guideline. The

stability conditions of 40±2° C/70±5% RH for 90 days. The physical stability of cream

composition was observed periodically. The cream composition was evaluated after one

month for the pH, drug, viscosity and mosquito repellency test.

Table no. 7: pH of polyherbal based compositions after stability studies

Table no. 8: Assay of active ingredient after stability study

Table 9: Mosquito repellency testing after storage for 90 days

Primary skin irritation study:

Skin irritation was carried out on 3 albino mice on their back skin and result according to

Draize scale showed no erythema and no edema formation (no irritation or no redness) on

mice skin (Figure 1).

Mosquito repellent activity study:

Table 10: % Repellency data of Optimized cream composition

Table 11: Mosquito repellent activity of the composition of this invention as compared

with compositions already available in the market

MPl- A Cream; a prior art composition available in market for applying to body

surface comprising 12% DEBA along with citronella oil as a fragrance.

MP2- A prior art composition available in market for applying to fabric and garments

comprising of 0.25ml Eucalyptus oil and Citronella oil.

MP3- A prior art composition available in market for applying to fabric and garments

comprising of Citronellal cone not mentioned.

Stability study:-

Table 12: Stability of Cream composition after stability study

Table 13: Drug content post- Stability Study of F4 cream composition

Claims :

1 . A herbal composition for repellency of at least Anopheles, Aedes and Culex

mosquitoes for at least eight hours when applied to the body.

2 . The herbal composition of claim 1 comprising three or more than three essential oils.

3 . The herbal composition of claim 2 wherein the three essential oils comprise

Citronella oil, Catnip oil and Kaffir lime oil.

4 . The herbal composition of claim 3 comprising a cream.

5 . The herbal composition of claim 3 comprising -5% w/w citronella oil, 2% w/w

catnip oil and 2.4% w/w Kaffir lime oil; and excipients.

nternat ona app cat on o.

PCT/IN2017/050119

A CLASSIFICATION OF SUBJECT MATTERA01N65/00 Version=2017 .01

According to International Patent Classification (IPC) or to both national classification and IPC

B FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)

A01N

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)

Patseer, IPO Internal Database, TKDL

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No

A AMER: "Repellency Effect of Forty-one Essential 1,2Oils Against Aedes, Anopheles, and CulexMosquitoes", Parasitology Research, 2006September (Epub 2006 April 27), vol. 99, no. 4 ,

pages: 478-90 .See page 480, figures 1-4, tables 2 , 3 , 5

A AMER: "Repellency Effect of Forty-one Essential 3-5Oils Against Aedes, Anopheles, and CulexMosquitoes", Parasitology Research, 2006September (Epub 2006 April 27), vol. 99, no. 4 ,

pages: 478-90 .See page 480, figures 1-4, tables 2 , 3 , 5

A TAWATS N et. al .: "Repellency of Volatile 3-5from Plants against Three Mosquito Vectors'Journal of Vector Ecology, 2001 June, vol.no. 1 , pages: 76-82. .See abstract, pages 77-79, figure 1

A TKDL (Traditional Knowledge Digital Library)entry: "MA2/455", "Tila -e- Anjadaan", known

X Further documents are listed in the continuation of Box C. i See patent family annex

; Special categories of cited documents: later document published after the international filing date or priority"A" document defining the general state of the art which is not considered date and not in conflict with the application but cited to understand

to be of particular relevance the principle or theory underlying the invention

"E" earlier application or patent but published on or after the international document of particular relevance; the claimed invention cannot befiling date considered novel or cannot be considered to involve an inventive

"L" document which may throw doubts on priority claim(s) or which is step when the document is taken alonecited to establish the publication date of another citation or otherspecial reason (as specified) document of particular relevance; d claimed invention cannot be

considered to involve an inventive step when the document is"O" document referring to an oral disclosure, use, exhibition or other combined with one or more other such documents, such combination

means being obvious to a person skilled in the art"P" document published prior to t international filing date but later than document member of the same pate family

the priority date claimed

Date of the actual completion of the international search Date of mailing of the international search report

22-06-2017 22-06-2017

Name and mailing address of the ISA/ Authorized officer

Indian Patent Office Meenakshi YadavPlot No. 32, Sector 14,Dwarka, ew Delhi-110075

Facsimile No. Telephone No. +91-1125300200

Form PCT/ISA/210 (second sheet) (January 2015)

nternat ona app cat o o.

PCT/IN2017/05011 9

C (Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of docmrieut, with indication, where appropriate, of the relevant passages Relevant to claim No.

since 1000 years (accessible a t 1-5www . tkdl . res . in/ ) .Whole document

Form PCT/ISA/210 (continuation of second sheet) (January 2015)