Sustainable manufacture of insect repellents derived from Nepeta cataria

Abstract

Malaria devastates sub-Saharan Africa; the World Health Organization (WHO) estimates that 212 million people contract malaria annually and that the plasmodium virus will kill 419 000 in 2017. The disease affects rural populations who have the least economic means to fight it. Impregnated mosquito nets have reduced the mortality rate but the Anopheles mosquitoes are changing their feeding patterns and have become more active at dusk and early morning rather than after 22h00 as an adaptation to the nets. Everyone is susceptible to the Anopheles at these times but infants and pregnant women are the most vulnerable to the disease. Plant-based mosquito repellents are as effective as synthetic repellents that protect people from bites. They are sustainable preventative measures against malaria not only because of their efficacy but because the local population can produce and distribute them, which represents a source of economic growth for rural areas. Here, we extract and test the essential oil nepetalactone from Nepeta cataria via steam distillation. Families in endemic areas of Burundi found them effective against bites but commented that the odor was pungent. An epidemiological study is
required to establish its clinical efficacy.

Full text

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Scientific REPORtS | (2018) 8:2235 | DOI:10.1038/s41598-017-18141-z
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Sustainable manufacture of insect
repellents derived from Nepeta
cataria
Gregory S. Patience1, Ginette Karirekinyana2, Federico Galli
1,4, Nicolas A. Patience1,5,
Cariton Kubwabo2, Guy Collin3, Jean Claude Bizimana2 & Daria C. Boto1
Malaria devastates sub-Saharan Africa; the World Health Organization (WHO) estimates that 212
million people contract malaria annually and that the plasmodium virus will kill 419 000 in 2017. The
disease aects rural populations who have the least economic means to ght it. Impregnated mosquito
nets have reduced the mortality rate but the Anopheles mosquitoes are changing their feeding patterns
and have become more active at dusk and early morning rather than after 22h00 as an adaptation to
the nets. Everyone is susceptible to the Anopheles at these times but infants and pregnant women
are the most vulnerable to the disease. Plant-based mosquito repellents are as eective as synthetic
repellents that protect people from bites. They are sustainable preventative measures against malaria
not only because of their ecacy but because the local population can produce and distribute them,
which represents a source of economic growth for rural areas. Here, we extract and test the essential oil
nepetalactone from Nepeta cataria via steam distillation. Families in endemic areas of Burundi found
them eective against bites but commented that the odor was pungent. An epidemiological study is
required to establish its clinical ecacy.
Malaria is a leading cause of human mortality. In 2015, the World Health Organization (WHO)1 estimated that
212 million cases of malaria occurred, leading to 419 000 deaths – 82% of casualties are in sub-Saharan Africa.
Children under the age of 5 account for 64% of victims. Malaria’s mortality rate dropped by 31% between 2010
and 2015 in Africa and the life expectancy of children under 5 increased by 1.2 years. However, in 80% of malaria
endemic countries, mosquitos developed resistance to at least one insecticide. Furthermore, in Burundi, deaths
have increased by 13% in the last year2, stunting economic growth3 where the annual per capita income is lower
than 400 USD4 and farmers hire labour at 1 USD a day. As with the mass drug administration of chloroquine,
which contributed to the resurgence of malaria in Peru aer 30 years of low incidence5, Plasmodium falciparum
protozoa may develop resistance to artemisinin monotherapy6. Mutations of the K13 gene are markers of arte-
misinin resistance but tetraoxane-based compounds have inhibitory characteristics against various strains of the
protozoa7. Artesunate is more eective than quinine with fewer side eects but a child administered a high dose
rectally died due to its toxicity8. Moreover, Burke et al.9 reported that Anopheles vaneedeni became a new malaria
vector complicating the ght against the disease.
Preventing rather than treating the disease is a better approach; sleeping under nets impregnated with insec-
ticides has reduced the world mortality rate from 2 million a year; they save 5.5 per 1000 children yearly10.
Insecticides are losing their ecacy due to increasing mosquito resistance11. Furthermore, weather patterns like
La Niña can cause unexpected peaks in mosquito populations12. Graves and Gelband13 tested SPf66, CS-NANP,
RTS,S, MSP/RESA vaccines and their ability to prevent dierent stages of malaria. eoretically, SPf66 and
MSP/RESA protect against the asexual stages of plasmodium parasites whereas the other two target the sporo-
zoite stages. CS-NANP and MSP/RESA oered no protection against malaria, SPf66 was ineective and RTS,S
reduced malaria episodes by 58%. Unfortunately, vaccines’ mass production and distribution is too expensive for
sub-Saharan Africa14. Furthermore, the mosquitos are changing their feeding patterns to adapt to the mosquito
1Département de Génie Chimique, École Polytechnique de Montréal, 2900, boul. Édouard-Montpetit, H3C 3A7,
Montréal, QC, Canada. 2Agence consultative en éthique de coopération internationale (ACECI), 11 Rue Mugamba,
Quartier Rohero II, Bujumbura, Burundi. 3Département des sciences fondamentales, UQAC, G7H 2B1, Saguenay, QC,
Canada. 4Università degli Studi di Milano, Dipartimento di Chimica, via Golgi 19, 20133, Milano, Italy. 5Department
of Bioresource Engineering, McGill University, 3610 University Street, H3A 2B2, Montreal, Canada. Correspondence
and requests for materials should be addressed to G.S.P. (email: gregory-s.patience@polymtl.ca)
Received: 21 July 2017
Accepted: 6 December 2017
Published: xx xx xxxx
OPEN
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nets. Moiroux et al.15 demonstrated that mosquitos have adapted to insecticide treated nets by changing their
biting habits. Proportion of outdoor biting increased from 45% to 68%16. us, nets will be less eective for young
children and pregnant women, who are the most vulnerable to the disease.
Cutaneous mosquito repellents (MR) are one means to reduce the frequency of mosquito bites.
N,N-diethyl-meta-toluamide (DEET) is an eective mosquito repellant17–20 but is cost prohibitive for rural
sub-Saharan populations. Targeting indigenous plants as a source of mosquito repellents will stimulate local
economies and at the same time protect the population. Odalo et al.21 extracted and tested the topical repellency
of essential oils indigenous to Kenya from Conyza newii (Compositae), Plectranthus marrubioides (Lamiaceae),
Lippia javanica (Verbenaceae), Tetradenia riparia (Lamiaceae), as well as Tarchonanthus camphoratus (Asteraceae).
e oils, under their experimental conditions (3 min, forearm exposure), repelled mosquitoes better than DEET.
N. cataria, commonly known as catnip or catmint, is a species of the Lamiaceae family. It is native to temper-
ate and tropical zones in Asia and in Europe and is widely cultivated22. Nepeta cataria (N. cataria) has many
uses in traditional medicine including treatment of chills, colds, constipation, headaches, infections, inamma-
tions, rheumatism, sore throats, spasms, and stomach aches23. N. cataria’s essential oil possesses antibacterial
and antimicrobial properties24,25. Nepetalactone (NPL) is the major constituent of this oil26–29. DEET, applied to
the forearm, repelled Aedes aegypti, Anopheles quadrimaculatus, and Anopheles albimanus for 426 min, 96 min,
and 87 min, respectively29. A 25% volume fraction of DEET in ethanol repelled Aedes aegypti for 8 h30. Whereas,
Bernier found that a dose of 0.5 mg cm−2 of DEET applied to a muslin cloth patch was active for 24 h. Catnip’s oil
is a better spatial repellent than DEET and demonstrated eective topical repellency properties31.
NPL is as active as DEET and its hydrogenated form – dihydronepetalactone – is two times more active
than DEET when formulated with isopropyl alcohol (1% w/v)32. Among 41 dierent essential oils applied to
skin, Catnip’s oered protection for 480 min33. Moreover, N. cataria exhibits a more favorable safety prole than
DEET34. Local communities widely accept essential oil based MR. In Ghana, 97% of the studied population
desired to continue applying the MR aer the 3-month trial35. Mn g’o ng ’o et al.36 found that six repellent plants
essential oil were widely accepted by the population studied due to their ecacy.
Introducing plant based mosquito repellents to vulnerable populations faces economic challenges but also
societal and governmental hurdles. Furthermore, they must be nontoxic with respect to dermal and eye contact,
ingestion, and inhalation. Unlike synthetic repellents, the chemical composition of essential oils, like N. cataria,
contain dozens of compounds (Supplemental information S1). e active ingredient must demonstrate its e-
cacy versus alternatives but they must also be stable with respect to UV exposure, oxidation and high temper-
ature (>40 °C). Moreover, to achieve the largest possible distribution at the lowest cost, local populations must
grow, harvest and extract the active ingredients. Birkett and Pickett discovered the ecacy of nepetolactone and
nepetolactol in repelling aphids. ey distilled the catnip withsteam and cyclohexane vapors then reduced the
oil with NaBH4. In their concluding remarks, they emphasize the potential of plants as source of commercialy
vailable products37. Here we address the challenges of cultivating N. cataria in Burundi and developing a topical
mosquito repellent. We compare the composition of the essential oil versus those cultivated throughout the world
and measure the thermal stability and its stability versus UV. Finally, we demonstrate that the population is ready
to test the mosquito repellents formulated with vegetable oil or as Pickering emulsions based on an acceptability
study we conducted in a rural and urban region of the country.
Results
Nepeta cataria production. N. cataria (purchased from http://mckenzieseeds.com) is a robust herbaceous
short lived perennial plant grown around the globe. Its essential oil is encapsulated in glandular trichomes38
measuring 50 µm in diameter (Fig.1). e plant is cultivated at least three times a year in Burundi and even twice
a year in New Jersey where Park et al. reported as much as 7.7 t/ha (dryplant matter yield) and an essential oil
yield of 12.5 kg/ha39. In Burundi, essential oil yield reaches 3 kg/t (dryplant matter), which is double the 1.6 kg/t in
New Jersey. e number of trichomes increases until the full owering stage40, while their size remains constant.
Figure 1. N. cataria forms glandular trichomes 50 µm in diameter. Leaves sampled at 20 cm from a 40 cm tall
planthad 100 trichomes per cm2 (10 optical images from 100 mm2 samples). Le red marker: 300 µm; right red
marker 10 µm.
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Essential oil characterization. e three principal compounds extracted from N. cataria in Burundi
were 4aα,7α,7aβ-nepetalactone (72%), β-caryophyllene (10%), and trans-β-ocimene (4%). e data from this
study compare well with other published results describing the composition of catnip oil from various geograph-
ical regions around the world. Asgarpanah et al.41 isolated the essential oils of the Nepeta genus and found that
34% of the species contained 4aα,7α,7aα-nepetalactone while 20% (including N. cataria) contained the epimer
4aα,7α,7aβ-nepetalactone and 10 plants of 41 contained 4aβ,7α,7aβ-nepetalactone. A GC-MS measured the
composition of the essential oil of N. cataria grown in Burundi(TableS1, supplementary material). Chalchat and
Lamy42 did not nd nepetalactone in the essential oil isolated from wild catnip N. cataria L. cv. citriodora grown in
the Drôme region: the major constituents were nerol, geraniol and citronellol. Gilani et al.43 reported 1,8-cineole,
α-humulene (14%) and α-pinene as the 3 major constituents in commercial catnip oil. Other compounds found
at relatively higher concentration included α-pinene, limonene and trans-β-ocimene. e dierences in catnip
oil composition (Table1) are due to extraction techniques, and environmental and agricultural factors like chem-
otype, soil, growing region, meteorology, pests, drying and extraction methods, etc.
Essential oil extraction. Batch steam distillation is the standard technology to extract the oil (Fig.2).
However, it is energy intensive since about 1000 kg of water vapour extracts 1 kg of oil. A major program element
to develop a process suited for local populations is to identify energy sources.
Much of the forests in Burundi, for example, have disappeared as the rural population relies on wood to cook.
Alternative energy sources to extract oil include coee husks (Fig.3), bagasse from sugar cane, tea, rice husks
and other farm residues44,45. ese lignocellulosic based sources may require torrefaction and pelletization46 to
increase their energy density. Micro-wave and ultrasound are alternatives to steam distillation with solar energy
as the vector to produce electricity. Tests with ultrasound at temperatures from ambient to 60 °C with water and
ethanol extracted chlorophyll together with some oil but yields were low. Furthermore, sonication at 20 W (fre-
quency of 20 kHz) destroyed the leaf, which indicates that too much power was applied and that it is not selec-
tively activating the trichomes. erefore, here we extracted the essential oil with steam distillation.
Stability study. Producing mosquito repellents locally minimizes transportation, distribution, and storage
costs. NPL is susceptible to degradation due to UV, heat and oxygen, which requires additional packaging to
reducelight exposure. Average temperatures in north Africa hover well over 30 °C while the average temperature
in the central Burundian plateau is 20 °C47. Sun radiation heats surface temperatures of inanimate objects well
above 50 °C but plant transpiration through stomatal apertures maintains leaves and the trichomes at a lower
temperature (Fig.4). NPL degradation will be most prevalent aer the extraction process.
Temperature, light, and oxygen deteriorate essential oils’ integrity and lower the active component concentra-
tion. As a bicyclic, monounsaturated terpenoid, nepetalactone undergoes primary oxidation to unstable products,
i.e. hydroperoxides, which then convert into stable secondary oxidation products such as alcohols, aldehydes,
Ref. Plant matter (Country) Compound 1 Compound 2 Compound 3
is study Flowering aerial parts
(Burundi) 4aα,7α,7aα-NPL (72.4 %) β-Caryopyllene (10.2 %) trans-β-Ocimene (3.8 %)
58 Aerial parts (USA) NPL (77.6 %) Epinepetalactone (15 %) Caryophyllene (2.8 %)
59 Flowering aerial parts
(France) 4aα,7α,7aβ-NPL (56.9 %) Caryophyllene oxide (18.2 %) β-Caryophyllene (6.2 %)
60 Aerial parts, bloom stage
(Argentina) NPL (57.3 %) Caryophyllene oxide (19.4 %) β-Caryophyllene (8.1 %)
61 Aerial parts (Balkan
mountain, Bulgaria) 4aα,7α,7aβ-NPL (78.0 %) 4aβ,7α,7aα-NPL (56.9 %) Nepetalic acid (1.6 %)
42 Flowering aerial parts
(France) Nerol (28.2 %) Geraniol (27.6 %) Citronellol (15.1 %)
62 Flowering aerial parts
(USA) (Z,E)-NPL (54.6 %) (E,Z)-NPL (31.9 %) β-caryophyllene (11.6 %)
63 Flowering aerial parts
(Iran) 4aα,7α,7aβ-NPL (28.8 %) 1,8–Cineole (13.5 %) 4aα,7β,7aα-NPL (11.9 %)
64 Flowering aerial parts
(Turkey) 4aα,7α,7aβ-NPL (70.4 %) 4aα,7α,7aα-NPL (6 %) 4aα,7β,7aα-NPL (2.5 %)
43 - (Pakistan) 1,8-Cineole (21 %) α-Humulene (14.4 %) α-pinene (10.4 %)
40 4-aerial develop-mental
stages (Iran) 4aα,7α,7aβ-NPL (55–59 %) 4aα,7β,7aα-NPL (30–31.2 %) α-Pinene (2.7–4.6%)
65 Flowers
(UK) Chemotype
A: Chemotype B:
4aS,7S,7aR-NPL (92 %)
4aS,7S,7aR-NPL (17 %) (4aS,7S,7aS)-NPL
(4aS,7S,7aS)-NPL (69.8 %) (E)-(1R,9S)-Caryophyllene (8 %)
(E)-(1R,9S)-Caryophyllene (13.2 %)
25 3-aerial develop-mental
stages (Iran) 4aα,7α,7aβ-NPL (55–58 %) 4aα,7β,7aα-NPL (30–31.2 %) α-Pinene(2.7–4.6%)
66 Flowering stage
(Morocco) 4aα,7α,7aβ-NPL (77.4 %) Dihydronepetalactone (5 %) Limonene (4.1 %)
67 Aerial parts (Turkey) NPL (27.5 %) 1,8-Cineole (10.8 %) Germacrene D (9.2 %)
Table 1. e main components of N. cataria species depends on agricultural practices, soil, age of the plant,
collection period, drying, extraction methods, climate and geographic origin (NPL – nepetalactone).
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Figure 2. Steam distillation schematic of Nepeta Cataria to extract its essential oil.
Figure 3. Mounds of coee husks generated by the SODECO hulling factory, Burundi.
Figure 4. Infrared image of a small branch whose end is immersed in water under the sun at noon (Montréal,
July 2017). e ambient temperature was 25 °C. e leaf in the white square was cut from a branch 1 h prior
to the photo. e centre of this leaf approaches 45 °C while the edges of the top leaves on the twig are closer to
30 °C.
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ketones, epoxides, peroxides, but especially acids48. Heat and light promote the cleavage of the unique double
bond in nepetalactone by epoxidation or allylic oxidation into alcohols, ketones, and aldehydes49. Many of these
constituents may be skin irritants, which would reduce their desirability as a topical treatment.
Water is an inexpensive solvent to dilute the active ingredients for cutaneous applications; however, NPL is
poorly soluble in water. Blending it with vegetable oil or another solvent increases the overall cost of the lotion.
An alternative is to generate a Pickering emulsion stabilized with silica50. is may also reduce NPL’s volatility and
thereby increase the eectiveness of a single application. We tested nepetalactone stability with respect to temper-
ature at 60 °C and to light with a UV-A lamp at λ = 365 nm (Spectroline EA-160) for both pure N. cataria oil (7 d)
and a 10% N. cataria essential oil emulsion over 140 d and measured their iodine and acid values.
Iodine value (IV) measures the number of unsaturations, i.e. both the tendency of a substance to oxidize, as
well as the number of unsaturations loss from oxidation, thus being an indirect measure of the essential oil light
and heat stability51. e IV does not dierentiate between primary and secondary oxidation. e IV of the refer-
ence oil, preserved from heat and light, was 59 g I2/100 g oil. e IV of the N. cataria Pickering emulsion before
heat and/or light exposure was 2.7 (Fig.4). Besides the obvious decrease of the IV because of dilution, moisture
may saturate double bonds52. e IV of the 10% N. cataria Pickering emulsion50 decreased by 68% in 140 days,
whereas the IV of the emulsion exposed to heat and-or light decreased by over 90% in 7 days, indicating a more
severe oxidation of the double bonds (Fig.5).
e acid value (AV) of pure N. cataria oil reaches 4% in 7 days, while it increases much slower for the emul-
sion. e AV of the N. cataria Pickering emulsion increases the most in the presence of both light and heat.
e AV of the reference sample rises 4-fold in 140 days (Fig.6). It increases 6-fold in 140 days for the emulsion
exposed to either heat or light and 9-fold in 3 weeks for the sample exposed to light and heat. For an ester terpe-
noid such as nepetalactone, organic acids are either the products of ester hydrolysis, or the secondary oxidation
products. Rajeswara Rao et al.53 did not report signicant physicochemical changes in essential oils stored in
water, even at 20% (v/v). erefore, double bond oxidation is the most probable pathway to organic acids, con-
rmed by a concomitant decrease in the IV.
Acceptability study. During a one-month trial, the majority of the participants (52/60) applied the lotion
correctly while 7 individuals applied the lotion sporadically: 5 individuals applied the lotion 3 times before sleep-
ing, 40 applied it 2 times and 15 applied it only once. In general, most of the individuals (91.6%) apply lotions aer
a shower to keep skin hydrated while 5 men do not routinely apply lotions. Among the adolescents and adults
(31 participants), most found the odor tolerable while 10 stated that it was strong smelling (pungent). e side
eects (Table2) included sneezing, feeling nauseous, and vomiting, but only 6 individuals reported these eects.
Figure 5. NPL Pickering emulsion stability with respect to IV and 60 °C over 20 weeks. Error bars represent
standard deviation, n = 3.
Figure 6. NPL Pickering emulsion stability with respect to acid value and 60 °C over 20 weeks (error bars are
smaller than the size of the symbols).
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Almost everyone agreed that the lotion reduced how oen they were bitten. Only one individual said that it
was ineective and that they were bitten as oen with the repellent; 55 individuals said that they were not bitten
aer applying the lotion (Table3). Here we assign a value of 2 to individuals nding the essential oil eect eective
and 1 to those who did not. Assuming the responses are normally distributed, the p-value was 0.001 for a null
hypothesis that the essential oil was ineective, which allows us to conclude that it was eective.
Discussion
To introduce NPL as a strategy to reduce mosquito bites requires governmental approval from several ministries’
(Health, Agriculture, Science and Education, Planning and Reconstruction), and collaborations from university
(faculty of medicine, agriculture), UN agencies (WHO, Global Fund, UNICEF), foreign donors, governments,
and agricultural cooperatives who devised the implementation strategy. Working with the media acquaints the
population to the opportunities and challenges and plays an important role mobilizing the decision makers at the
local and political sphere.
Applying the mosquito repellent frequently demonstrates a high degree of acceptability. Rather than just
lotions, the 31 individuals from the study also suggested incorporating the essential oil in perfumes (21), soaps
(25), and indoor sprays (20). Rowland et al.54 reported that adding DEET to soap reduced the number of cases of
malaria by 50%.
ese results are encouraging considering that, on average, people may be bitten 75 times per night55. e
combination of mosquito repellents and insecticide impregnated nets reduced malaria 80% more than for indi-
viduals that only slept under nets56.
Mosquitos are developing resistance to insecticide treated nets and they are changing their feeding habits
to adapt to the reduced availability of their prey during the night. ese adaptations by the Anopheles mosquito
requires new strategies to ensure that the world malaria death rates continue to decline. We recommend that
local populations grow and produce their own mosquito repellent with sustainable resources like coee husks,
bagasse, or other waste lignocellulosic feeds stocks. More work is required to identify alternative energy sources
and extraction technologies that are more ecient to isolate the essential oil. Furthermore, we recognize that
mosquitos may also develop a tolerance for any repellent and therefore, we must continue to develop other agri-
culturally based compounds to protect vulnerable populations, particularly children and pregnant women.
Methods
Steam distillation. KarireProducts cultivated N. cataria in the Moso region in the province of RUTANA,
Burundi, picked the leaves at full bloom, and air-dried them for 30 hours. In a 5 hour process, a stainless steel
steam distillation system isolated essential oil from chopped dried plant material (20 to 25 kg/batch). We stored
the essential oil in amber containers at low temperature (4 °C). A propane burner boiled water at the bottom of
a tank and the steam it generated passed through a metal mesh that was supporting the weight of the leaves. e
steam rst heats the biomass. Aer one hour, both oil and water vapour broke through to the top of the distillation
column and passed through a pipe at the top lead to a condenser. e condensed solution separated in a second
vessel and we collected both the hydrosol and essential oil.
Ultrasound extraction. A Sonics Vibracell Ultrasound probe delivered 20 W (500 W nominal power) to
20 mL of water and ethanol in which we added 1 leaf (approximately 0.2 g) of catnip collected before the plant
owered. Sonication lasted 1 min. A thermocouple measured the temperature of the liquid. We collected all sam-
ples in 2 mL glass vials. e reference samples were stored in a 500 mL hermetically sealed dark brown glass bottle
that we covered in aluminum foil and stored in a chemical cabinet maintained at ambient temperature.
Scanning electron microscopy. A eld emission scanning electron microscope (FE-SEM-JEOL JSM-
7600F) with a voltage of 2 kV imaged catmint leaves (LEI detector). e distance between sample and detector
was 13 mm.
Undesirable Eects Individuals, n = 60
Sneezing 3
Nausea 2
Vomiting 1
Tot al 6
Table 2. Reported side eects.
Comments Individuals, n = 60
Ineective 1
Lotion reduces bites 4
No bites aer application 55
Table 3. Lotion eectiveness according to the survey.
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Gas chromatography. N. cataria oil is a clear pale yellow liquid, with a refraction index of 1.4882 ± 0.0005
(23 °C). We diluted the samples in HPLC grade pentane (1:200). A gas chromatograph equipped with a ame ion-
isation detector (GC-FID) and another GC coupled to a mass spectrometer (GC-MS) quantied the main com-
ponents of the essential oil. e GC-FID was equipped with a DB-5 column (30 m × 0.25 mm × 0.25 m; Agilent
Technologies, Santa Clara, CA, USA), while the GC-MS had a Solgel-Wax column (30 m × 0.25 mm × 0.25 m;
SGE Analytical Science, Austin, TX, USA). We set both GCs injection port and detector temperature at 220 °C
and 260 °C, respectively. Helium carried analytes at a ow rate of 1.4 mL/minute. Temperature remained at 40 °C
for 2 minutes, then increased to 210 °C at a rate of 2 °C/minute. e split ratio was 50:1 and the injection volume
was 3 microlites. e mass spectrometer operated in electron-impact (EI) mode at 70 eV, with a scan range of
40–550 amu and a scan speed of 1458.6 amu/s. e temperature of the MS interface was 300 °C. We computed
FID peak areas without a correction factor. e retention times of n-alkanes with an even number of hydrocar-
bons (C8–C24) injected using the same analytical conditions determined the retention indices of the essential oil
constituents. We qualitatively identied all the compounds from the NIST library57. To identify the geometrical
isomers we injected standards, purchased from Sigma Aldrich.
Stability tests. For the light stability tests we exposed the samples to a UV-A lamp (Spectroline EA-160),
while for the heat stability we placed the samples in an oven at 60 ± 3 °C. In the combined light and heat stability
tests we placed the oil containing vials at the bottom of a thermic bath (ISOTEMP 205) at 60 ± 3 °C surmounted
with the UV-A lamp.
We applied ASTM-D5768 to measure the iodine value of the essential oil, which represents the degree of
unsaturation: the iodine reacts with the double bonds, also known as the Wijs procedure.
We reported the acid value as g of KOH required to neutralize 100 g of sample.
Acceptability study. Aer meeting with presidential sta and ministers of health and agriculture, students
in the faculty of medicine developed a strategy to assess the population’s acceptability of applying mosquito repel-
lents daily. We produced a lotion that contained a mixture of 5% nepetalactone in vegetable oil, almond oil and
citronella. In 2011 from March 3rd to April 3rd, the students followed 8 families in Kamenge (an urban neigh-
borhood of Bujumbura) as selected by the non-governmental agency ALUMA respecting pre-established selec-
tion criteria and 4 families in the rural community of Cibitoke (selected by the local hospital). Both were hyper
endemic malaria regions. Each family received 2 bottles with 100 mL of the lotion. e volunteers included one
pregnant woman, 10 children younger than 5 y, 19 children from 5 to 11 y, 11 adolescents from 12 to 20 y, and 20
adults older than 20 y of which most of these were farmers (17). ere were 32 males and 28 females in the study.
Most individuals (45%) were in bed under impregnated mosquito nets by 21h00, while 32% were in bed by 22h00,
and 10 children were in bed before 20h00.
Data availability. All data generated or analyzed during this study are included in this published article (and
its Supplementary Information les).
Ethics statement. All participants and guardians were enrolled from March 3rd to April 3rd 2011 and pro-
vided written informed consent. Action de Lutte contre la Malaria au Burundi (ALUMA) and the hospital of
Cibitoke selected the individuals. e hospital of Cibitoke and ALUMA approved the experimental protocol and
it was carried out in accordance to all relevant guidelines and regulations.
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Acknowledgements
We thank the French Ambassador for a nancial contribution and his personal implication in the project, the
Batwa of Matara, ABEM, Aliksir, ALUMA, ACVE, and all the organizations that participated directly or indirectly
in the nancing. We would like to thank Mr. Marco Giulio Rigamonti for the optical microscope image.
Author Contributions
G.S.P. and G.K. developed the program to cultivate N. cataria in Burundi: imported seeds, identified land,
imported an extraction unit, engaged the population, communicated with the press, sought government
approvals, and solicited support from international governments and organizations. G.S.P. prepared Figures 2–6,
supervised the work, participated writing and editing all sections. F.G. and G.S.P. took the SEM image. F.G and
N.A.P. wrote the introduction, performed the ultrasound extraction of catnip essential oil and revised the nal
version of the manuscript. D.C.B. participated in the analytical parts (GC-FID and GC-MS), wrote the Methods
section and the section on stability, and supervised undergraduate students testing the stability. C.K. and G.C.
developed the analytical techniques to identify the essential oil compounds and wrote the sections summarizing
the data. J.C.B. designed, conducted and wrote the acceptability study and N.A.P. translated the document. All
authors reviewed the manuscript.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-017-18141-z.
Competing Interests: G. Karirekinyana is now marketing products derived from the N. Cataria essential oil in
Burundi. All the work reported in the document was completed before commercialization. KarireProducts has
10 employees. All other authors declare that they have no nancial interests.
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