KR100407604B1 - Repellent agent for mosquito - Google Patents

Abstract

The present invention relates to a mosquito repellent, and more particularly, fennel oil, fenchon ((+)-fenchone), and octadecene noic acid (( E) -9-octadecenoic acid) relates to a hygienic pest repellent comprising at least one selected from the group consisting of. Fennel oil, fenchon ((+)-fenchone), and octadecenoic acid ((E) -9-octadecenoic acid) of the present invention can be provided as a repellent of sanitary pests such as mosquitoes without harm to the human body. have.

Description

Mosquito repellent {Repellent agent for mosquito}

The present invention relates to mosquito repellent, and more particularly to fennel oil, fenchon ((+)-fenchone), and octadecenoic acid ((E) -9-octadecenoic acid) extracted from fennel fruit. .

There are more than 3,000 mosquitoes in the world. Among them, hygiene harms human beings. There are about 500 Aedes , 300 Culex , and 350 Anpheles (Harwood). 1979. Entomology in Human and Animal Health.Macmillan.) There are 51 known mosquitoes in Korea, including 20 Aedes , 18 Culex , and Anpheles . (Yoo Hyo-Seok, 1994. Korea Insect Collection, Konkuk University Press)

Mosquitoes not only suck blood from humans and livestock, but also carry many diseases such as malaria, encephalitis, yellow fever, and dengue fever, causing many lives to die. Today, two-thirds of the world's population is exposed to mosquito-borne diseases. Mosquito vampires are also pests that directly or indirectly cause serious damage by acting as stress sources for sleep disturbances and outdoor activities. (Kettle, DS 1991. Medical and Veteninary Entomology.Wieley Interscience Publication.New York )

Among the mosquitoes known in Korea, Chinese spotted mosquitoes ( Anopheles sinensis ) are malaria and inland brugian filariasis , and Aedes togoi are southern filamentous ( culex tritaeniorhynchus ). Is a mediator of Japanese encephalitis, and the other two groups , Culex pipiens pallens and Culex pipiens molestus , which have been collected in recent years, have recently developed in apartment complexes, causing blood-sucking problems. (Han-il Lee, 1988. Hygiene and Insectology (4th edition). Advisor, Seoul, 340 pp.)

Conventionally, in order to control mosquitoes, chemical control by insecticides was mainly used, but the use of chemical synthetic insecticides caused side effects such as drug resistance, killing of useful organisms, toxicity to human body, residual and various environmental pollution. (Georghiou, GP and Saito, T. 1983. Pest Resistance to Pesticides.Plenum Press New York and London; National Research Council. 1986. Pesticide Resistance: Strategies and Tactics for Management.National Academy Press, Washington, DC .; Brown ,, AWA 1978. Ecology of Pesticides. Academic Press, New York.) Therefore, there is an urgent need to develop alternatives and control methods for pesticides that can minimize human and ecosystem impacts and effectively protect humans from mosquitoes.

The repellent, which was developed under the new concept as an alternative to insecticides, uses natural substances that hinder the behavior of living organisms as a pest control agent, which can prevent insect-borne diseases for humans and has no side effects such as environmental pollution. .

However, repellents are demanding because they are intended for human use and must include the function of controlling them to stimulate the chemosensory of insects. Necessary requirements for repellent should be harmless to human body, long lasting effect, no irritation to skin, no unpleasant odor, chemically stable, effective without treatment all over skin And so on.

Recently, as research on plant extracts and plant-derived compounds has been actively conducted, terpenes, phenols, and alkaloids that plants contain as secondary metabolites (Harborne, JB 1993.Introduction to Ecological Biochemistry, 4th Ed.Academic Press, New York) Is a low toxicity to the human body, simple to treat like conventional pesticides and effective for limited pests such as pests, but does not affect natural enemies (Jacobson, M. and Crosby, DG 1971. Naturally Occurring Insecticides. Marcel Decker, New York; Elliot, M. 1977. Synthetic pyrethroids, pp. 1-28, in M. Eliott (ed.) Synthetic Pyrethroids.ACS Symp.No. 42, Amer.Chem.Soc ., San Francisco, CA; Hedin , PA 1982. J. Agric Food Chem 30 :....... 201-215; Arnason, JT, Philogene, BJ, and Morand, P. 1989. ACS Symp Ser No. 387, Amer Chem Soc, Wachington, DC, 1989; Isman, MB 1995. Rev. Pestic. Toxicol. 3: 1-20.), Has been recognized as an alternative to pesticides. Mosquito repellent St. plants been reported so far, there are naemyu (neem oil), lanthanide Camara (Lantana camara), Arte Misia vulgaris (Artemisia vulgaris) and eucalyptus (Eucalyptus) in the plant. (Dua, VK, NC Gupta , AC Pandey and VP Sharma. 1996. J. Am. Mosq. Control Assoc. 12: 406-408; Sharma, VP and MA Ansari. 1994. J. Med. Entomol. 31: 505-50).

Insect repellent (PMD), derived from eucalyptus, is produced by extracting oil from lemon eucalyptus plants in China. Although PMD is not as effective as DEET ( N, N -diethyl- m -toluamide), it has been shown to be effective against anopheles and effective for ticks, flies and mosquitoes. In addition, unlike DEET, PMD has no reactivity with plastics and synthetic fibers, so it can be applied to various products, and its toxicity is very low (oral test LD ₅₀ 2,408 mg / kg in rats, transdermal LD ₅₀ 2,000 mg / kg). Trigg, JK 1996. J. Am. Mosq.Control Asso c. 12: 243-246)

The above-mentioned DEET is the most widely used repellent among the repellents developed so far, and more than 30 million are consumed each year because 30% of the nation uses DEET in the United States (as of 1994). Not only does it affect species, it is used throughout pests such as mosquitoes, flies, teeth, fleas, ticks, and also to protect livestock.

On the other hand, in Korea, malaria, which completely disappeared in the 1970s, began again in 1993, and 3,330 malaria cases occurred in late 1998 by the end of September. The infection rate increased further, and the area where the patient developed was widened from the vicinity of the DMZ to the northern part of Gyeonggi-do toward Seoul. In addition, in case of Japanese encephalitis, the number of patients is currently reduced, but it is not yet a relief phase.

In the United States, mosquito-borne diseases such as encephalitis and yellow fever, which had long been a problem, re-emerged since the 1990s, as well as mediating diseases like never before, such as West Nile encephalitis.

In addition, due to the increase in temperature and environmental changes due to global warming, the occurrence of mosquitoes and blood-sucking activity increases in summer as well as in winter.

In this situation, the use of repellents that can protect humans from mosquito-borne diseases, along with conventional mosquito control by synthetic insecticides is increasing, but the development of repellents that can supplement the problems of conventional repellents is sluggish.

As a typical repellent agent, DEET has an unpleasant odor and irritates the skin and causes problems such as causing damage to glasses or watch bands by reacting with some plastics or synthetic rubbers, as well as fast and strong skin penetration. Due to the restrictions on use. In the DEET skin penetration test conducted in 1982, DEET 300 ㎍ / ㎠ was applied to the skin, and after 1 hour, it had 30% penetration rate and 12% after 36% penetration rate. The use of DEET was restricted, and adults were also restricted from applying more than 20% of the body surface.

Therefore, the development of materials that can reduce excessive DEET usage and maintain the mosquito avoiding effect is required, the development of DEET substitutes harmless to the human body is in progress centering on plants. However, the development of repellents that must meet the demanding requirements due to the nature is still insufficient.

Accordingly, an object of the present invention is to provide a sanitary pest repellent, in particular a mosquito repellent, which is harmless to the human body and can replace the synthetic repellent DEET.

Figure 1 shows a process for separating the hygiene pest repellent active ingredient by chromatography the hexane fraction of the fennel fruit methanol crude extract of the present invention.

In order to achieve the above object, the present invention provides a hygienic pest repellent comprising an extract of Fennel ( Fenicinum vulgare ) fruit.

The present invention also provides a sanitary pest repellent agent comprising a compound selected from the group consisting of phenchon mother compound, and octadecenoic acid mother compound.

Hereinafter, the present invention will be described in detail.

The fennel (fennel or Foeniculum vulgare) is a unique scent as a perennial plant with umbel umbel reconciled and are being used as stomach, anthelmintic and expectorant in one shot. In addition, the oil obtained by distilling the fennel fruit is called fennel oil, and is used as a scent of a liqueur, and is used as an aroma therapy using incense.

The repellent of the present invention is preferably used for hygiene pests. The sanitary insects are insects that directly or indirectly harm humans, such as bloodsucking insects, parasitic insects, pathogen propagation insects, gyophilic insects, venotoxic insects, and unpleasant insects. Vampire insects include mosquitoes, mosquitoes on the back, teeth and bedbugs. Parasitic insects include fleas and teeth. Pathogen propagation insects include flies, mosquitoes, wheels, and teeth. ), Bee, sting bug, etc., poisonous insects include blue-winged wings, sky cattle paste, and rosacea, and unpleasant insects include midget, worms and stink bugs. In addition, taxonomically, arthropods such as mites, spiders, centipedes, and scorpions may be included. The most preferred sanitary pest of the present invention is mosquitoes.

In addition, the extract available for avoiding the hygienic pest of the present invention is a methanol crude extract of fennel fruit extracted with methanol. The crude crude extract contains all the substances that can come out of the cell wall of the fennel fruit, and the extraction method is different from the fennel oil obtained through distillation, and the components are not exactly the same, but oil components obtained from the fennel oil are obtained in the same manner. Therefore, the preferred extract for avoiding sanitary pests of the present invention is fennel oil, and the fenchon ((+)-fenchone) or octadecenoic acid (( E ) -9-octa decenoic acid) isolated from the crude methanol extract is most preferred. desirable.

Fenchon ((+)-fenchone) and octadecenoic acid ((E) -9-octadecenoic acid) are substances separated from fennel that are used as medicinal or spice, and fenchon is a substance whose function is known as a spice. to be.

The repellent of the present invention preferably comprises 65% to 70% by weight of methanol crude extract, more preferably 35% to 40% by weight of bovine oil, 15% to 20% by weight of fenchon or 30% by weight To 35 weight percent octadecenoic acid. However, the content of phenchon or octadecenoic acid included in the repellent is naturally adjusted according to the method of using the repellent or the formulation presented.

Repellents comprising the fennel fruit extract of the present invention are preferably in the form of a spray, a liquid, a solid, and a gel, most preferably a liquid.

On the other hand, the experimental procedure performed to extract the fennel-derived compound of the present invention is as follows.

In the present invention, the fennel fruit is extracted with methanol, fractionated into hexane, chloroform, ethyl acetate, and water layer, and then mosquito repellent function is carried out by patch test to perform chromium layer of hexane layer having repellent ability to sanitary pests. Separation was performed and in the final step, two kinds of repellent compounds were separated. The structure of the compound was analyzed to be fenchon ((+)-fenchone) and ( E ) -9-octadecenoic acid.

However, methanol extract and fennel oil, which are crude extracts of fennel, have repelling ability against sanitary pests, and also phenchon ((+)-fenchone) and octadecenoic acid ((E) -9-octadecenoic acid) are also hygienic. It was found to have repelling ability against pests. By preparing and using a repellent containing such fennel oil, fenchon ((+)-fenchone) or octadecenoic acid ((E) -9-octadecenoic acid) as an active ingredient, damage from hygienic pests can be prevented.

In addition, the fennel oil used in the present invention has been proved to be harmless to humans because it has already been used in medicinal or skin-care, penchon ((+)-fenchone) and octadecenoic acid ((E) -9-octadecenoic acid of the present invention ) Can solve instability and detoxification problems of the existing synthetic repellent DEET.

In addition, by modifying the chemical structure based on the phenchon ((+)-fenchone)) or octadecenoic acid ((E) -9-octade cenoic acid) of the present invention, the repellent activity is higher and has a variety of different effects It can be used as a parent compound to develop a compound. A parent compound refers to a new substance that removes or enhances the effects of a problem by adding or removing hydroxyl groups or other chemical structures through chemical synthesis based on the structure of a known substance. In the case of pyrethroid-based insecticides, a representative example of the parent nucleus compound, an insecticide newly synthesized from the original plant essential oil as a parent nucleus compound is used to manufacture a similar substance with less photolysis and lower fish toxicity than the original ingredient.

Therefore, it is possible to provide an insecticide comprising the Fenchon ((+)-fenchone) parent nucleus compound or octadecenoic acid ((E) -9-octadecenoic acid) mother nucleus compound of the present invention.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example 1 Sample Preparation and Identification of Active Ingredients

13 kg of Foeniculum vulgare fruits were purchased, dried in an oven at 60 ° C. for 3 days, then finely pulverized, extracted twice with 100 L of methanol at room temperature and filtered with Tokyo filter paper No2 (Japan). After that, the resultant was concentrated under vacuum at 35 ° C. to yield a yield of 6% to obtain a crude methanol extract. The yield of crude methanol extract was calculated as the weight of methanol extract compared to the weight of crushed fennel plants. 780 g of the extract was fractionated using the polarity and specific gravity difference of the organic solvent. When 20 g of methanol fraction was fractionated, hexane (18.6 g) fraction, chloroform (0.8 g) fraction, ethyl acetate (0.1 g) fraction and water layer (0.5) were fractionated. g).

Experimental Example 1

The repellent activity of the crude extract, hexane fraction, chloroform fraction, ethyl acetate fraction and water layer fraction extracted in Example 1 was analyzed.

Test mosquitoes used in the experiment

Egyptian forest mosquitoes ( Aedes aegypti ) used in the present invention for mosquito repellent assay was used by the National Institutes of Health, was selected and tested only indoor insects that have not been exposed to insecticides or any other drugs. Mosquito breeding was put in a 30 × 35 × 10 cm container of tap water for 3 days and then used for larval rearing. The larvae were fed by sifting the calf feed and sifting the fine grains with yeast and 5 to 5 The larvae were fed, and the thick ones were used for the larvae at the 3-4 age stage with a 3 to 7 reduction in the yeast ratio. The pupa mosquitoes were placed in a paper cup with an eyedropper, and then placed in a breeding vessel with a mesh of 35 × 35 × 35 cm wire structure and allegorized as an adult. Adult food was soaked in cotton with 8% sugar water. Breeding conditions were bred under the conditions of light conditions 16: 8 (Note: night), temperature 27 ± 3 ℃, relative humidity 80 ± 10%.

Patch test

As a method of testing the mosquito repellent effect of a sample, there are a method using an animal and a method using a human. It is difficult to test the mosquito avoidance effect quickly and accurately because the test results using the animal may be different from the test results using the human, and the test procedure can be cumbersome and damage to the test assistant.

In the present invention, a human patch test using a patch test of Schreck (Schreck, CE, K. Posey, and D. Smith. 1977. Tech. Bull. US Dep. Agric . 1549: 215.) is used. The experiment was conducted indoors so as not to damage the experimenter.

As an experimental container, a mesh was used to cover a wire structure of 35 × 35 × 35 cm. 60 Egyptian female mosquitoes, 7 to 10 days old, were placed in each container, and the crude extract of Example 1 and four fractions dissolved in 100 µl of ethanol were uniformly treated with gauze having a diameter of 50 mm, and then 2 Allow to dry for a minute and place on rubber gloves with holes of the same diameter. The rubber gloves are designed to maintain the distance between the fabric and the skin at a 5 mm height on the rim, so that they cannot be vamped even if the mosquitoes are attracted during the test.

The mosquito repellent effect was determined by the number of mosquitoes settled on the gauze for 5 minutes, and used as a control by putting gauze treated with 95% ethanol solution only. The avoidance effect was calculated by the following formula (1).

(Calculation 1)

In addition, in order to confirm the mosquito repelling effect, the existing synthetic repellent DEET was assayed in the same manner to compare the activity.

The repellent effect of methanol extract of the fennel fruit of Example 1 and Egyptian forest mosquitoes of each fraction is shown in Table 1 below, it was found that the remarkable repellent effect in the methanol extract and hexane fraction.

sample Concentration (mg / cm 2) % Rejection ^a Crude Extracts (MeOH Extracts) 0.1 ＞ 80% Hexane fraction 0.1 ＞ 80% Chloroform Fraction 0.1 <60% Ethanol Fraction (EtOAc) 0.1 <60% Water layer fraction 0.1 <60% DEET 0.1 ＞ 80%

Example 2

In Experimental Example 1, an experiment for separating the active ingredient contained in the hexane (720 g) fraction having a strong repellent activity was performed.

Figure 1 shows the process of separating the active ingredient of the hexane fraction.

Primary Silica Gel Chromatography

Hexane fraction 187 g was adsorbed on a silica gel column (berck 70-230 mesh, 600 g, 5.5 × 70 cm), and then primary chromatographed with a stepwise gradient of hexane and ethyl acetate. The gradient used was hexane: ethyl acetate ratio in the order of 90:10, 85: 15,70: 30, 50:50, 0: 100 and the fractions of each ratio were eluted with H1, H2, H3, H4, H5. It was. The five fractions confirmed the mosquito repellency by the patch test of Experimental Example 1, and as a result, two fractions H1 187 g and H4 57 g were separated.

Secondary Silica Gel Chromatography

Silica gel chromatography was further performed on the active fraction separated from the primary.

First, the H1 fraction eluted three fractions by fixing the ratio of hexane: ethyl acetate to 20: 1, and named H11, H12, and H13 according to the eluted order. In addition, the patch test was carried out to confirm the active fraction H11 fraction was the active fraction.

At the same time, the H4 fraction was eluted with the ratio of hexane: ethyl acetate at 2: 1, and the three fractions were eluted and ordered H41, H42 and H43 in the order of eluting. In addition, the patch test was carried out to confirm the active fraction, H41 fraction was the active fraction.

3rd Chromatography

Fractions H11 and H41 having secondary activity were further purified.

The H11 fraction was eluted with 30: 1 hexane: ethyl acetate ratio in silica gel chromatography to separate the active fraction H111 and the inactive fraction H112.

The H41 fraction was eluted with chloroform in silica gel chromatography to separate the active fraction H412 and the inactive fractions H411 and H413.

4th Chromatography

Fractions H111 and H412 having activity in the third phase were further purified.

The H111 fraction was eluted with a hexane: ethyl acetate ratio of 35: 1 in silica gel chromatography to separate the active fraction H1111 and the inactive fraction H1112.

H412 fractions were chromatographed by reverse phase HPLC for semiprep. Final active fraction (compound II) 1.5 as detected by 7.8 ID × 300 mm (Phenomenex, C ₁₈ ) using tetrahydrofuran (THF) -methanol-distilled water (1: 8: 1) at a column flow rate of 4.0 mL / min. g was separated.

5th Chromatography

Fraction H1111 having activity in the fourth phase was further purified.

The H1111 fraction was eluted with a hexane: ethyl acetate ratio of 40: 1 in silica gel chromatography to separate the active fraction H11113.

In addition, the H11113 fraction was separated into 19 ID × 300 mm μPorasil silica (Waters) using a concentration gradient hexane: ethyl acetate ratio of 98: 2 at a column flow rate of 4.0 mL / min. The final active fraction (compound I) 4 g was obtained.

Example 3: Component Determination of Active Fraction

The structure of the active eluting material of the final active fraction (compound I, compound II) separated in Example 2 was based on spectroscopic analysis. ¹ H and ¹³ C-NMR spectra were recorded on a JNM-LA 400F7 spectrometer and chemical shifts in ppm. UV spectra were obtained from the UVIKON 922 spectrometer, and mass spectra were obtained from the JEOL GSX400 spectrometer. The active substance isolated in Example 2 was represented by Fenchon ((+)-fenchone)) and ( E ) -9-octadecenoic acid as represented by the following formulas (I) and (II). .

(Formula 1)

(Formula 2)

Experimental Example 2

Patch test

Table 1 shows the repellent activity of the fennel-derived compounds Fenchon ((+)-fenchone) and ( E ) -9-octadecenoic acid on the Egyptian forest mosquitoes by the patch test carried out in Experimental Example 1. Shown in

Concentration (mg / cm 2) Evasion rate (mean ± standard deviation,%) ^a DEET Fenchone ( E ) -9-octadecenoic acid 0.04 100a 100a 100a 0.02 100a 100a 98.6 ± 0.9 a 0.01 96.4 ± 0.5b 93.9 ± 0.6 b 91.1 ± 0.2b 0.005 84.6 ± 1.3c 82.1 ± 0.7c 73.4 ± 2.3c

The same letters among the a, b, c, and d marks shown in the columns of the ^a table of Table 2 indicate that there is no difference in the mean statistical statistics ( P = 0.05, Sheffe's test), and the evasion rate was transformed into the arc sine square root.

When treated with Fenchon ((+)-fenchone) and ( E ) -9-octadecenoic acid (Example 2) in Example 2 each at 0.01 mg / cm 2, the repellent effect was similar to that of the control compound DEET. When 0.005 ㎎ / ㎠ treatment, it was lower than DEET (84.6%), but maintained a similar avoidance effect (82.1%, 73.4%).

Skin test

In order to investigate the mosquito repellent effect more accurately, a skin test was performed directly on the skin of the sample confirmed the repellent effect. As an experimental container, a mesh of a wire structure of 35 x 35 x 35 cm was used. In each container, 60 female Egyptian mosquitoes, 7 to 10 days old, were placed in 100 μl of ethanol, and then treated evenly on the skin of a rubber glove with a diameter of 50 mm. It was dried and experimented for 5 minutes. The repellent effect of the drug was judged by the number of mosquitoes vampires for 5 minutes, and the treated group was treated with only 95% ethanol solution. The avoidance effect was calculated by the following formula.

(Calculation 2)

In addition, DEET was assayed in the same manner to compare the activity is shown in Table 3.

Concentration (mg / cm 2) Evasion rate (mean ± standard deviation,%) ^a DEET Fenchone (E) -9-octadecenoic acid 0.4 100a 100a 80.4 ± 0.2 0.2 100a 100a 52.3 ± 0.9 0.1 100a 91.7 ± 0.9 b ^A 0.04 98.4 ± 0.5b 76.2 ± 0.2c ㆍ

The skin test, which is applied directly to the skin, required higher throughput than the results of the patch test. Fenchon ((+)-fenchone) treated with 0.4 mg / ㎠ showed the same perfect repelling effect as DEET, and ( E ) -9-octadecenoic acid also showed an excellent repelling effect of 80.4%.

As mentioned above, in the present invention, methanol crude extract, fenchon ((+)-fenchone), and octadecenoic acid ((E) -9-octadecenoic acid) having mosquito repelling ability were isolated from the fennel. Fennel oil, fenchon ((+)-fench one), and ( E ) -9-octadecenoic acid of the present invention are harmless to humans and have repelling ability against hygienic pests. It can be used as a substitute for DEET which is a synthetic repellent of DEET.

Claims (2)

A mosquito repellent containing phenchon or octadecenoic acid as an active ingredient. The mosquito repellent of claim 1, wherein the fenchon or octadecenoic acid is included in a fennel fruit extract selected from the group consisting of methanol extract of Foeniculum vulgare fruit, hexane fraction of fennel oil and fennel fruit methanol extract.