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CHEMICAL COMPOSITION AND INSECTICIDAL ACTIVITY OF THE ESSENTIAL OIL OF ANETHUM GRAVEOLENS L.

Byline: Rauf Attique Babri, Irshad Khokhar, Zaid Mahmood and Shahid Mahmud

ABSTRACT: The essential oil of seeds of Anethum graveolens L. growing in Pakistan was obtained by hydrodistillation. It was analysed by GC-MS. Components were identified and R-(-)-carvone(38.899%), apiol (30.812%), limonene(15.938%) and trans-(+)-dihydrocarvone (10.999%) were the main components. The essential oil was found to be toxic to Periplanata americana L., Musca domestica L. and Tribolium castaneum. The mean mortality ranged from 25 to 100% for the different insects. In conclusion, the essential oil of A.graveolens may have the potential to be developed as a natural insecticide against various insects.

Keywords: Anethum graveolens L., insecticidal activity; essential oil; R-(-)-carvone; apiol

INTRODUCTION

Natural products have proved to be excellent insecticides and pesticides after a great deal of research [1]. Synthetic insecticides may initiate various problems to our environment and health. These insecticides are neurotoxic to man as well as domestic animals. This fact leads us towards the application of natural insecticides such as essential oils as good alternative to chemical insecticides. Modern research has proved that natural insecticides are safe to use against different insects and pests and do not cause any environmental damage [2].

Anethum graveolens, commonly known as dill, is an annual medicinal plant with tiny yellow flowers belonging to the plant family Umbelliferae. The plant grows in Pakistan, India, Afghanistan, Middle East, Russia, Iran and Egypt [3]. Its seeds have a strong spicy odor and are therefore used as a flavouring agent in the food industry. Dill seeds are commonly used for aches in the stomach and intestines, dyspepsia, bladder inflammation, liver diseases, cramps, and insomnia [4]. The essential oil and extracts of dill seeds have shown varying degrees of antimicrobial activities [4]. The literature survey points out that several studies have been done on chemical composition of the essential oil of dill seeds [5-10].

The present study was undertaken to investigate the chemical composition and insecticidal activity of the essential oil of the seeds of A. graveolens against Periplaneta americana (Linnaeus) and Musca domestica L., important vector of diseases, and Tribolium castaneum, an insect causing serious damage to stored grain products.

MATERIALS AND METHODS

Plant Material

The seeds of Anethum graveolens were purchased from local market of Lahore. Voucher specimens (LAH#1009) of the seeds were deposited at the Herbarium, Department of Botany, University of the Punjab, Lahore.

Extraction of the essential oil

The seeds were ground and submitted to hydrodistilltion using Clevenger-type apparatus for 6 hours. The extracted essentialoil were dried over anhydrous sodium sulphate and stored in amber-coloured bottles in a refrigerator at 4oC until use.

Gas Chromatography/Mass Spectrometry analysis

The essential oil was analyzed on a Hewlett-Packard 6890NGC-MS system (Agilent Technologies) coupled to a HP5973 mass-spectrometer. Separations were carried out using a DB-5 capillary column (30 m x 0.25 mm i.d., 0.25 um film thickness). The injector and detector temperatures were maintained at 220 and 2800C, respectively. Column temperature was initially kept at 600C for 2 minutes and then gradually increased to 2400C at the rate of 50C min-1. Helium was used as carrier gas at a constant flow of 1.0 mL min-1 and an injection volume of 1 uL was employed. The MS scan parameters included electron impact ionization voltage of 70 eV, a mass range of 40-750 m/z and a scan interval of 0.5 s. Samples diluted in n-hexane were injected manually in the splittless mode. The identification of the components was based on comparison of their mass spectra with those of NIST3.0 Libraries provided with the computer-controlling GC-MS system as well as from the published literature. Insects.

Adult American cockroaches, house flies and Tribolium castanum were used in the bioassays. American cockroaches were reared in plastic cans at 28(+-20 C), 50(+-10) % relative humidity on water and dog food. Adults of Tribolium castanum were obtained from PCSIR laboratories, Lahore, Pakistan. They were reared on rice flour at 28(+-20 C), 50(+-10) % relative humidity in darkness. The Musca domestica adults originated from Tollinton market in Lahore in Punjab province, Pakistan and were kept in plastic cages (15cmx15cmx15cm). They were reared on sugar solution and liver of chicken at 28(+-20 C) and 50(+-10) % relative humidity in 12hr light-12 hr dark photo-period. Continuos exposure bioassays. Serial dilutions of essential oils were made in acetone. These bioassays were conducted by pipetting 0.5 ml of essential oil concentration (or 0.5 ml acetone in case of control) into a 9cm diameter glass Petri plate and by allowing the acetone to evaporate in fumehood for forty five minutes.

Four cockroaches were used for each experiment. Mortality was assessed after 15 minutes, 30 minutes, 60 minutes, 120 minutes, 240 minutes, 6 hours, 12 hours and 24 hours.

Fumigant toxicity bioassays

Fumigant toxicity of the essential oil was determined by sealing four adult cockroaches or ten adult flies in a 120ml jar with a 1 cm diameter cotton ball treated with 100 ul of 100% essential oil. Essential oil was injected into the centre of each ball to allow its volatilization while preventing the insects from coming in contact with the oil. Mortality was recorded at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours and 24 hours, respectively for the cockroaches and at 15 minutes, 30 minutes, 1 hour and 2 hours, respectively for the house flies. Control jar had insects and an untreated cotton ball.

In case of T.castaneum, fumigant toxicity of the essential oil was determined by sealing ten adults in a 120ml jar with a 1 cm diameter disc of filter paper treated with 100 ul of 100% essential oil. The disc was attached to the upper side of the jar. Mortality was recorded at 1 hour, 6 hours and 24 hours, respectively. Control jar had insects and an untreated filter paper. For each insect, the experiment is repeated thrice. Statistical analysis of Data Every experiment was performed in triplicate. The results were recorded as means +- standard deviation. Statistical analysis was performed using one-way analysis of variance (1-way ANOVA) using SPSS statistical package (SPSS 16) and the statistical significance was determined at P(less then) 0.05.

RESULTS AND DISCUSSION

The essential oil was extracted from the seeds of Anethum graveolens L. by hydrodistillation with a yield of 1.45% and was analyzed by GC-MS as shown in figure 1.

The composition of the essential oil is given in table 1 and mainly consists of R-(-)-carvone (38.89%), apiol (30.81%), limonene (15.93%) and trans-dihydrocarvone (10.99%) whereas gamma-terpinene (0.19%), m-cymene (0.29%), S- (+)-carvone (0.06%) and myristicin (0.86%) were present as

Table 1. Composition of essential oil of seeds of Anethum graveolens

No.###Constituenta###RTb###Percentage

###(min.)

1.###E,E-2,6-dimethyl-3,5-octatetraene###5.963###0.583

2.###Limonene###6.770###15.938

3.###Gamma-terpinene###7.204###0.192

4.###m-cymene###7.977###0.294

5.###Trans-dihydrocarvone###11.124###10.999

6.###R-(-)-carvone###12.909###38.899

7.###S-(+)-carvone###13.081###0.064

8.###1-methoxy-4-[1-propenyl]-benzene###13.401###1.354

9.###Myristicin###19.078###0.864

10.###Apiol###21.853###30.812

Compounds, identified on the basis of comparison with NIST(2005) MS database spectra and Adams(1995) libraries are listed in order of elution from a HP-5MS column.

RT, retention time on a HP-5MS column in minutes.

Ashraf et al [6] analyzed the dill seed essential oil by GLC and found the oil to be rich in carvone (52.25%), dill apiole (28.28%) and limonene (9.34%). Singh et al [7] reported carvone (55.2%), camphor (11.44%), limonene (16.6%) and dill apiole (14.4%) to be the key components present in the essential oil extracted from the seeds of A.graveolens L.

Dill apiole and camphor which were the key components in the previous studies were not found in the present investigation. Our results are in good agreement with Jirovetz et al [5] who reported carvone (50.1%) and limonene (44.1%) as the major constituents of the A.graveolens essential oil from Bulgaria.

The contact toxicity bioassay of the essential oil against P.americana resulted in mortality which ranged from 25 to 100% during the first 3 hours for the various concentrations of the essential oil.

Table2: Contact toxicity of the essential oil of A.graveolens to Peripalaneta americana(L.)

Essential###Conc.###Mean mortalitya (%)

oils###(%)###

###15###30###60###2###3###6###12###24

###min###min###min###hr###hr###hr###hr###hr

A.graveolens

L.###5###0###0###25###75###100###100###100###100

###10###0###25###50###100###100###100###100###100

###100###50###100###100###100###100###100###100###100

Control###0###0###0###0###0###0###0###0###0

a Results are mean of three experiments, four insects per set The mortality ranged from 25 to 100%during the first 12 hours in the fumigant toxicity bioassay of the essential oil against P.americana. In case of the house fly, mortality ranged from 33.3 to 70% during the first 3 hours, whereas the mortality ranged from 58.3 to 100% during 24 hours for the red flour beetle.

Table3:Fumigant toxicity of the essential oil of A.graveolens to various insects

Time###Mean mortality(%) of

###P.americana###M.domestica###T.castaneum###Control

15 min###0###33.3###-###0

30 min###0###53.3###-###0

60 min###0###63.3###58.3###0

2 hrs###0###70.0###-###0

3 hrs###25###-###-###0

6 hrs###50###-###83.3###0

12 hrs###100###-###-###0

24 hrs###100###-###100###0

a Results are mean of three experiments

Anethum graveolens essential oil has shown good insecticidal activity due to appericiable concentration of carvone, limonene and trans-dihydrocarvone in its composition as shown in the table 1. Carvone was observed to be more toxic than bicyclic compounds such as verbinol and thujone [10]. Lee et al [11] reported the insecticidal activity of carvone against the house fly. Previously, Jang et al [12] studied the fumigant toxicity of carvone to the German cockroach with an LC50 value of 0.25 mg/cm3. Carvone and dihydrocarvone were found to be toxic to larvae and adults of T.castanum, S.oryzae L. and R.dominica F by Tripathi et al [13]. Previously, the insecticidal activity of A.graveolens essential oil against T.castanum with an EC50 value of 7.86uL was reported by Chaubel et al [14]. Limonene and sesquiterpenes posses weak to moderate toxicity to various insects [15].

CONCLUSION

Although, the insecticidal activity of essential oil is much less than that of synthetic insecticides such as dichlorvos, propuxor and permethrin, they are quite harmless. They are not toxic to mammals. Their volatility as well as their non-toxicity makes them an ideal candidate to be used in resteraunts, hospitals and schools against various insects. Most of the essential oils have pleasant smell and can be used to protect food from stored product insects such as T.castanum and maize weevil. There is a need to assess various essential oils for insecticidal activity against insects that cause diseases and damage food products.

REFERENCES

1. Isman, M.B. Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world. Annu. Rev. Entomol. 51, 45-66, (2006).

2. Tripathi, A.K., Upadhyay, S., Bhuiyan, M. and Bhattacharya, P.R.. A review on the prospects of essential oils as biopesticides in insect pest-management., Journal of Pharmacognosy and Phytotherapy, 1(15), 52-63, (2009).

3. Nasir, E. and Ali, A.I. Flora of West Pakistan, No. 20 Umbelliferae, Stewart Herbarium Gordon College Rawalpindi, (1972).

4. Kaur G.J., Arora D.S. Antibacterial and phytochemical screening of Anethum graveolens, Foeniculum vulgare and Trachyspermum ammi. BMC Complement. Altern. Med.., 9: 30, (2009).

5. A Jirovetz L, Buchbauer G, Stoyanova AS, Georgiev EV, Damianova ST.Composition, quality control and antimicrobial activity of the essential oil of long time stored dill (Anethum graveolens L.) seeds from Bulgaria. J. Agric. Food Chem. 18: 3854-3857, (2003).

6. Asraf, M., Aziz, J. and Bhatty, M.K..Studies on the essential oils of the Pakistani species of the family Umbelliferae. Part VI. Anethum graveolens (Dill, Sowa) seed oil. Pakistan J. Sci. Ind. Res., 20(1), 52-54, (1977).

7. Singh, G., Maurya, S., Lampasona, M.P.D. and Catlan, C..Chemical Constituents, Antimicrobial Investigations, and Antioxidative Potentials of Anethum graveolens L. Essential Oil and Acetone Extract: Part 52. Journal of food science, Volume 70, issue 4, Pages M208 - M215, (2005).

8. Yili, A., Yimamu, H., Maksimov, V. V., Aisa, H. A Veshkurova, O. N. and Salikhov, S.I.. Chemical composition of essential oil from seeds of Anethum graveolens cultivated in China. Chemistry of Natural Compounds, Vol. 42, No. 4, 491-492, (2006).

9. Kruger, H., Hammer, K. A. new chemotype of Anethum graveolens L Journal of Eessential oil Research, volume 8(2), 205-206, (1996).

10. Rice, P.J., Coats, J.R. Insecticidal properties of several monoterpenoids to the house fly (Diptera: Muscidae), red flour beetle (Coleoptera: Tenebrionidae), and southern corn rootworm (Coleoptera: Chrysomelidae)., J. Econ. Entomol., 87(5), 1172-1179, (1994).

11. Lee S, Tsao R, Peterson C, Coats JR. Insecticidal activity of monoterpenoids to western corn rootworm (Coleoptera: Chrysomelidae), twospotted spider mite (Acari: Tetranychidae), and house fly (Diptera: Muscidae) . J. Econ. Entomol., 90(4):883-92, (1997).

12. Jang, Y.S., Yang, Y,C., Choi,D.S. Vapour-phase toxicity of marjoram oil compounds and their related monoterpenoids to Blattella germanica( Orthoptera: Blaattellidae). J. Agric. Food Chem., 53: 7892-7898, (2005).

13. Tripathi AK, Prajapati V, Kumar S. Bioactivities of l-carvone, d- carvone, and dihydrocarvone toward three stored product beetles". J Econ Entomol. , Oct; 96(5):1594-601, (2003).

14. Chaubey, M.K. Insecticidal activity of Trachyspermum ammi (Umbelliferae), Anethum graveolens (Umbelliferae) and Nigella sativa (Ranunculaceae) essential oils against stored-product beetle Tribolium castaneum Herbst (Coleoptera: Tenebrionidae). African Journal of Agricultural Research, Vol. 2 (11), pp. 596-600, (2007).

15. Ibrahim, M.A., Kainulainen, P. Aflatuni, A. Tiilikkala, K.; Holopainen, J. K. Insecticidal, repellent, antimicrobial activity and phytotoxicity of essential oils: With special reference to limonene and its suitability for control of insect pests (Review), Agricultural and Food Science in Finland, vol 10; part 3, 243-260, (2001).

e-mail: raufattique@gmail.com

1 Institute of Chemistry, University of the Punjab, Quaid-e-Azam Campus, Lahore-54590, Pakistan.

2PCSIR Laboratories Complex, Ferozepur Road, Lahore-54600, Pakistan.
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