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Chemical composition and antifungal properties of the essential oil and various extracts of Mikania scandens (L.) Willd
⁎Corresponding author. Tel.: +88-071-74910-20x2252/2266; fax: +88-071-74905. marahman12@yahoo.com (Atiqur Rahman) atiq@acct.iu.ac.bd (Atiqur Rahman)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
This study was undertaken to assess the antifungal potential of the essential oil and various extracts of Mikania scandens (L.) Willd. The hydrodistilled leaf essential oil of M. scandens was analysed by GC–MS. Twenty-four compounds representing 97.45% of the total leaves oil were identified, of which β-caryophyllene (16.98%), δ-cadinene (12.22%), α-cubebene (11.33%), 1,2-benzenedicarboxylic acid (10.17%), caryophyllene oxide (7.74%), β-himachalene (4.68%), T-cadinol (3.98%), tetratetracontane (3.83%), 1H-cyclopropa[a]naphthalene (3.56%), β-farnesene (3.08%) etc. were the major compounds. The essential oil and extracts (chloroform, ethyl acetate and methanol) of M. scandens were tested for antifungal activity, which was determined by disc diffusion and minimum inhibitory concentration (MIC) determination methods. The essential oil and various extracts displayed a great potential of antifungal activity as a mycelial growth inhibition against the tested phytopathogenic fungi such as Rhizoctonia solani AG-1 (IB) KACC 40111, R. solani AG-2-2 (IV) KACC 40132, Pythium graminicola KACC 40155, Tricoderma harzianum KACC 40791 and Fusarium oxysporum KACC 40052, in the range of 40.0–75.4% and the minimum inhibitory concentration ranging from 125 to 500 μg/ml. The present results demonstrated that M. scandens mediated oil and extracts could be potential sources of natural fungicides to protect crops from fungal diseases.
Keywords
Mikania scandens (L.)
Essential oil
GC–MS
Different extracts
MIC
Antifungal activity
1 Introduction
Mikania scandens (L.), a medicinal plant is popularly used as a herbal remedy for various ailments in Bangladesh. The genus Mikania is a member of the family Asteraceae (Compositae). Within that family, Mikania is a member of the tribe Eupatorieae of the subfamily Asteroideae. Some species of Mikania are M. scandens, Mikania cordifolia (L. f.) Willd, Mikania cordata (Burm. f.) B. L. Robins. In Bangladesh, M. scandens is known as “Jarmany lota” (Moon et al., 1993). Besides, this vining species can be found in the southeast corner of Missouri. The plant is very easy to identify in the field because of its vining habit, opposite, sagittate leaves, and umbels of whitish flower heads. This is a weedy species which can grow very long very quickly. It is probably not the best choice to plant around a water garden. The plant is, however, frequently visited by many different types of flying insects. The genus Mikania is a large cosmopolitan genus confined mostly to the tropics.
Fungi have long been recognized as causal agents of plant diseases such as root rot symptom (Rhizoctonia solani), pythium root rot (Pythium Graminicola), soil green mould (Tricoderma harzianum), and vascular wilt (Fusarium oxysporum) (Hakuno et al., 2002; Waterhouse and Waterston, 1964; Harman et al., 2004; Agrios, 1988). Chemical fungicides are made up of many different volatile compounds and have been shown to possess fungicidal properties (Ahmet et al., 2005). Essential oils and plant extracts are gaining increasing interest because of their relatively safe status, their wide acceptance by consumers and their exploitation for potential multi-purpose functional uses. So, essential oils and plant extracts are one of the most promising groups of natural compounds for the development of safer anti-fungal agents. M. scandens leaf is used as its analgesic and antioxidant properties (Hasan et al., 2009). However, there is no report available in the literature on the detailed analyses of the essential oil of M. scandens and its antifungal property. Therefore, we undertook investigations of the chemical composition of the essential oil by GC–MS and the antifungal properties of the essential oil and extracts from M. scandens occurring in Bangladesh and the results are reported in this communication.
2 Materials and methods
2.1 Plant materials
The leaves of M. scandens (L.) were collected from the local area of Muksudpur, Gopalganj, Bangladesh, in June and July 2010. The taxonomic identification of the plant was confirmed by Dr. Oliur Rahman, Professor, Department of Botany, University of Dhaka, and a voucher specimen (DACB 37894) has been deposited at the Bangladesh National Herbarium.
2.2 Isolation of the essential oils
The air-dried leaves (250 g) of M. scandens were subjected to hydro distillation for 3 h using a Clevenger type apparatus. The oil was dried over anhydrous sodium sulphate and preserved in a sealed vial at 4 °C until further analysis.
2.3 Preparation of organic extracts
The air-dried leaves M. scandens were first pulverized into a powdered form. The dried powder (50 g) was then extracted with chloroform, ethyl acetate and methanol separately at room temperature for 7 days and the solvents were evaporated by a vacuum rotary evaporator at a temperature of 50 °C. The extraction process yielded chloroform (6.2 g), ethyl acetate (7.4 g) and methanol (6.5 g) extracts, respectively. Solvents (analytical grade) for extraction were obtained from commercial sources (Sigma–Aldrich, St. Louis, MO, USA).
2.4 GC–MS/MS analysis
The GC–MS was carried out using a total ion monitoring mode on Varian 3800 gas chromatograph interfaced to a Varian Saturn ion trap 2200 GC–MS spectrometer. The temperatures of transfer line and ion source were 280 and 275 °C, respectively. Ions were obtained by the electron ionization mode. The ZB-1 capillary column (30 m length, 0.25 mm I.D. and 0.25 μm film thickness) was used. A 20% split injection mode was selected with a solvent delay time of 3 min with an injection volume of 0.2 μl. The initial column temperature was started at 50 °C for 1 min, programmed at 8 °C/min–200 °C and heated until 280 °C at 10 °C/min. The injection port was set at 250 °C. Helium was used as the carrier gas at a constant flow rate of 1.0 ml/min. Molecular ions (mass range: 40–500 m/z) were monitored for identification. The relative percentage of the oil constituents was expressed as percentage by peak area normalization. The identification of components of the essential oil was based on their retention indices, relative to a homologous series of n-alkane (C8–C20) on the ZB-1 capillary column under the same operating conditions and computer matching with the GC–MS spectra from the Wiley 6.0 MS data and literature data (Adams, 2007).
2.5 Microorganisms
The plant pathogenic fungi used in the experiment were R. solani AG-1 (IB) KACC 40111, R. solani AG-2-2 (IV) KACC 40132, P. graminicola KACC 40155, T. harzianum KACC 40791 and F. oxysporum KACC 40052. The fungal pathogens were kindly provided by Prof. Yong Se Lee, Department of Bioresource Technology, College of Agriculture and Environmental Science, Daegu University, Korea. Cultures of each fungal species were maintained on potato-dextrose agar (PDA) slants and stored at 4 °C.
2.6 Preparation of spore suspension and test samples
The spore suspension of R. solani AG-1, R. solani AG-2-2, P. graminicola, T. harzianum and F. oxysporum was obtained from their respective 5–10 day old cultures, mixed with sterile distilled water to obtain a homogenous spore suspension of 105 spore/ml. Essential oil, chloroform, ethyl acetate and methanol extract of M. scandens were dissolved in methanol separately to prepare the stock solution with their respective known weights, which were further diluted to prepare test samples.
2.6.1 Determination of antifungal activity of essential oil and crude extracts
Petri dishes (9 cm diameter) containing 20 ml of PDA medium were used for antifungal activity assay, performed in solid media by the disc diffusion method (Duru et al., 2003). Sterile Whatman paper discs of 6 mm diameter were pierced in the agar plates, equidistant and near the border, where the essential oil 10 μl (1000 μg/disc) and all extract samples 15 μl (1500 μg/disc) were used separately. A disc of fungal inoculums 6 mm in diameter was removed from pre-grown cultures of all the fungal strains tested and placed upside down in the centre of the Petri dishes. The plates were incubated at 28 ± 2 °C for 7 days, until the growth in control slates reached the edges of the plates. Growth inhibition of each of the fungal strains was calculated as the percentage of inhibition of radial growth relative to the control along with antifungal effect on fungal mycelium. The plates were used in triplicates for each treatment. Growth inhibition of treatment against control was calculated by percentage, using the following formula:
Inhibition ratio (%) = {1−mycelium growth of treatment (mm)/mycelium growth of control (mm)} × 100
2.6.2 Minimum inhibitory concentration (MIC)
The minimum inhibitory concentration of essential oil and crude extract was determined by the twofold dilution method (Murray et al., 1995). Samples were dissolved in methanol according to their respective known weights. The solutions were serially diluted with methanol and were added to PDB to final concentrations of 125, 250, 500, and 1000 μg/ml, respectively. A 10 μl spore suspension of each test strain was inoculated in the test tubes in PDB medium and incubated for 5–7 days at 28 ± 2 °C. The control tubes containing PDB medium were inoculated only with fungal suspension. The minimum concentration at which no visible growth was observed was defined as the MIC, which was expressed in μg/ml.
2.7 Statistical analysis
The essential oil and different organic extracts were assayed for antifungal activity. Each experiment was run in triplicate, and mean values were calculated. The statistical analysis was carried out employing a one way ANOVA (p < 0.05). A statistical package (SPSS version 11.0) was used for the data analysis.
3 Results
3.1 Chemical composition of essential oil
GC–MS analyses of the oil of M. scandens (L.) led to the identification of twenty-four different compounds, representing 97.45% of the total leaf oil. The identified compounds are listed in Table 1 according to their elution order on a ZB-1 capillary column. The oil contains a complex mixture consisting of mainly oxygenated monoterpene and sesquiterpene hydrocarbons. The major compounds detected in the essential oil were β -caryophyllene (16.98%), δ-cadinene (12.22%), α-cubebene (11.33%), 1,2-benzenedicarboxylic acid (10.17%), caryophyllene oxide (7.74%), β-himachalene (4.68%), T-cadinol (3.98%), tetratetracontane (3.83%), 1H-cyclopropa[a]naphthalene (3.56%) and β-farnesene (3.08%).
Sl. No.
Compound
RIa
% RAb
Identificationc
1.
O-Decylhydroxylamine
1100
1.95
RI, MS
2.
Myrtenol
1201
1.40
RI, MS
3.
Dodecamethyl cyclohexasiloxane
1321
tr
RI, MS
4.
α-Cubebene
1351
11.33
RI, MS
5.
Naphthalene, 1,2,3,4-tetrahydro-1,6-dime
1353
tr
RI, MS
6.
α-Copaene
1379
1.82
RI, MS
7.
β-Caryophyllene
1414
16.98
RI, MS
8.
1H-cyclopropa[a]naphthalene
1417
3.56
RI, MS
9.
cis-β-Farnesene
1448
0.70
RI, MS
10.
γ-Muurolene
1460
1.24
RI, MS
11.
δ-Cadinene
1524
12.22
RI, MS
12.
Caryophyllene oxide
1578
7.74
RI, MS
13.
2-Pentadecanone, 6,10,14-trimethyl
1601
1.04
RI, MS
14.
1,2-Benzenedicarboxylic acid
1603
10.17
RI, MS
15.
Humulene epoxide-II
1607
1.67
RI, MS
16.
β-himachalene
1614
4.68
RI, MS
17.
T-Cadinol
1634
3.98
RI, MS
18.
T-Muurolol
1641
2.47
RI, MS
19.
β-Farnesene
1672
3.08
RI, MS
20.
Tonalide
1850
1.34
RI, MS
21.
Nonadecane
1900
2.64
RI, MS
22.
Phthalic acid, butyl hexyl ester
1970
1.19
RI, MS
23.
(-)-Spathulenol
2464
1.92
RI, MS
24.
Tetratetracontane
4395
3.83
RI, MS
Total identified
97.45%
3.2 Antifungal activity of essential oil and crude extracts
The oil of M. scandens exhibited a moderate to high antifungal activity against all the tested fungi except R. solani (AG-1). A low inhibition effect was observed against R. solani (AG1-1). At the concentration of 10 μl (1000 μg/ml), the essential oil showed a potent inhibitory effect on the growth of R. solani (AG1-1 (62.8–63.4%), R. solani (AG-2-2) (53.2–54.6%), P. graminicola (70.3–71.3%), T. harzianum (54.8–56.0%) and F. oxysporum (74.9–75.9%) as shown in Table 2. Also, the crude chloroform, ethyl acetate and methanol extract (1500 μg/ml) showed mycelium growth inhibition against some of the phyto-pathogens but not for all. According to the results reported in Table 2, the ethyl acetate extract showed a lower antifungal effect than essential oil against R. solani (AG-1) (53.1–54.7%), P. graminicola (56.4–57.4%) and F. oxysporum (53.2–54.6%). The crude methanol, chloroform and ethyl acetate extract did not show any inhibitory effect against R. solani (AG-2-2). Besides, the chloroform extract showed moderate antifungal activity against some of the fungi tested. Also, the chloroform extract showed relatively better antifungal effect against F. oxysporum (54.7–56.1%) as compared to ethyl acetate extract. nd: not detected of antifungal activity. Solvents (Chloroform, Methanol, and Ethyl acetate).
Fungal strains
Radial growth inhibition
Essential oil
CHCl3
MeOH
EtOAc
Percent a
Percent a
Percent a
Percent a
Rhizoctonia solani AG-1 (IB) KACC 40111
63.1 ± 0.7b
40.0 ± 0.6b
nd
53.9 ± 0.8b
Rhizoctonia solani AG-2-2 (IV) KACC 40132
53.9 ± 0.7b
nd
nd
nd
Pythium graminicola KACC 40155
70.8 ± 0.5b
52.3 ± 0.6b
nd
56.9 ± 0.5b
Tricoderma harzianum KACC 40791
55.4 ± 0.6b
nd
nd
nd
Fusarium oxysporum KACC 40052
75.4 ± 0.5b
55.4 ± 0.7b
nd
53.9 ± 0.7b
3.3 Minimum inhibitory concentration
According to the results given in Table 3, MIC of essential oil was found more effective against P. graminicola and F. oxysporum (125 μg/ml) as compared to those of R. solani (AG-1) and T. harzianum (250 μg/ml). The ethyl acetate extract displayed potent antifungal activity against F. oxysporum, P. graminicola and R. solani AG-1 with MIC values of 250–500 μg/ml, whereas the F. oxysporum and P. graminicola were 250 μg/ml. Besides, the MIC values of chloroform and methanol extract against F. oxysporum, P. graminicola and R. solani AG-1 were found within the range of 250–500 μg/ml. nd: not detected.
Fungal strains
Minimum inhibition concentration (MIC)a
Essential oilb
Leaf extractsc
CHCl3
MeOH
EtOAc
Rhizoctonia solani AG-1 (IB) KACC 40111
250
500
500
500
Rhizoctonia solani AG-2-2 (IV) KACC 40132
nd
nd
nd
nd
Pythium graminicola KACC 40155
125
500
250
250
Tricoderma harzianum KACC 40791
250
nd
nd
nd
Fusarium oxysporum KACC 40052
125
250
500
250
4 Discussion
The increasing social and economic implications caused by fungi means there is a constant striving to produce safer food crops and to develop new antifungal agents. In general, plant-derived essential oil is considered as non-phytotoxic compounds and potentially effective against plant pathogenic fungi. In recent years, interests have been generated in the development of safer antifungal agents such as plant-based essential oils and extracts to control phytopathogens in agriculture (Costa et al., 2000). Several publications have documented the antifungal activities of essential oil and plant extracts (Rahman et al., 2010, 2011). Thus essential oil is a promising natural antifungal agent with potential applications in agro-industries to control plant pathogenic fungi causing severe destruction in crops. The hydro distillation of the M. scandens (L.) gave dark yellowish oil with the major components having oxygenated monoterpenes and sesquiterpenes, and their respective hydrocarbons. In recent years, several researchers have reported that monoterpene and sesquiterpene hydrocarbons and their oxygenated derivatives are the major components of essential oil of plant origin, which have enormous potential to strongly inhibit microbial pathogens (Cakir et al., 2004). The essential oil of M. scandens showed a remarkable antifungal effect against all the fungi which could be attributed to the presence of phenolic compounds and oxygenated monoterpenes and sesquiterpene hydrocarbons (Guillen and Manzanos, 1998). In our opinion, major components of M. scandens essential oil such as β-caryophyllene (16.98%), δ-cadinene (12.22%), α-cubebene (11.33%), 1,2-benzenedicarboxylic acid (10.17%) and caryophyllene oxide (7.74%) have the key roles for their antifungal activities (Cheng et al., 2004; Gazim et al., 2008; Tolouee et al., 2010; Ogunlesi et al., 2009; Abi-Ayad et al., 2011). Also, the components such as β-himachalene (4.68%), T-cadinol (3.98%), tetratetracontane (3.83%), 1H-cyclopropa[a]naphthalene (3.56%) and β-farnesene (3.08%) contributed to the antifungal activity of the oil (Daoubi et al., 2005; Cheng et al., 2006; EL Mehalawy, 2004; Guo et al., 2008).
5 Conclusion
Therefore, it would also be interesting to study the effects of essential oil and different extracts of M. scandens (L.) against other important fungi for developing new antifungal agents to control serious fungal diseases in plant, animal and human beings. Thus, M. scandens could become an alternative to synthetic fungicides for use in agro-industries and also to screen and develop such novel types of selective and natural fungicides in the treatment of many microbial plant pathogens causing severe destruction to crop, vegetable and ornamental plants.
References
- J. Med. Plants Res.. 2011;5:5433-5436.
- Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy (fourth ed.). USA: Carol stream, Illinois, Allured Publishing Corporation; 2007.
- Plant Pathology (third ed.). New York: Academic Press, Inc.; 1988. (p. 803)
- Biochem. Syst. Ecol.. 2005;33:245-256.
- Flavour Frag. J.. 2004;1:62-68.
- J. Chem. Ecol.. 2004;30:1957-1967.
- Bioresour. Technol.. 2006;97:306-312.
- J. Ethnopharmcol.. 2000;72(1–2):111-117.
- J. Agric. Food Chem.. 2005;53(17):6673-6677.
- Fitoterapia. 2003;74:170-176.
- Int. J. Agric. Biol.. 2004;06(2):310-316.
- J. Microbiol.. 2008;39:61-63.
- Flavour Frag. J.. 1998;13:259-262.
- Molecules. 2008;13:2114-2125.
- J. Jpn. Soc. Turfgeass Sci.. 2002;30(2):105-114.
- Nat. Rev. Microbiol.. 2004;2(1):43-56.
- Am J. Pharmacol. Toxicol.. 2009;4(1):1-7.
- Funct. Ecol.. 1993;7(5):610-615.
- Manual of Clinical Microbiology (sixth ed.). Washington, DC: ASM; 1995.
- Afr. J. Biotechnol.. 2009;8(24):7042-7050.
- Control of phytopathogenic fungi by the essential oil and methanolic extracts of Erigeron ramosus (Walt.) B.S.P. Eur. J. Plant Pathol.. 2010;128:211-219.
- [Google Scholar]
- J. Am. Oil Chem. Soc.. 2011;88:573-579.
- Int. J. Food Microbiol.. 2010;139(3):127-133.
- IMI Des. Fun. Bac.. 1964;4:38.