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Antifungal Capacity of Lippia Sidoides Agains Rizhopus Stolonifer

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American Journal of Plant Sciences
Vol.06 No.07(2015), Article ID:56044,10 pages
10.4236/ajps.2015.67110

GC-MS Analysis and in Vitro Antimicrobial Susceptibility of Foeniculum vulgare Seed Essential Oil

Ravi Kant Upadhyay

Department of Zoology, DDU Gorakhpur University, Gorakhpur, India

Email: rkupadhya@yahoo.com

Copyright © 2015 by author and Scientific Enquiry Publishing Inc.

This work is licensed under the Artistic Commons Attribution International License (CC By).

http://creativecommons.org/licenses/by/4.0/

Received 10 February 2015; accepted 26 Apr 2015; published xxx Apr 2015

ABSTRACT

Essential oil from seeds of Foeniculum vulgare was extracted on Clevenger apparatus. Essential oil was analyzed on Gas-Chromatography-Mass spectrometry (GC-MS) from which thirty vi components were identified, amidst which 6 major and thirty small components having different structural formulae and molecular weight representing total 99.98% of oil. Essential was investigated for its antibacterial and antifungal action against seven infectious microbial pathogens. Paper disc diffusion and serial micro-dilution assays were performed for the conclusion of inhibition zone (DIZ) diameters and minimal inhibitory concentration, respectively. The Foeniculum vulgare essential oil showed the Diameter of Inhibition Zone (DIZ) ranging from 19.4 ± 0.07 - 26.4 ± 0.09 mm at a concentration level of 28 μg/disc in all the x strains tested. The minimum inhibitory concentration (MIC) of essential oil against bacterial and fungal strains was obtained in the range of 7.0 - 56 μg/ml. Antibacterial and antifungal action of Foeniculum vulgare essential oil is due to the presence of certain secondary plant metabolites such every bit terpenoids, steroids and flavonoids, esters and acids which are identified in the essential oil. The oil components can exist further studied for their biological activity and overcome the trouble of drug resistance in microbes.

Keywords:

Antimicrobial Activity, Foeniculum vulgare, Essential Oil, GC-MS Analysis, MIC, MBC, MFC, DIZ

1. Introduction

Constitute essential oils are effluvious oily liquids, having highly specific volatile odor or flavors and show broad- spectrum antimicrobial activity against various groups of pathogens [ane] . These are obtained from various plant parts such every bit flowers, buds, seeds, leaves, twigs, barks, woods and roots [2] . More than than sixty families of angiosperms mainly Lamiaceae, Rutaceae, Geraniaceae, Apiaceae, Aspetaceae, Lauraceae, Fabaceae, and Poaceae possess essential oils in different plant species. Essential oils are circuitous mixtures of various chemic constituents such every bit lectins, polypeptides, alkaloids, phenols, quinines, flavones, flavonoids, terpenes, tannins, coumarins, benzene derivatives, various hydrocarbons and straight concatenation compounds. Plant essential oils multiple biological activity such every bit antibacterial [3] , antifungal [four] , anti-cancer [5] [half dozen] and anti-oxidant [7] . Essential oils are obtained in pure form or a complex mixture of several components without making any change in their chemical composition [8] . These are used for a wide variety of purposes [9] such as flavoring, perfuming [x] aromatherapy and food preservation [11] - [thirteen] . These are obtained past fractionation or rectification and steam distillation of various plant parts in Clevenger apparatus [xiv] . Essential oils possess mixed functional groups, too complex in their structure and are highly volatile at a very low temperature. Due to high volatility essential oils hands spread in the environs and medium. These act more efficiently confronting drug resistant microbes in civilisation medium due to their fast diffusion and volatile action. Treatment with establish essential oils shows least residual event in the body, but these severely inhibit growth and metabolism of a variety of infectious pathogens mainly microbes. These are also used alternative medicine in aromatherapy for treatment of cancer, illness pathogens and dermal infections [fifteen] [sixteen] .

Saunf (Foeniculum vulgare ) is an annual or biennial institute belong ing to family Umbelliferae family. It has sparse, fine, feathery leaves and pinkish/white flowers, which are followed by greenish seeds. Fennel oil has a sweet, spicy, warm smell, is most colorless to stake yellow and has a watery viscosity. Fennel seeds are rich source of essential oil which is extracted past steam distillation and yields 0.eight% - one.0% oil. Due to stiff flavor, essential oil extracted from seeds is used for medicinal purposes such every bit aphrodisiac, antispasmodic, carminative, depurative, deodorant, digestive, fungicidal, lipolytic, stimulant and stomachic substance. Information technology is used in aromatherapy and helps to ease the heed and fight fatigue. Saunf oil application warms and calms the digestive system, relieves rheumatism and arthritic pain, muscular spasms and detoxifies the body [9] . Information technology is also used for massage, relieve mental tensionand fatigue. Oil vapors provide freshness and testify warming effect on the tummy, relieve wind and cramps, and revitalizing the glandular system. Fennel is famous worldwide as a spice and is regularly used in vegetables, and has multiple medicinal properties also, such as its digestive and stomachic properties. Fennel seeds are often used every bit a spice in Indian cuisine and cooked well till the flavor blends with the dish. It has lemony, citrusy flavour when crushed, and information technology contains pinene and linalool terpenes. Fennel seeds are at their best when used fresh. Often, it is dry out roasted and grounded before being added to a dish. Fennel oil improves appetite, regulates endocrinal secretions, cures nausea and eliminates vomiting as a symptom of many conditions. In the present report, essential oil (E.O.) from Foeniculum vulgare was isolated from ripe seeds and screened for its antimicrobial activity in vitro by applying various bioassays. For evaluation of antimicrobial susceptibility potential various tests and growth inhibitory bioassays were conducted by setting controls and treatments done in seven bacterial strains (i.e. Escherichia coli, Bacillus cereus, Klebsiella pneumoniae, Lactobacillus acidophilus, Staphylococcus aureus and Streptococcus pneumoniae and Micrococcus luteus) and 3 isolates of fungi (i.eastward. Candida albicans, Aspergillusniger, Rhizopus stolonifer). MIC, MBC, MFC, inhibition zone diameters (DIZ) were adamant in bacterial and fungal strains were adamant in presence of each varying concentrations of essential oil. Antimicrobial susceptibility obtained in Fennel essential oil was compared with wide-spectrum antibiotics drugs.

2. Experimental

ii.1. Instrumentation

Gas-Chromatography-Mass Spectrometry (GC-MS)

GC/MS analyses of Foeniculum vulgare Eastward.O. was carried out on a Shimadzu GC-MS-QP2010 apparatus equipped with a −5 cavalcade (60 m × 0.25 mm i.d., film thickness 0.25 µm). Helium was used as carrier gas at a constant column catamenia ane.2 ml/min at 173 kpa inlet pressure and injector volume was 1.0 µL. The examination was performed according to a set temperature programming which was maintained from 100˚C to 200˚C with constant rise of v˚C/min and then held isothermal at 200˚C for 6 min. Farther, the temperature was increased past 10˚C/min upward to 290˚C and once more held isothermal at 290˚C for 10 min. The injector and ion source temperatures were 270˚C and 250˚C, respectively. The crude and active bands (AB-1) and (AB-2) (2 mg/ml) were dissolved in methanol (HPLC grade, Merck, India) and are injected with a split up ratio of 1:x. Mass spectra were taken at seventy eV; a scan interval of 0.5 south and fragments from 40 to 950 Dalton. The identification of the essential oil components was based on the comparing of their relative retention time (tR) and mass spectra with those of commercial standards (for the chief components) and on the GC retentiveness indices (RT) determined rel. to n-alkanes (C8 - C32). The final confirmation of constituents was made by computer matching of the mass spectra of peaks with the Wiley and National Establish Standard and Engineering (NIST) libraries mass spectral database. The relative percentages of the essential oil constituents were calculated from the GC acme areas. Further, quantitative analysis was performed past means straight peak expanse intention technique based on the total ion chromatogram (TIC).

two.2. Institute Cloth

The seeds of Foeniculum vulgare were purchased from local market at Gorakhpur, Uttar Pradesh, Bharat. The identification of plant material was fabricated by plant taxonomist. A voucher specimen is deposited for further confirmation in the departmental laboratory.

2.3. Extraction and Isolation of Essential Oil

Seeds of Foeniculum vulgare were grounded by using domestic mixer and powdered material was hydro-dis- tilled in Clevenger appliance continuously for v hrs to yield essential oil [13] . The crude pulverization was extracted with pure methanol twice and dried residue was dissolved in known volume of fresh solvent (w/v) before testing the antimicrobial activeness.

two.4. Identification of Major Constituents

The chemical constituents of essential oil from Foeniculum vulgare are listed in Table ane. Thirty half-dozen components were identified past using GC representing 99.98% of the oil. The master constituents of essential oil were identified 9-octadecenoic acrid (18.56%), 8Z)-xiv-methyl-8-hexadecenal (7.75%), pentad ecanecarboxylic acid (iv.25%), o-benzenedicarboxylic acrid (14.47%), 1,3,3-trimethyl-2-vinyl-1-cyclohexene (10.77%), 2-methyl-3-oxoestran- 17-yl acetate (v.46%), 1H-benzocycloheptene (10.71). The dominant components are ii-hydroxy-ane-(hydrox- ymethyl) ethyl ester (1.84%), and 9-octadecenoic acid (18.56%) but in dissimilar ratios [Effigy 1, Tabular array one] together with few compounds found in minor concentrations. Compounds were identified by comparing of memory fourth dimension and MS peaks. Both formula and molecular weights were established in each case.

two.five. Determination of Antimicrobial Susceptibility

2.5.1. Source of Microorganisms

Cultures of seven pathogenic bacterial strains each of Escherichia coli (ATCC 25922), Bacillus cereus (ATCC 11778), Lactobacillus acidophilus (ATCC 53103), Micrococcus luteus (ATCC 9341), Staphylococcus aureus (ATCC 25923), Klebsiella pneumoniae (ATCC 15380) and Streptococcus pneumoniae (ATCC 12755) were maintained in the laboratory in Luria Goop (2% due west/v) regularly for four days at 37˚C before use in experiments. For experiments, a portion (100 µl) of the overnight civilization was mixed in the tests and control for inoculation. For activeness, testing bacterial cultures were stored at 4˚C and sub cultured after every eightth twenty-four hours in solid agar plates. For determination of antifungal action of constitute latex, fungal strains of Aspergillus niger MTCC 1344, Candida albicans MTCC 227, Rhizopus stolonifer MTCC 3789 were grown in the laboratory. Moreover, each test fungi was maintained in strain specific agar medium mainly Sabouroud's Agar and Potato Dextrose Agar and its pure cultures were established by using single spore isolation technique.

2.v.two. Disc Diffusion Assay

The in vitro antimicrobial action of Foeniculum vulgare essential oil was evaluated in vitro Agar Disc Diffusion Assay. In each analysis inhibition zone diameters were measured in presence and absence of essential oil. For treatments essential oil was diluted by applying serial micro-dilution method by adding Luria Broth media separately. Six dissimilar concentrations of essential oils (1 - 32 μg) (West/V) were coated on sterile filter newspaper discs (Whatmann No. 1) of 6 mm size and oil impregnated discs were dried nether laminar flow cabinet. Earlier starting experiments inoculums size was determined and adjusted to prepare a final colony number every bit 108 colony

Table 1. Major and minor constituents isolated from Fennel (Foeniculum vulgare ) essential oil.

Figure 1. GC-MS spectrum of essential oil obtained from Foeniculum vulgare.

forming units (CFU/ml) in sterile agar plates. Bacterial inoculums were spread evenly on to the surface of agar plate by using a sterile rubber pad spreader. Later which essential oil coated discs were positioned on the inoculated agar surface in the heart. Essential oil was assayed in triplicate for antibacterial activity testing. All treated and untreated plates were incubated for 24 hrs at 27˚C. DMSO was used equally negative command while ampicillin was used as standard (positive command) to compare the bacterial growth and Griseofulvin was used to compare fungal growth in negative command. The radial growth of fungi was measured after 12 hours interval up to 36 hours of initial inoculation. The average percentage inhibition of growth in presence of various essential oils was calculated past using post-obit formula,

where C = bore of fungus colony in command plates; T = bore of fungus colony in tested plates.

2.five.3. Minimum Inhibitory Concentration Determination (MIC)

Bacterial growth inhibition was accessed in the presence of different increasing concentrations of essential oil in Luria Broth culture medium and MIC values were determined for each bacterial and fungal strain. For this purpose, essential oils were diluted in a concentration range from 48 µg/ml to 0.0058 µg/ml by using series micro dilution method. Essential oil was added to fresh media suspension afterward following the series dilutions upwards to x−10. Fennel essential oil was assayed in triplicate. Before conducting experiments all the conditions for in vitro anti-microbial activeness were standardized to determine MIC and MBC values. The MIC values were considered as the lowest concentration of essential oil, in which no turbidity in the culture flask was visualized after 24 hrs of incubation at 37˚C. The turbidity in the civilization flasks was considered as visible growth of microorganisms. Further, it was standardized in terms of absorbance at 600 nm in a visible spectrophotometer. For decision of minimum bacterial concentration (MBC) growth inhibitory assays were performed. For this purpose inoculums' size was adjusted to prepare a terminal colony number as 108 colony forming units (CFU/ml in sterile agar plates. The incubation of test and command cultures was too performed at 37˚C for 24 hours. For comparing, both negative and positive command was set and bacterial colony number was counted in all test and control discs. For comparison broad-spectrum antibiotics i.eastward. ampicillin was used as standard to compare the bacterial growth while Griseofulvin for comparing of fungal growth. Results were interpreted by using a standard tabular array that relates to the degree of microbial resistance prescribed past NCCLS (National Committee for Clinical Laboratory Standards). A plot of MIC on a logarithmic calibration versus zone inhibition diameters (arithmetics scale) was prepared for essential oil and antibody to know the susceptibility level. These plots were used to find the zone inhibition diameters respective to the drug concentrations and that of essential oils. The depression MIC value was considered equally susceptibility of essential oils/drugs to the pathogen, while loftier MIC value (with a minor zone inhibition bore) was considered as resistant.

2.5.4. Statistical Analysis

All statistical calculations are expressed as mean ± SE of three replicates. Data were analyzed past ane way ANNOVA to locate pregnant variations in oil activity in various bacterial and fungal strains followed by the Duncan's multiple range tests.

3. Results

Chemical Composition of Oil

Fennel essential oil is extracted from the seeds of coriander with the help of steam distillation. The scientific proper noun of Fennel (Saunf) is Foeniculum vulgare. Plant origin natural products are known to have more than antimicrobial activity against drug resistant microbes. This activity could act as chemical defence confronting pathogenic diseases. Nonetheless, in the present fourth dimension, both the traditional and folk medicines have been considered as alternatives of synthetic drugs for healthcare to the patients. However, for screening pharmaceutical and therapeutic potential of these natural products various bioassays are adult to discover and confirm the anti-pathogenic effects and establish a good correlation with disease pathogens. For obtaining broad-spectrum drugs, essential oils are found to be proficient therapeutic-targeting molecules. The present study emphasizes the limerick of essential oil isolated from Foeniculum vulgare and its upshot on inhibition of bacterial and fungal growths.

Few major components are identified as ii-hydroxy-1-(hydroxymethyl) ethyl ester (1.84%), and 9-octadece- noic acrid (18.56) simply in dissimilar ratios [Tabular array ane] together with few compounds found in minor concentrations. The ascendant components are ii-hydroxy-i-(hydroxymethyl) ethyl ester (i.84%), and 9-octadecenoic acrid (18.56%) but in different ratios [Table ane] together with few compounds found in minor concentrations. Disc diffusion assays were conducted with Foeniculum vulgare essential oil to measure growth inhibition zone bore and screen anti-microbial potential. The essential oil of Foeniculum vulgare has shown college range of inhibition zone bore 20.2 ± 0.18 - 26.5 ± 0.xiv mm at a concentration level of 24 μg/disc. The positive control has shown diameter of inhibition zone (DIZ) ranging from 15.1 ± 0.xxx - eighteen.eight ± 0.37 mm at concentration of 24 μg/disc. All DIZ respective to examination organisms are mentioned in Tabular array two. The results of MIC obtained against all the bacterial strains take been given in Table 3. Lower MIC values presented have shown very high antimicrobial susceptibility of Foeniculum vulgare to Eastward coli, Bacillus cereus, L. acidophilus and Due south. pneumoniae are in a range of half-dozen - 48 μg/ml.

Lower MIC values presented have shown very high antimicrobial susceptibility of Foeniculum vulgare to E coli, Bacillus cereus, L. acidophilus and Southward. pneumoniae are six, 12.0, 24, 24 μg /ml respectively.

Tabular array ii. Zone of inhibition of essential oil from methanolic extract of Foeniculum vulgare.

Values are expressed as mean ± SD (n = 3) and values followed by aforementioned letter of the alphabet are not significantly unlike at the P < 0.05; Determined by Duncan's Multiple Range test. Positive command = Ampicillin/Griseofulvin, negative control = DMSO.

Table iii. Antimicrobial activities of essential oil from methanolic extract of Foeniculum vulgare on different microbes and their corresponding MIC .

Positive command is Ampicillin/Griseofulvin.

4. Discussion

In the present investigation, Foeniculum vulgare essential oil GC-MS analysis showed presence of 36 compounds representing more than than 99.98% of the essential oil [Tabular array ane]. The inhibition zones of the essential oils on tested organism show a significant correlation with MIC values (P < 0.05). Based on growth inhibition zone diameters obtained in tests, results were divided into three categories i.east. resistant (>7 mm), intermediate (>12 mm), and susceptible (>18 mm). Maximum growth inhibition diameter was obtained 26.5 ± 0.14 mm against Micrococcus luteus followed by 25.9 ± 0.14 mm confronting Lactobacillus acidophilus. Foeniculum vulgare oil has shown significantly higher growth inhibition zone diameters in Aspergillus niger 22.7 ± 0.40 mm, Candida albicans 20.5 ± 0.15 mm and 23.0 ± 0.49 mm in Rhizopus stolonifer than the broad spectrum antifungal drug griseofulvin 17.4 ± 0.thirty mm [Tabular array 2]. Similar growth-inhibition zone diameters were reported in P. aeruginosa 33.3 mm, B. subtilis 29.ix mm P. vulgaris 29.iv mm, One thousand. pneumoniae twenty.eight mm and S. aureus each [xvi] , Clostridium ferfringens, E. coli and Lactobacillus acidophilous [17] , Bacillus species [18] , Staphylococcus aureus [19] [20] , and Salmonella enteritidis in presence of dissimilar essential oils [21] . Coriander oil (Foeniculum vulgare) has shown 6.0 µg/ml MIC valueagainst E. coli, 12.0 µg/ml against, Bacillus cereus, 24.0 µg/ml Lactobacillus acidophilus [Tabular array 3]. Similarly, it has shown MIC value in a range of 6 - 24 µg against Aspergillus niger, Candida albicans, Rhizopus stolonifer [Table 3]. Similar MIC 6 µg/ml was reported in allicin and diallyl sulfur compounds against Helicobacter pylori [22] . Similarly luteolin [23] , thymus [24] , phenolics [17] (2006) and Cavacrol [3] , di-terpenoids [25] isolated from diverse essential oils have too shown strongeranti-microbial action against few bacteria mainly against oral pathogens [26] and Escherichia coli O157:H73. Similarly, juniper oil extracted from Juniperus communis (L) has shown strong bactericidal activity against both Gram-positive and Gram-negative bacteria with MIC values between viii% and 70% v/v [27] . Same oil has as well shown stronger fungicidal activity against Candida sp. (MIC from 0.78% to 2% v/v). Sardinian juniperus essential oil was establish active against foodborne pathogens and spoilage microorganism [7] . Similar, antimicrobial activity was exhibited by different Mentha species i.e. Mentha longifolia 50., Mentha aquatica and Mentha piperita L. against E. coli with very low MIC value (four µL/ml) [4] and Hypericum species such equally Hypericum scabrum, Hypericum scabroides and Hypericum triquetrifolium essential oils [28] . Moreover, di-terpenoids isolated from Sagittaria pygmaea has shown antibacterial activity against Streptococcus mutans (ATCC 25175) with MIC value of 15.6 µg/ml [25] . Essential oils from Coriandrumsativum (L) [29] Cinnamomum osmophloeum [30] . Besides this, Dracocephalum foetidum essential oil besides exhibited strong antibacterial activity against methicilin-resistant Staphylococcus aureus (MRSA) [31] . For comparison of antimicrobial activity of essential oils certain wide spectrum antibiotics were also tested against same bacterial strains, which have shown marginal activity orintermediate effect. In various bioassays, Foeniculum vulgare essential oil has shown very high anti-bacterial and anti-fungal activities in vitro. Similar, antimicrobial activity of essential oils of Laserpitium latifolium and Fifty. ochridanum confronting 1 Gram-positive and three Gram-negative bacteria and two Candida albicans strains. Essential oil showed a high antimicrobial potential against Staphylococcus aureus, S. epidermidis, Micrococcus luteus, or Candida albicans (minimal inhibitory concentrations of thirteen.0 - 73.0 μg/ml [32] . Essential oils (East.O.s) from Zataria multiflora Boiss. (zataria) and Origanum vulgare (oregano) showed extensive antimicrobial activity in a broad range of nutrient spoilage or pathogenic fungi, yeast and bacteria, and on hepatitis A virus [33] , Opposite to this, essential oils showed weak inhibitory activeness against the Gram-positive pathogens. Essential oils of Pereskia aculeata Mill. and P. grandifolia Souza et al., 2014) [34] , Similarly Piper species: Piper abbreviatum, P. erecticaule and P. lanatum displayed weak action towards Gram-positive bacteria with MIC values in the range 250 - 500 μg/ml. P. erecticaule oil showed the best activity on Aspergillus niger (MIC 31.3 μg/ml), followed by P. lanatum oil (MIC 62.5 μg/ml) [35] . Juniperus excelsa Bieb. leaf essential have showed high activity towards: Staphylococcus aureus, Streptococcus pyogenes and Haemophilus influenzae (MIC = 125 μl/ml). The pinene-blazon of essential oil showed moderate action against Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Corynebacterium spp. and Campylobacter jejuni (MIC > 50%) [36] . Essential oils isolated from the leaves of Cosmos bipinnatus showed effects against both Gram-negative and Gram-positive bacteria isolates. The MIC of Gram-positive strains ranged between 0.sixteen and 0.31 mg/ml while those of Gram-negative bacteria ranged between 0.31 and 0.63 mg/ml. The Gram-positive bacteria were more susceptible to the essential oil than the Gram-negative bacteria [37] .

Due to presence of volatile components, i.e. phenolic compounds in higher concentration [17] and its diffusion at room temperature fennel essential oil displayed loftier susceptibility against both Gram-negative and Gram-positive bacteria. In addition, it may also increment the plasma membrane permeability that results in higher leakage of fluid material from bacterial cells [32] and inhibit microbial respiration [38] . Therefore, major antimicrobial activity seems to be post diffusion action of essential oils on growth and metabolism of both the bacterial and fungal strains [39] [xl] . No uncertainty, Saunf (Foeniculum vulgare) essential oil contains and so many promising molecules, which can be used for therapeutic purposes mainly pharmacological potential [41] . Like other plant, natural products essential oils possess broad-spectrum antimicrobial activity against pathogenic microbial strains [42] . Equally high antimicrobial susceptibility obtained in tests incomparison to drugs, olive essential oil and its components tin be used for conception of highly active non-antibiotic drug that may be less toxic and testify lesser side effects. The antibacterial action tin exist attributed to effects of the combination of several components of the oil. The results bespeak that the Foeniculum vulgare might exist exploited as natural antibacterial amanuensis and accept application in the treatment of several infectious diseases caused by these bacteria.

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