Plants, families, part used and rendement.
The antibacterial activity of essential oil of five Indonesian herbs has been studied. The essential oil produced from different parts of plants (lime, lemon, Surinam cherry, fennel, and toothache plants) were extracted by water steam or hydro distillation and then examined by GCMS. The antibacterial activities of the essential oils were determined by measuring MIC (minimum inhibitory concentration), whereas some bacterial strains were used in this study such as follows: Staphylococcus aureus FNCC 0047, Bacillus subtilis ATCC 6051, B. cereus FNCC 0057, Escherichia coli IFO 0091, Pseudomonas cepacia FNCC 0063, and P. aeruginosa FNCC 0063, respectively. All the five samples used in this experiment have antibacterial activity against Gram-positive and Gram-negative bacteria. Gram-negative bacteria appear more resistant than Gram-positive bacteria. By using MIC measurement the Gram-positive and Gram-negative bacteria showed different sensitivities toward essential oils. Among the material study, the essential oil of Eugenia uniflora L (Surinam cherry) showed the highest antibacterial activity.
- essential oil
- Indonesian herbs
- Eugenia uniflora
- Spilanthes paniculata
Essential oil has been used since ancient times as perfumery in ritual ceremony and folk medicine by native countries. Together with the time passed, essential oil utilizing has been developed not only in folk medicine but also in food industry as flavoring, cosmetics as fragrance, and additives . These oil have many biological activities especially rich in mono and sesquiterpene which are known as antimicrobial. Indonesia has around 28,000 plant species and was predicted that more than 7000 species have potency as medicinal plants. Unfortunately just less than 300 species were used in pharmaceutical industry, whereas the rest still need the evidence . One of the important compounds in Indonesian herbs is essential oil, a volatile oil from the plants composed of many phenolic compounds and responsible for strong antibacterial effect.
Essential oil is aromatic oily liquid obtained from different parts of the plants (root, bark, leaf, bud, flower, fruit, and seeds). This oil name bears the name of the plant species from which it is derived. This oil has a sharp smell that is produced as secondary metabolite and variable mixtures of terpenoid, monoterpene (C5), sesquiterpene (C15), and diterpene (C20). Another functional groups present in the molecule formed another molecules such as aldehydes, ketones, acids, lactones, etc. The amount of components varies from approximately 10–100, although usually the main part of the oil is composed of only a few components .
As a tropical country, Indonesia is rich with aromatic plants, and it is natural for this country to be one of the essential oil world supplier. Approximately 70 kinds of essential oil were trading in the international market and 40 kinds among them come from Indonesia . There are a lot of methods to produce essential oil, and it can be obtained by expression, enfleurage, solvent extraction, and distillation (hydro distillation and steam distillation). Distillation is most commonly used for commercial production . Plant materials cut in small pieces were placed in distillation apparatus and hydro distilled/steam distilled for 3–6 hours. The hot water or vapors contact with material and bring the essential oil inside the sample, and the next step solvent was evaporated. The system of distillation instrument will cool down and condense vapors to produce essential oil and water mixture. Distillate separation will give essential oil and water.
Although the essential oils have different aroma and big variation, but this oil shows similar physical properties as color and solubility in water, for example essential oil are immiscible with water but quite soluble in most organic solvents. These characters can be used as basic criterion of quality of essential oil.
The increasing demand of essential oil in the world was due to the potential of essential oil in pharmacological therapeutic.
The major problem in antimicrobial chemotherapy is the increasing occurrence of resistance to antibiotic. A lot of essential oils are known to exert antimicrobial activity, but the mechanism of action is often not entirely understood. The overuse of antibiotics is the most important factor contributing to the appearance of many kinds of resistant microbes [6, 7]. The aim of this study was to determine the antibacterial activity of five Indonesian herbs and to analyze the dominant component of each essential oil.
1.1. Antibacterial activity of essential oil
The bioactivity of essential oils has been known since ancient times. This compound has been known to have various bioactivities, including antibacterial, antiviral, anti-inflammatory, antifungal, antimutagenic, anticarcinogenic, and antioxidant, as well as other miscellaneous activities [8, 9].
Essential oil/volatile oil is produced from plant materials and showed an important role in plants by acting as protector of the plants from herbivores, microbial, and insects. On the other side, essential oil also has a role in the pollination and seed distribution because the strong smell of the oil attracts some insects to carry out both processes .
Antibacterial activity of essential oil depends on their chemical composition and the amount of each compound. The composition, structure, as well as functional groups of the oils play an important role in determining their antimicrobial activity [18, 19].
The mixture of various chemical substances that belong to different chemical families, including terpenes, aldehydes, alcohols, esters, phenolic, ethers, and ketones, gives the antibacterial activity [20, 21].
In general, essential oil is easier to attack Gram-positive bacteria than Gram-negative bacteria due to the differences of the strength of the cell membrane. The cell wall of Gram-positive bacteria is more simple than Gram-negative bacteria, because the big part of the cell wall is peptidoglycan, and so hydrophobic molecules are able to penetrate the cell. On the contrary, the cell wall of Gram-negative bacteria is more complex, and it has peptidoglycan layer thinner than Gram-Positive bacteria but the peptidoglycan linked to lipopolysaccharide. This is the reason that the cell wall of Gram-negative bacteria stronger than the other and relatively resistant to hydrophobic compounds .
Generally, the chemical characterization of many essential oils reveals the presence of only 2–3 major components at a fairly high concentration (20–70%) compared to other components present in trace amounts. Most essential oils are composed of terpenes, terpenoids, and other aromatic and aliphatic constituents with low molecular weights . Essential oil contains a wide series of secondary metabolites that can inhibit or slow the growth of bacteria, yeasts, and molds. The essential oil and their components have a variety of targets, particularly the membrane and cytoplasm, and in certain situations, they completely alter the morphology of the cells [24, 25, 26].
2.1. Material and methods
Five essential oils were used which are as follows: Lime oil (
All chemical reagents made by E-Merck, Germany, Nutrient Broth, Mueller-Hinton Agar and Tetracycline (PA, Oxoid, England), and paper disk (PA, Whatman, England).
Isolation and physicochemical analysis of essential oil were subjected to steam distillation using clevenger-type apparatus. GCMS analysis of essential oils was done in Laboratory of Organic Chemistry, Faculty of MIPA Universitas Gadjah Mada Yogyakarta.
2.1.1. Isolation of essential oil
The essential oils were obtained from the aerial part of plants, such as peel (
One kilogram of each plant part was cut into small pieces and subjected to steam distillation apparatus, which is completed with Clevenger apparatus for 6 hours. The next step, essential oils were isolated by extraction of the distillate used diethylether, and its percentage yield was calculated.
2.1.2. Direct bioautographic test
The essential oil was evaluated in vitro by thin-layer chromatography (TLC) method. Before using, the plates were activated at 105°C for 10 minutes. The plate of silica gel F254 4 × 10 cm (Merck) as a solid phase and toluene:ethylacetate (93:7) as a mobile phase were used. Afterward, plate was sprayed with bacterial suspension in Mueller-Hinton Broth (MHB), and plate was stored in a water-vapor chamber at 37°C in 24 hours. Iodonitrotetrazolium chloride 5 mg/ml was used to visualize the antibacterial spot . This activity was used as an effort in the beginning for check and recheck after measurement antibacterial activity.
2.1.3. Determination of antibacterial activity
The antibacterial activity was detected by minimum inhibitory concentration (MIC) of five essential oils. Bacteria was inoculated to nutrient broth (NB), incubated at 30°C for 24 hours. Inoculum was diluted by using physiological solution (NaCl 0.9%) to match 0.5 Mc Farland standard. The bacterial suspension was diluted and measured by UV–Vis Spectrophotometer to obtain Optical Density (OD) 0.4–0.5 at 550 nm .
A paper disk was dropped 20-μl essential oil in certain concentration and put the disk in a petri dish with medium content bacteria inside. The petri dish was incubated at 30°C for 24 hours. Inhibition area diameter (IAD) was measured as a middle line start from the clear spot around the disk. The lowest concentration which shows the clear spot around the paper disk is the minimum inhibitory concentration (MIC).
2.1.4. Data analysis
Antibacterial activity data of each plant were analyzed by using randomized completely block design (RCBD) sub-sampling, five treatments, three subsamples, and five replications, whereas the blocks are the analysis time .
2.2. Result and discussion
2.2.1. Essential oil
The percentage yield (rendement) of essential oil obtained from samples less than 3% except Foeniculi oil (3%), and the smallest amount was Spilanthes oil around 0.1% (Table 1).
|Scientific name||Plant family||Part used||Rendement (% weight)|
Plant volatiles constitute about 1% of plant secondary metabolites and are mainly represented by terpenoids, phenylpropanoids/benzenoids, fatty acid derivatives, and amino acid derivatives . A lot of monoterpenes demonstrated their potent aromas, and these compounds are known as essential oil composer. Essential oil containing monoterpenes is responsible for the fragrant and biological properties of aromatic and medicinal plants .
Essential oil obtained from samples was further analyzed by GCMS. A total of more than 20 compounds were found in each essential oil sample. Table 2 informed five dominant compounds of each essential oil.
|No||Scientific name||Five dominant compounds|
Citrus aurantifoliaSwingle (lime) and C. limon(L) Burm
According to the result of this study, the percentage yields of essential oil of the peel of lime and lemon were 0.4 and 0.5%, respectively. Limonene was major component of both Citrus oils, but limonene in lemon oil (43.40%) was higher than lime oil (29.29%). The presence of β–pinene in big amount (24.54%) in lime oil makes the aroma of lime oil quite different from lemon oil. Limonene is the main component of Citrus essential oil. The major compound in the lime essential oil is
Ref.  found that limonene is the dominant compounds in lime essential oil (49.657%), and this compound was also observed at every stages of maturation, which indicates that limonene could be used as a functional index of ripeness. The peel of Sicilian lemon variety was reported to have d-limonene concentration around 70 and 0.84% of bisabolene . Ref.  also found that monoterpenes are the dominant component (86–88.79%) of
Oil of lemon is one of the most important flavoring oils, used widely in all kind of beverages, soft drink, baked goods, such as cakes, pastries, gelatin dessert, ice cream, etc. This oil can also be applied in perfumes, toilet waters,
Eugenia unifloraL. (Surinam cherry)
Foeniculum vulgareMill. (fennel)
In this work, the composition of essential oil of fennel seeds obtained by hydro distillation composed of 30 compounds and the highest amount is
Spilanthes paniculatawall (toothache plant)
According to this study, the essential oil of fresh flower of
A comparison of oil composition of this study with those reported from different places in the world show differences not only in the kind of the compounds but also the percentage content of some of the mayor and minor constituents.
2.2.2. Antimicrobial activity
Table 3 demonstrated the antimicrobial properties of the five essential oils (lime, lemon, Surinam cherry, fennel, and toothache plants). The strength of antibacterial activity was declared as minimum inhibitory concentration (MIC); Low (L) if the inhibitory area diameter (IAD) less than 0.7 cm, Medium (M) if the IAD 0.7–0.8 cm, and Strong (S) The IAD >0.8 cm .
|Material||Minimum inhibitory concentration (MIC) μg|
According to Tables 2 and 3, essential oil of every plants used in this study had significant antibacterial activities against some of bacterial. The diameters of inhibitory area (IAD) or the diameters of growth inhibition zone were measured including the diameter of disk 6 mm.
The strength of antibacterial activity of the essential oil is presented inTable 3.
220.127.116.11. Lime and lemon essential oil
Antibacterial activity for Medium level of Lime and lemon oil showed the similar antibacterial strength against Gram (+) bacteria (1000 μg), but for Gram (−) bacteria, the higher concentration was needed. The antibacterial activity of lemon oil was higher than the lime oil, because 3000 μg concentration of lemon oil gives strong level and lime oil need 5000 μg. The higher the concentration of the essential oil, the lower is the antibacterial activity obtained .
Both of essential oils (lime and lemon) showed the pale yellow color, bitter taste, and fresh piquant odor. But the odor of each essential oil (lime and lemon) is not exactly the same. The different of the odor between lime and lemon oil relates the profile of chemical compounds. Although limonene is the dominant compound of both essential oil, but the amount is different and followed by different compounds. Lime oil showed
Eugenia unifloraL (Surinam cherry) essential oil
The essential oil was collected from leaf; the oil has yellowish white color, unidentified odor, and the aroma is quite difficult to express it. This oil also shows good antibacterial activity because in low concentration give medium to strong level MIC in 200–600 μg. The profile of essential oil composed of
Foeniculum vulgareMill (fennel) essential oil
This essential oil of
The GC–MS analysis of essential oils of
Spilanthes paniculatawall (toothache plant) essential oil
The essential oil was obtained from toothache plant leaf; the oil has a yellowish color. This essential oil showed antibacterial activity in medium level at 1000 μg and strong level at 1500 μg against Gram positive, whereas negative test bacteria except against
All the essential oil samples used in this study indicated broad antibacterial spectrum because of its show antibacterial activity against Gram-positive and negative bacteria. These data conform with [83, 84] that essential oil exhibits antibacterial activity against a large number of Gram-positive and Gram-negative bacteria. It has been observed that the mode of action of essential oil is based on their ability to disrupt cell wall and cytoplasmic membrane, leading to lysis and leakage of intracellular compounds . The disturbance of the cell membrane will disturb many vital processes such as energy conversion, nutrient processing, the synthesis of structural macromolecules, and the secretion of growth regulators . Essential oils of various plants were reported to cause increased bacterial cell membrane permeability, leading to the leakage of cellular components and loss of ions [86, 87].
The strength of antibacterial activity of essential oil is able to be a basic potent to reduce antibiotic consuming, although many antibiotics are available for treating various bacterial pathogens. The increased multidrug resistance has led to the increased severity of diseases caused by bacterial . The use of several antibacterial agents at higher doses may cause toxicity in human, so that the researcher needs to explore alternative new molecules against bacterial strains. Plant essential oils are potential candidates as antibiotic/antibacterial agents. The main advantage of natural agents is that they do not enhance the antibiotic resistance, a phenomenon commonly happened in long termed use of synthetic antibiotics .
In conclusion, all the five samples used in this experiment have antibacterial activity against gram positive and negative bacteria. Gram-negative bacteria appear more resistant than Gram-positive bacteria. Gram-positive and Gram-negative bacteria showed different sensitivities to essential oil. Among these samples antibacterial activity of
Conflict of interest
The authors declare that there is no conflict of interest.