Abstract: Background and Objective: Dryobalanops lanceolata (D. lanceolata) Burck is one of the plant species from Dipterocarpaceae family which have several medicinal purposes such as mouth ulcers, abscesses, boils and cold sores. The stem barks of D. lanceolata have antibacterial and cytotoxic properties against human breast cancer. While, the tree produce a clear yellow-aromatic resin, scientific support for the essential oil from this plant and its uses is still limited. This research was aimed to obtain the essential oil from leaves of D. lanceolata and to evaluate the antibacterial activities as well as antioxidant activity by in vitro research. Materials and Methods: The essential oil from D. lanceolata leaves was characterized by gas chromatography mass spectrometry (GC-MS). This oil was obtained by steam distillation and presented eugenol (28.73%), gamma-terpinene (15.60%), 2-beta-Pinene (9.80%) and 1-Limonene (8.09%) as the major compounds. The antibacterial activity and antioxidant effect of essential oil from the leaves of D. lanceolata were determined. The antibacterial activities of D. lanceolata oil were observed in vitro on Streptococcus sobrinus (S. sobrinus) and Streptococcus mutans (S. mutans) strains. The oil was investigated against two standard references using agar well diffusion method. The antioxidant activity was assayed by DPPH (1,1-Diphenyl-2-picryhydrazyl) and using ascorbic acid as positive control. Results: The yield of Dryobalanops lanceolata oil obtained in the present study was 0.12%. The oil was active against S. sobrinus and S. mutans. The Dryobalanops lanceolata oil also has potency to inhibit the free radicals at concentration 1.5625-25 ppm, which the IC50 was 14.28 ppm. Conclusion: These results demonstrate that Dryobalanops lanceolata oil has high antimicrobial activity for bacterial that cause dental caries disease and are antioxidant.
INTRODUCTION
Essential oils are volatile oils, which derived from leaves, stems, flowers or twigs of plants that usually carry flavor or taste. The content of typically aroma or fragrance of crops usually characterizes that the essential oil of a plant that is biologically active as an antioxidant and antimicrobial1. In general, the function of essential oils is as a perfume but the essential oil can also be used as an ingredient for the treatment of various diseases such as asthma, headache and cough2. Several studies have been proved that the essential oil contained compounds that could scavenged free radical and inhibit the growth of bacteria and fungi. Essential oils are derived from aromatic plants like cloves3, cinnamon4,5, ginger6, turmeric and other spices potentially as antimicrobial agents against microbial pathogens such as Escherichia coli, Staphylococcus aureus, Salmonella typhi and the fungus Candida albicans, even also function as a natural insecticide7. The role of essential oils as antimicrobials can be developed more widely into antimicrobial preparations such as disinfectants as well as antiseptics.
Dryobalanops lanceolata Burck. was an endemic plant of Kalimantan but common and widespread in Sabah, which has been named by vernacular as "Kapur paji". The genera of Dryobalanops were have large trees, shade tolerant, fast growing and found in clay-rich and sandy soils8,9. Wibowo et al.10 investigated that the oligostilbenoid derivatives from stem bark of D. lanceolata have an antibacterial activity against Staphylococcus aureus, S. epidermidis and S. xylosus. Our previous study11 investigated that Dryobalanops lanceolata oil from leaf has the potency as an antimicrobial agent against S. aureus and Candida albicans.
To the best of our knowledge, no report on the antibacterial activity of essential oil from leaves of D. lanceolata against dental caries pathogen (Streptococcus sobrinus and Streptococcus mutans) has been published so far. Thus, in the present study, the essential oil was extracted from leaves plant collected in East Kalimantan and its antibacterial as well as antioxidant activities were determined.
MATERIALS AND METHODS
The study was conducted between April and November, 2014. The study was carried out at the Forest Products Chemistry Laboratory, Mulawarman University, Samarinda, East Kalimantan, Indonesia.
Materials and chemicals: Leaves of D. lanceolata were collected from Education Forest of Mulawarman University, East Kalimantan, Indonesia. The leaves were dried and prepared for 1 day. Anhydrous sodium sulphate, glucose and nutrient broth were obtained from Merck (Darmstad, Germany). DPPH (1,1-Diphenyl-2-picrylhydrazyl) was purchased from Wako Pure Chemical Industries, Ltd (Japan). Other chemicals were commercially available.
Procedures
Steam distillation method: The essential oil was collected by the steam distillation method. The steam distillation method adopted from Wong et al.12 with slight modification (kettle were used for distillation process). The leaves were packed into the kettle sitting on a perforated plate above the boiling water. The essential oil was volatilized with boiling water at temperature 100°C for 2 h or more. After the steam distillation process, the oil was collected and separated used the separatory funnel which can be used to separate the immiscible liquids of two layers such as oil and water. The oils which have higher density than water will sink at the bottom but some of it did not sink, it was at the top and stuck to the separatory funnel. Wait to the water and oil separated and formed two layers and oil was collected. The process of separation will do several times until no oil was left in the separatory funnel and can be separate anymore. Sodium sulfate (Na2SO4) was used to adsorb the residual of water that fused with the oil. The quantity of the oil was determined according to the yield. The yield of essential oil was calculated using the equation13:
Gas chromatography-mass spectrometry (GC-MS) analysis: The essential oil was analysed by GC-MS (Shimadzu-QP-5050A). Column: HP-5MS, 60 m×250 μm ID×0.25 μm film thickness. Temperature program: from 70-290°C (40 min) at 15°C min–1. Injection temperature: 290°C. The injection port temperature was 290°C and that of the detector was 250°C. Injection mode: split (50:1). Inlet pressure: 18.03 psi. The carrier gas was helium with a flow rate of 1 mL min–1. The mass spectrometer conditions were as follows: Ionization voltage 70 eV. The MS source temperature at 250°C, MS quadruple temperature at 150°C, interface temperature at 290°C, electron ionization mass spectra were acquired over the mass range 40-800 m/z. Compounds were identified using their MS data compared to those from the NIST and Willey Mass Spectral Library and published mass spectra.
Antibacterial assays: Antibacterial assays were done by the agar diffusion method14 with slight modification (the different size of petri dish diameter and volume of media agar were used in this study). The microorganisms used in this study included Streptococcus sobrinus and Streptococcus mutans. All materials were sterilized by auto claving at 15 lbs pressure (121°C) for 15 min15. Microbes were inoculated in nutrient agar (NA). About 5-7 mL sterile media were poured into sterile petri dishes with 6 cm of diameter. After the media were altered to solidify, 25 μL of suspension microbial were inoculated by sterile swab and spreaded all over the surface of the media plates three times16. The wells were bored in solidified media using sterile cork borer with 6 mm diameter. The final concentrations used in this test were 100% (pure of essential oil), 10 and 1% which diluted in 40% ethanol. Twenty microliter of the sample were adding into the well. Chlorhexidine and chloramphenicol were a synthetic standard as antibiotic for positive control at the concentration of 10 μg/well and 40% ethanol as negative control. All the plates were incubated at 37°C for 18-24 h. After incubation, the plates were observed for formation of clear inhibition zone around the well indicated the presence of antibacterial activity. The zone of inhibition was calculated by measuring the diameters of the inhibition zone around the well. All test were performed in duplicate. The diameter of inhibition zones were measured in mm taken with a ruler17. Activity index of sample was calculated using following equation18:
Antioxidant assay: The effect of essential oil on the DPPH radical was determined by DPPH radical scavenging method according to the method of Amiri19. The samples were dissolved in methanol (concentration of stock solution were 25, 12.5, 6.25, 3.125 and 1.5625 ppm). Ascorbic acid was used as positive control. Absorbance measured at 517 nm using Shimadzu UV-VIS 1200 spectrophotometer (Shimadzu Corp., Kyoto, Japan). Percent inhibition of the DPPH radical was calculated by the sample. The 50% inhibition (IC50) of antioxidant activity was calculated as the concentrations of samples that inhibited 50% of scavenging activity of DPPH radicals.
Data analysis: The mean results of the percentage inhibition of antioxidant against concentrations was plotted using the Microsoft Excel computer program, which also gives the regression equations. The regression equations were used to calculate IC50.
RESULTS
In this study, the essential oil of D. lanceolata leaves was obtained by steam distillation method. The steam distillation yielded clear and yellowish essential oil (Fig. 1). The oil from D. lanceolata leaves isolated by steam distillation method obtained yield and yellow refractive index 1.340 and 0.12%, respectively.
The components of D. lanceolata oil have been evaluated by GC-MS. The compounds were identified comparing by retention time, molecular formula, molecular weight and area (Table 1). In the essential oil from D. lanceolata leaf oil, 22 components were identified, which were dominated by monoterpene hydrocarbons. The main constituents of the sample were Eugenol, g-Terpinene, 2-β-Pinene and 1-Limonene (28.30, 15.60, 9.80 and 8.09%, respectively). According to these results, eugenol was mainly present in D. lanceolata essential oils. The structure of the major bioactive compound is presented in Fig. 2.
The anti-bacterial activity of D. lanceolata essential oils against S. mutans and S. sobrinus were summarized in Table 2. The antimicrobial potential of the D. lanceolata essential oils was evaluated according to their zone of inhibition against S. mutans and S. sobrinus and the results (zone of inhibition) were compared with the activity of the standards, chlorhexidine and chloramphenicol (10 mg mL–1). The zone of inhibition above 6 mm in diameter was taken as positive result. Generally, the tested organisms were sensitive to D. lanceolata essential oils with varying magnitudes. When the activity index is considered (Table 1), antibacterial effect of D. lanceolata leaf oil at concentration 1, 10 and 100% present in the following order: S. sobrinus>S. mutans. It can be seen that 100% of D. lanceolata oil possess a higher antibacterial effect than chlorhexidine and chloramphenicol (at concentration 10 μg/well) against S. sobrinus and S. mutans.
| Fig. 1(a-b): | (a) Tree and leaves of D. lanceolata and (b) Color of essential oil of D. lanceolata |
| Table 1: | Chemical constituents of the essential oil of D. lanceolata by GC-MS |
| Table 2: | Antibacterial activity of essential oil of D. lanceolata |
Data are means of three measurements±SD | |
| Fig. 2: | Structure of the Eugenol, major compounds of D. lanceolata essential oil |
A highly significant decrease of the DPPH radical concentration due to the scavenging activity of each oil concentration and standards as illustrated in Fig. 3. The activity to scavenge DPPH radical increases significantly with increasing oil concentration had been found. Antioxidant activity of the sample was determined by concentration of sample that could scavenge 50% free radical (IC50) and the sample showed strong antioxidant activity on DPPH. The oil had a good radical scavenging (2.71-70.56%).
| Fig. 3: | Free radical scavenging activity of D. lanceolata essential oil |
Scavenging activity was measured using the DPPH radical assay, Data are means of three measurements±SD |
|
Highest percentage shown in concentration 25 ppm was 70.56% and it was inhibited the 50% DPPH radicals (IC50) in concentration 14.28 ppm.
DISCUSSION
The most popular method for extraction process of essential oil was steam distillation which the raw materials were applied in this method. The final process of steam distillation was gain the liquid form (the mixture of oil and water) and it separated using proper method20,21. The range of the refractive index values indicated that the components were neither degraded nor polymerized and remained as mono or sesquiterpenoids and their derivatives22. Guenther23 suggested that the refractive index is affected by the length of the carbon chain and the number of double bonds. If the carbon chain was longer, the refractive index would bigger but it will decrease with the addition of double bonds.
In general, essential oil components can be subdivided into two distinct groups of chemical constituents, the hydrocarbons which are made up almost exclusively of terpenes and the oxygenated compounds (oxygenated terpenoids). Studies showed that oxygenated terpenoids such as alcoholic and phenolic terpenes have more antimicrobial activity than the other constituents. Several reports on the antimicrobial activity of monoterpenes have shown that the number of double bonds in a structure and the acyclic, monocyclic and/or bicyclic structure have no significant influence on their activity, although higher inhibitory activity is seen in aromatic compounds such as carvacrol, thymol and eugenol. However, oxygenated terpenoids show characteristic and distinct activity patterns towards microorganisms, terpenoids that contain alcohols possess higher activity than the corresponding carbonyl compounds24-26.
Previously study reported that the leaves of D. lanceolata contained monoterpenes such as β-Pinene, Limonene, β-Myrcene27. The monoterpenes and sesquiterpenes contained in D. lanceolata also found in the D. aromaticum leaves such as Camphene, α-Pinene, α-Copaene, β-Caryophyllene, α-Amorphene, Aromadendrene, α-Caryophyllene, γ-Cadinene, β-Selinene, Germacrene and Selina-3,7(11)-diene28. Hopeaphenol and vaticaphenol A were isolated from methanolic crude extract of D. lanceolata29 and malaysianol B and C, stenophyllol, nepalensinol B, vaticanol B and C, upunaphenol D, flexuosol A, nepalensinol E, ε-viniferin, ampelopsin F and laevifonol found in the acetone extract of the stem bark of D. lanceolata30,31. Different chemical compounds in the leaf of D. lanceolata might be due to the preparation method of extract.
The degree of the antibacterial activities of the essential oil could be attributed to the hydrophobicity of the components. The hydrophobicity of essential oils might enable them to partition in the lipid component of bacterial cell membrane, rendering them permeable and leading to leakage of bacterial cell contents32,33. The GC-MS study revealed eugenol to be the major constituent of D. lanceolata oil. The antimicrobial and antioxidant capacity of D. lanceolata essential oil could be mainly related to the occurrence of eugenol as assessed by Chen et al.34 and Politeo et al.35. Eugenol possesses various biological abilities, including antioxidant activity, antiinflammatory action, anticarminative, anti-spasmodic, antiparasitic activities, antibacterial and antiviral effects. In medicine, eugenol is used as an antiseptic and an anesthetic. Eugenol-producing dental materials are used in clinical dentistry36,37.
The D. lanceolata oil has antioxidant activity against DPPH (1,1-Diphenyl-2-picryhydrazyl) as free radicals. DPPH is used to test the ability of the compound to act as a free radical trap or hydrogen donor and evaluate the antioxidant activity38. The antioxidant activity of essential oils could be attributed to their hydrogen donating ability. Antioxidant properties are very important in counteracting the deleterious role of free radicals in foods and biological systems39. The potential antioxidant activity of the essential oils was determined by the scavenging activity of the stable free radical DPPH.
CONCLUSION
In conclusion, essential oil of D. lanceolata leaves showed a good in vitro antioxidant and antibacterial activity against S. sobrinus and S. mutans. This activity could be attributed to the eugenol compounds present in this oil. This study proved that essential oil of D. lanceolata leaves can be used as an alternative anti-bacterial agent to prevent and treat dental caries diseases.
SIGNIFICANCE STATEMENTS
This study discovers the essential oil from the leaves of Dryobalanops lanceolata that can be beneficial for medicinal purposes. This study will help the researcher to uncover the critical areas of essential oil from Dryobalanops lanceolata that many researchers were not able to explore. Thus a new theory on the bioactivity of Dryobalanops lanceolata essential oil may be arrived at.
ACKNOWLEDGMENT
The authors gratefully acknowledged to Directorate General of Higher Education, Indonesia Ministry of National Education for the financial research support. (Grant Number: 36i/H17.16/PG/2014).