- Original article
Antioxidant, anti-hyaluronidase and antifungal activities of Melaleuca leucadendron Linn. leaf oils
Journal of Wood Science volume 58, pages 429–436 (2012)
The present study examined the chemical composition, in vitro antioxidant, anti-hyaluronidase and antifungal activities of essential oils of Melaleuca leucadendron Linn. from Gundih-Central Java, Indonesia in different plant ages of 5, 10 and 15 years old. The Chemical composition of essential oils were analyzed by GC/MS. Twenty-six components were identified, of which 1,8-cineole (49.22–55.04 %), α-terpineol (8.79–10.70 %), d-limonene (5.58–6.39 %), and β-caryophyllene (5.03–7.64 %) were the main compounds in these oils. The antioxidant assay and anti-hyaluronidase assay showed that M. leucadendron leaf oils possess mild antioxidant activity with IC50 between 7.21 and 9.23 mg/ml and anti-hyaluronidase activity with IC50 between 1.94 and 3.03 mg/ml. The antifungal assay showed the effectiveness of these essential oils against Fomitopsis palustris (IC50 0.12–3.16 mg/ml), Trametes versicolor (IC50 0.01–0.06 mg/ml), Cladosporium cladosporioides (IC50 0.03–0.49 mg/ml), and Chaetomium globosum (IC50 0.06–0.15 mg/ml).
Essential oils and their components have recently been of great interest because of their relatively safe status, wide acceptance by consumer and the possibility of their exploitation for potential multi-purpose functional uses . Many researchers have reported utilization of Melaleuca essential oils, mainly used in the manufacture of cosmetics, germicides, as clinical efficacy and medicinal. These essential oils are also used as antiseptic, antibacterial, antiviral, antifungal, antiprotozoal, antioxidant, anti-inflammatory agents and oral cleaner [2–5]. Genus Melaleuca is currently represented by approximately 250 species, one species that is found in Indonesia and can produce commercial essential oil is Melaleuca leucadenron Linn. This species is one of the most important essential oil producing species. Leaves and stems of this species produce strongly scented essential oils, some of which have useful medicinal properties . Essential oil from this species is widely used in Indonesia as an expectorant for throat preparations and as ointments for stomach upsets and mosquito bites. However, there is no sufficient scientific information about bioactivities of M. leucadendron leaf oil from Indonesia.
In the previous study , we investigated the chemical components of M. leucadendron Linn. leaf oils from Java, Indonesia. The results showed that leaf essential oils of M. leucadendron from Java, Indonesia contained 26 compounds which were mostly monoterpenes, sesquiterpenes and related alcohols. GC/MS analyses have shown the presence of 1,8-cineole, α-terpineol, d-limonene, and β-caryophyllene as major compounds in these oils. The aforementioned study indicates that some important compounds which can be considered as bioactive agents are contained in these oils.
The antioxidant activity of plant extracts has been recently highlighted [8, 9] due to the fact that free radicals such as reactive oxygen species (ROS) are responsible for various diseases and salinity . The human health promoting effects of plants were elucidated to be due to some of bioactive substances such as essential oils having antioxidant activity . Several chemical constituents from essential oils also have been reported to inhibit hyaluronidase activity and display anti-inflammatory effects; they also have significant ecological functions for the protection against fungal infections .
Therefore, this present study was carried out to investigate M. leucadendron leaf oils from Gundih, Central Java, Indonesia at 3 plant ages (5, 10 and 15 years) and its major components in order to evaluate their effectiveness as antioxidant, anti-hyaluronidase and antifungal agents. Antioxidant activities in this study were investigated in vitro by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, anti-hyaluronidase activities by using hyaluronidase enzyme and antifungal activities by agar diffusion method against six fungal species.
Materials and methods
Essential oils and GC/MS analysis
Fresh samples of M. leucadendron leaves were collected from plantations of M. leucadendron in Gundih, Central Java Province, Indonesia in different plant ages of 5 (A1), 10 (A2) and 15 (A3) years old. The 5–7 kg of fresh leaves from each sample was extracted by hydrodistillation method for 5 h. The oils produced were kept in a labeled bottle at approximately 0 °C until used.
The chemical analysis of the oils was conducted by a GC-17A gas chromatograph (GC) coupled to a QP5050A mass spectrometer (Shimadzu Co. Ltd., Kyoto, Japan) using a fused-silica capillary column TC-1701 (0.25 mm i.d. × 15 m, 0.25 μm film thickness; GL Sciences). GC/MS was performed using the following conditions: carrier gas He; flow rate 20.6 ml/min; splitless injection; injection volume 1.0 μl; injection temperature 230 °C; oven temperature programmed from 30 °C (5 min hold) to 100 °C at 10°C/min (5 min hold), and from 100 to 230 °C at 15°C/min (5 min hold); interface temperature 230 °C; and electron-impact ionization at 70 eV. The identification of oil components was confirmed by comparison of retention time with those of authentic samples (α-terpineol, d-limonene, 1,8-cineole, β-caryophyllene, eugenol), and with National Institute of Standards and Technology (NIST) database library, and Kovats retention index (RI)  which was calculated from the retention times of authentic aliphatic saturated hydrocarbons (C8–C22, Wako Chemicals Co.) and compared with literature data .
The antioxidant activities of M. leucadendron leaf oils were evaluated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. An ethanol solution (1 ml) of sample was added to a solution (10 ml) of 0.25 mM DPPH in ethanol. Absorbance of blank sample containing the same amount of ethanol and DPPH was measured as a control. The solution was rapidly mixed and was kept in water bath at 30 °C for 30 min and absorbance was measured at 515 nm by using U-2810 spectrophotometer (Hitachi High-Technologies Co., Tokyo, Japan). The percent of inhibition was calculated by the following equation:
where A c is absorbance of the control and A s is the absorbance of the sample tested. IC50 values, which represent the concentrations of the essential oils that cause 50 % inhibition is determined by linear regression analysis.
Anti-hyaluronidase activity was examined by the Lee and Choi  method with slight modifications. Sample solutions (1, 2 and 4 mg/ml) were prepared by using M. leucadendron leaf oils or authentic compounds dissolved in mixed solvent (5 % DMSO in ethanol). Fifty μl of ovine hyaluronidase (7900 U/ml) dissolved in 0.1 M acetate buffer (pH 3.5) was mixed with 100 μl of each sample solution, and then incubated for 20 min in a water bath at 37 °C. One hundred μl of 12.5 mM calcium chloride was added to the reaction mixture, and then the mixture was incubated for 20 min in a water bath at 37 °C. The hyaluronidase activated by Ca2+ was reacted with 250 μl of sodium hyaluronate (1.2 mg/ml) dissolved in 0.1 M acetate buffer (pH 3.5), and then incubated in a water bath at 37 °C for 40 min. 100 ml of 0.4 N sodium hydroxide and 100 μl of 0.4 M potassium borate were added to the reaction mixture, and then incubated in a boiling water bath for 3 min. After cooling to room temperature, 1.5 ml of dimethylaminobenzaldehyde (PDMAB) was added to the reaction mixture, and then incubated in a water bath at 37 °C for 20 min. Optical density (OD) at 585 nm of the reaction mixture was measured by using U-2810 spectrophotometer (Hitachi High-Technologies Co., Tokyo, Japan). The percentage of inhibition was calculated by the following equation:
where ODc is optical density of control and ODs is optical density of the sample tested. IC50 values, which represent the concentrations of the essential oils that cause 50 % inhibition is determined by linear regression analysis.
Antifungal activity was examined by Wang et al.  method with slight modifications against wood rot fungi of Fomitopsis palustris NBRC 30339 and Tramestes versicolor NBRC 4937, and household molds of Aspergillus niger NBRC 6342, Cladosporium cladosporioides NBRC 6348, Chaetomium globosum NBRC 6347, and Penicillium citrinum NBRC 6352. PDA (Potato Dextrose Agar, Difco) plates were prepared for fungal media using Petri dishes (9 cm diameter). The different concentration of the oil samples and major compounds were serially diluted with methanol and added to 20 ml of PDA. Petri dishes are kept in clean bench at room temperature for a day and methanol is removed by vaporization. Each agar mycelium plug was inoculated at the center of the Petri dish and incubated in dark at 25 °C. Colony growth diameter was measured every day for 14 days or while the fungal growth in the control treatment had completely covered the media in the Petri dishes. PDA plates used as a control contain only methanol without essential oil or authentic compound solutions. Growth inhibition was calculated by the following equation:
where S a is surface area of mycelium growth of treatment (cm2) and S b is surface area of mycelium growth of control (cm2). The IC50 value of antifungal activity for each sample was graphically estimated using logarithmic or linear regression analysis by extrapolation of percent inhibition versus concentration.
Data were analyzed using SPSS (IBM) and Excel program. All tests and analyses were run in triplicate and averaged. Data were compared using Scheffe’s test and expressed as mean ± SD. Results with p < 0.05 were considered to be statistically significant.
Results and discussion
GC/MS analysis of essential oil
Our previous study of M. leucadendron leaf oils from Java, Indonesia showed 26 compounds in these essential oils identified by GC/MS analysis . This study showed that the components of essential oils from Gundih, in different plant ages are similar to those in previous ones with a few differences in percentages of the contents. These oils were composed of hydrocarbons and alcohols, mainly monoterpenes and sesquiterpenes. Table 1 shows that M. leucadenron leaf oils from plant ages 5 (A1) and 10 years (A2) contained 26 compounds and from plant age 15 years (A3) contained 24 compounds. The oils were rich in 1,8-cineole (49.22–55.04 %), α-terpineol (8.79–10.70 %), d-limonene (5.58–6.39 %), and β-caryophyllene (5.03–7.64 %). The increase of tree age results in decrease of 1,8-cineole content, adversely increase of β-caryophyllene content. The highest quantities of α-terpineol and d-limonene were obtained from A2 and A1, respectively.
The DPPH radical scavenging activities of various concentrations of M. leucadendron leaf oils are shown in Table 2. The in vitro antioxidant activities of the oils were also compared with those of BHA (butylated hydroxyanisole) and eugenol.
Table 2 shows that the radical scavenging activities of A1 (IC50 9.23 mg/ml), A2 (IC50 7.21 mg/ml), A3 (IC50 7.71 mg/ml) and four major compounds of 1,8-cineole (IC50 4.92 mg/ml), d-limonene (IC50 4.58 mg/ml), β-caryophyllene (IC50 3.68 mg/ml), and α-terpineol (IC50 4.59 mg/ml) were relatively low if compared with the single compounds like BHA (IC50 25.68 × 10−3 mg/ml) and eugenol (IC50 3.25 × 10−3). BHA and eugenol, usually utilized as commercial antioxidant agents have strong radical scavenging activity. The essential oil of A1 had lower antioxidant activity than A2 and A3 (Table 2). This is probably due to the presence of phenolic compounds such as eugenol, A1 has lower eugenol content (3.27 %) than A3 (4.35 %) and A2 (4.55 %). Several studies also reported the phenolic compounds like eugenol exhibited high antioxidant activity [17, 18]. Previous studies on antioxidant properties of Eucalyptus camaldulensis leaf oils , Aframomum corrorima essential oil , Italian large leaf, purple ruffles, cinnamon, and lemon basil oils , show that the antioxidant activity of the essential oils are usually lower than those of commercial antioxidants, but the content of phenolic compounds like eugenol, thymol etc., largely influences this ability of essential oils. In this study M. leucadendron leaf oils possess variable radical scavenging activities, probably due to the different quantity of eugenol in these essential oils. Their mild antioxidant activity is assumed to be moderate for several purposes, such as ointments, plasters, etc.
Hyaluronidase is an enzyme that depolymerizes the hyaluronic acid (HA) in the extracellular matrix of connective tissue, it is found both in organs (testis, spleen, skin, eye, liver, kidney, uterus and placenta) and in body fluids (tears, blood and sperm) [22, 23]. The enzyme is known to be involved in allergic effects , migration of cancer , inflammation, petechial hemorrhages following its injection in mesentery preparations, and also an increase in the permeability of the vascular system . High molecular weight HA has an important role in the regulation of scarless repair in fatal wound healing by markedly diminishing the inflammatory response. Enzyme hyaluronidase degrades HA by lowering its viscosity and increasing the permeability. Degradation products of HA lead to increase inflammation, angiogenesis, fibrosis, and collagen deposition in wound healing .
The effect of M. leucadendron leaf oils as anti-hyaluronidase was investigated in this study. The IC50 values of anti-hyaluronidase assays of M. leucadendron leaf oils were from 1.94 to 3.03 mg/ml. The result showed that A1 (IC50 3.03 mg/ml) had lower anti-hyaluronidase activity than A3 (IC50 1.94 mg/ml) and A2 (IC50 2.67 mg/ml). Four major compounds of M. leucadendron leaf oil were also tested for their anti-hyaluronidase activity. The results showed that β-caryophyllene had high anti-hyaluronidase activity (IC50 4.16 × 10−3 mg/ml), 1,8-cineole possess low anti-hyaluronidase activity (IC50 1.17 mg/ml), and d-limonene and α-terpineol showed no anti-hyaluronidase activity (Table 3). Probably the content of β-caryophyllene in the oils affects the anti-hyaluronidase activity of A1 lower than those of A2 and A3. Several studies show that sesquiterpenes have a calming effect as well as inhibition of hyaluronidase (anti-inflammatory) and anti-infectious effects. β-Caryophyllene is a sesquiterpene widely distributed in essential oils of various plants and known to possess anti-inflammatory effect . Some studies also show phenolic compounds have high anti-hyaluronidase. [29–31] This study indicates that M. leucadendron leaf oils possess moderate anti-hyaluronidase activity caused from β-caryophyllene and slightly 1,8-cineole. Although anti-hyaluronidase activity of M. leucadendron leaf oil is not so large, it seems to increase in proportion to the content of β-caryophyllene.
The antifungal indices of the oil samples and the major compounds against 6 fungi are shown in Tables 4 and 5, respectively. The IC50 values of oils and major compounds were determined using various concentrations of these substances encompassed their values.
The antifungal activities of the M. leucadendron leaf oils against wood rot fungi are shown in Table 4, using the oil concentration of 2.5, 5 and 10 mg/ml for F. palustris and the oil concentration of 0.05, 0.1, 1 mg/ml for T. versicolor. Essential oils of M. leucadendron showed higher inhibitory effects against T. versicolor than against F. palustris, complete inhibition against T. versicolor was achieved at concentration of 1 mg/ml. Antifungal indices of major compounds showed α-terpineol was the highest inhibitory effect against F. palustris and T. versicolor, followed by 1,8-cineole, β-caryophyllene, and d-limonene. The result showed that α-terpineol completely inhibited the growth of F. palustris at concentration of 2.5 mg/ml and that of T. versicolor at 5 mg/ml, however, d-limonene had no effect on F. palustris (Table 5).
IC50 of M. leucadendron leaf oils against F. palustris ranged from 0.12 to 3.16 mg/ml and against T. versicolor ranged from 0.01 to 0.06 mg/ml. IC50 values of 1,8-cineole, β-caryophyllene, and d-limonene against T. versicolor and F. palustris are mostly higher than those of the leaf oils (Table 6), it means that the leaf oils may have synergic effects against T. versicolor and F. palustris.
The essential oils of M. leucadendron leaves also showed inhibitory effects against some molds. The essential oils showed strong activity against C. cladosporioides and C. globosum with complete inhibition at concentration of 5 and 1 mg/ml, respectively. The essential oils at concentration of 10 mg/ml revealed the values for P. citrinum ranging between 39.86 and 79.90 %. The perfect inhibition against A. niger was obtained at concentration of 20 mg/ml (Table 4). The other researchers also reported low antifungal activity of essential oils against A. niger [32, 33]. Among major compounds, α-terpineol had the antifungal indices against A. niger, C. cladosporioides, C. globosum and P. citrinum higher than 1,8-cineole, β-caryophyllene and d-limonene; the fungal growths were completely inhibited at concentrations of 1 mg/ml on A. niger and C. cladosporioides and 5 mg/ml on C. globosum and P. citrinum, respectively (Table 5). IC50 values of M. leucadendron leaf oils against A. niger, C. cladosprioides, C. globosum and P. citrinum range from 10.24 to 10.97 mg/ml, 0.03 to 0.49 mg/ml, 0.06 to 0.15 mg/ml, and 5.84 to 8.70 mg/ml, respectively. IC50 value of each compound indicates that α-terpineol shows the best inhibitory effect on A. niger and P. citrinum, and d-limonene shows the best inhibitory effect on C. cladosporioides and C. globosum, but no effect against F. palustris (Table 6).
This study shows that essential oils of M. leucadendron are effective against F. palustris, T. versicolor, C. cladosporioides and C. globosum, but they are low effective against A. niger and P. citrinum. The antifungal effectiveness of the essential oils is probably due to the existence of α-terpineol. Several studies show that essential oil contained α-terpineol as major compound is effective as an antifungal agent [34, 35].
In conclusion, M. leucadendron leaf oils possess multi-effective properties, such as antioxidant, anti-hyaluronidase and antifungal activities. Each compound in M. leucadendron has different properties and biological activities. The effective components for each property are different, i.e., eugenol for antioxidant, β-caryophyllene for anti-hyaluronidase, α-terpineol for antifungal. Each property of the oils is not so strong compared to those of the individual commercial agents, but it is rather moderate that may be why these essential oils are widely utilized in many countries for expectorants, ointments and so on. The synergic effects for each biological activity might be found among several compounds, but they were not investigated this time. Combination of several compounds probably provides the other natural activities such as antiseptic, antifeedant, insecticide, sedative etc. Therefore, further studies are necessary to obtain the detailed information on the practical effectiveness of this essential oil for natural multi-purpose uses.
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The authors wish to thank Perum Perhutani Unit I, Central Java, Indonesia for providing the leaf samples for our study.
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Rini, P., Ohtani, Y. & Ichiura, H. Antioxidant, anti-hyaluronidase and antifungal activities of Melaleuca leucadendron Linn. leaf oils. J Wood Sci 58, 429–436 (2012). https://doi.org/10.1007/s10086-012-1270-x
- Melaleuca leucadendron Linn.
- Essential oil