http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–9 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2015.1037003

ORIGINAL ARTICLE

Chemical composition and biological activities of essential oil of Beilschmiedia pulverulenta Wan Mohd Nuzul Hakimi Wan Salleh1, Farediah Ahmad1, Khong Heng Yen2, and Razauden Mohamed Zulkifli3 Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia (UTM), Skudai, Johor, Malaysia, 2School of Chemistry and Environmental Studies, Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM) Kota Samarahan, Sarawak, Malaysia, and 3Department of Biosciences and Health Sciences, Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia (UTM), Johor, Malaysia

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Abstract

Keywords

Context: The ethnopharmacological study of Beilschmiedia indicates that several species are used for the treatment of various ailments. Objective: This is the first study of the chemical composition of Beilschmiedia pulverulenta Kosterm (Lauraceae) essential oil and its antioxidant, antimicrobial, antityrosinase, antiinflammatory, and anticholinesterase activities. Materials and methods: The antioxidant activities were evaluated by b-carotene, 1,1-diphenyl-2picrylhydrazyl (DPPH) radical scavenging, ferric-reducing antioxidant power (FRAP), and phenolic content at different concentrations. The antimicrobial activities against Gram-positive and Gram-negative bacteria and fungi were revealed by disk diffusion and microdilution. The antityrosinase and anti-inflammatory activities were assayed against mushroom tyrosinase and lipoxygenase enzymes. The anticholinesterase activity was analyzed using acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. Results: Forty-two components were detected in B. pulverulenta oil with eugenol (45.3%) being the major component. The oil phenolic content and the FRAP were 660.1 mg gallic acid/g and 604.0 mg ascorbic acid/g, respectively. The oil gave an IC50 value of 94.5 mg/mL and an inhibition of 93.9% in DPPH and b-carotene, respectively. The antimicrobial activity showed that the oil had strong activity against all Gram-positive bacteria with an minimum inhibitory concentration (MIC) value each of 62.5 mg/mL and moderate against all fungi with MIC and minimum bactericidal concentration (MBC) values each of 125 mg/mL. The oil showed significant antityrosinase and anti-inflammatory activities with 67.6 and 62.5% inhibition, respectively. In addition, the oil had moderate AChE (56.5%) and BChE (48.2%) activities. Discussion and conclusion: The results show that the oil could potentially be used for nutraceutical industries, food manufactures, and therapeutic agents against various diseases such as inflammation and rheumatism.

Anticholinesterase, anti-inflammatory, antimicrobial, antioxidant, antityrosinase

Introduction Lauraceae is a large, predominantly tropical family of aromatic evergreen trees and shrubs. It is distributed worldwide, most commonly found in the tropics of America and Southeast Asia. The habitat is the lowland rain forest, but some also occur at high elevation in tropical montane forest. The Lauraceae family comprises 30 genera and over 2000 species. About 213 species and 16 genera of Lauraceae family have been reported from the Malaysian region (Kochummen, 1989; Nishida, 2008). Beilschmiedia species are known to produce many types of phytochemicals such as terpenoids, endiandric, acid derivatives, essential oils, fatty acids, epoxyfuranoid lignans, flavonoids, and alkaloids. Several isolates have shown antioxidant, antibacterial, antimalarial, Correspondence: Farediah Ahmad, Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia (UTM), 81310 Skudai, Johor, Malaysia. Tel: +607 5534317. Fax: +607 5566162. E-mail: [email protected]

History Received 28 December 2014 Revised 5 March 2015 Accepted 22 March 2015 Published online 16 April 2015

cytotoxic, anti-inflammatory, and antituberculosis activities (Chen et al., 2006, 2007; Chouna, 2010; Huang et al., 2011; Lenta et al., 2009). Some Beilschmiedia species are used in traditional medicines for the treatment of uterine tumors, rheumatism, pulmonary disorders, and as appetite stimulants and spices (Iwu, 1993). Several studies on the essential oil compositions of Beilschmiedia species have been reported, including Beilschmiedia alloiophylla (Rusby) Kosterm, B. brenesii Hook.f., B. chancho blanco (Beilschmiedia sp. near brenesii), B. costaricensis (Mez & Pittier) C.K.Allen, B. erythrophloia Hayata, B. madang Blume, B. miersii (Gay) Kosterm., B. pendula (Sw.) Hemsl., B. tarairie (A.Cunn.) Kirk, and B. tilaranensis Sachiko Nishida (Chaverri & Ciccio, 2010; Kumamoto & Scora, 1970; Salleh et al., 2015; Scora & Scora, 2001; Setzer & Haber, 2007; Su & Ho, 2013). In continuation of our research on the Beilschmiedia species, this is the first report on the chemical compositions and biological activities of the essential oil of Beilschmiedia pulverulenta Kosterm. The biological activities include antioxidant,

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antimicrobial, antityrosinase, anticholinesterase.

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anti-inflammatory,

and

Materials and methods Plant material A sample of B. pulverulenta was collected from mixed dipterocarp forests located in Kuching and Samarahan Districts of Sarawak in January 2010 and identified by Mohizar Mohamad. The voucher specimen (UiTMKS 4014) was deposited at the Natural Product Research and Development Center (NPRDC), Universiti Teknologi MARA Sarawak.

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Solvents and chemicals Antioxidant: b-Carotene, linoleic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH), gallic acid, ascorbic acid, and butylated hydroxytoluene (BHT) were obtained from Sigma-Aldrich (Munich, Germany). Folin–Ciocalteu’s reagent, anhydrous sodium sulfate, sodium carbonate, polyoxyethylene sorbitan monopalmitate (Tween-40), 2,4,6-tripyridyl-s-triazine (TPTZ), iron(III) chloride hexahydrate, 2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and potassium persulfate were purchased from Merck (Darmstadt, Germany). All other chemicals and solvents were of analytical grade. Antimicrobial: Nutrient agar, nutrient broth, sabouraud dextrose agar and sabouraud dextrose broth, stretopmycin sulfate, and nystatin were purchased from Oxoid (Milan, Italy). All tested microorganisms were purchased from Mutiara Scientific (Kuala Lumpur, Malaysia). Antityrosinase: Mushroom tyrosinase enzyme (EC1.14.18.1), kojic acid and L-DOPA were purchased from Sigma-Aldrich (Munich, Germany). Anti-inflammatory: Lipoxygenase inhibitor screening assay kit (item no. 760700) was purchased from Cayman Chemical Company (Ann Arbor, MI). Anticholinesterase: Acetylcholinesterase (Type-VI-S, EC3.1.1.7), butyrylcholinesterase (EC3.1.1.8), acetylthiocholine iodide, butyrylthiocholine chloride, 5,50 -dithio-bis(2-nitrobenzoic)acid (DTNB), and galantamine were purchased from Sigma-Aldrich (Munich, Germany). Extraction and analysis of essential oil Oil preparation The dried aerial parts (leaf and stem bark) were subjected to hydrodistillation in an all glass Dean-stark apparatus (SigmaAldrich, Munich, Germany) for 8 h. The oil obtained was dried over anhydrous magnesium sulfate and stored at 4–6  C. The oil yield (w/w) was calculated based on fresh weight basis. Gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS) GC analysis was performed on a Hewlett Packard 6890 series II A gas chromatograph equipped with an Ultra-1 column (Hewlett-Packard Company, Palo Alto, CA) (100% polymethylsiloxanes) (25 m long, 0.33 mm thickness, and 0.20 mm inner diameter). Helium was used as a carrier gas at a flow rate of 0.7 mL/min. Injector and detector temperatures were

set at 250 and 280  C, respectively. Oven temperature was kept at 50  C, then gradually raised to 280  C at 5  C/min and finally held isothermally for 15 min. Diluted samples (1/100 in diethyl ether, v/v) of 1.0 mL were injected manually (split ratio 50:1). The injection was repeated three times and the peak area percentage was reported as means ± SD of triplicates. The calculation of peak area percentage was carried out by using the GC HP Chemstation software (Agilent Technologies, Santa Clara, CA). GC–MS chromatograms were recorded using a Hewlett Packard Model 5890A gas chromatography and a Hewlett Packard Model 5989A mass spectrometer. The GC was equipped with Ultra-1 column (25 m long, 0.33 mm thickness, and 0.20 mm inner diameter). Helium was used as a carrier gas at a flow rate of 1 mL/min. Injector temperature was 250  C. Oven temperature was programmed from 50  C (5 min hold) at 10  C/min to 250  C and finally held isothermally for 15 min. For GC–MS detection, an electron ionization system with an ionization energy of 70 eV was used. A scan rate of 0.5 s (cycle time: 0.2 s) was applied, covering a mass range from 50 to 400 amu. Identification of components The chemical components of the oil were identified by comparing their mass spectra with reference spectra in the computer library (Wiley, Hoboken, NJ) and also by comparing their Kovat’s indices with those of authentic compounds or data in the literature (Adams, 2001). The quantitative data were obtained electronically from FID area without the use of correction factor. Biological activities – antioxidant activity -Carotene–linoleic acid bleaching assay The b-carotene–linoleic acid bleaching assay was determined by following the method described by Miraliakbari and Shahidi (2008). A mixture of b-carotene and linoleic acid was prepared by adding together 0.5 mg b-carotene in 1 mL CHCl3, 25 mL linoleic acid, and 200 mg Tween-40. CHCl3 was then completely evaporated under vacuum and 100 mL of oxygenated distilled water was subsequently added to the residue and mixed gently to form a clear yellowish emulsion. The essential oils and BHT (positive control) were dissolved individually in MeOH (2 g/L) and 350 mL volumes of each of them were added to 2.5 mL of the above emulsion in test tubes and mixed thoroughly. The test tubes were incubated in a water bath at 50  C for 2 h, together with a negative control (blank) that contained the same volume of MeOH. The absorbance values were measured at 470 nm on a UV–Vis spectrophotometer. The percentage inhibition (I%) of the essential oil was calculated using the following equation:   A carotene after 2h I% ¼ 100 Ainitial carotene where Ab-carotene after 2h is the absorbance value of b-carotene after 2 h assay remaining in the samples and Ainitial b-carotene is the absorbance value of b-carotene at the beginning of the experiments. All tests were carried out in triplicate and the

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DOI: 10.3109/13880209.2015.1037003

Chemical composition and biological activity of B. pulverulenta

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inhibition percentages were reported as mean ± SD of triplicates.

standard curves prepared with known concentrations of ascorbic acid (AA) and were expressed as mg AA/g.

DPPH radical scavenging assay

Antimicrobial activity

The free radical scavenging activity was determined using DPPH as previously described (Shimada et al., 1992). DPPH was first dissolved in EtOH to a concentration of 0.1 mM and a solution of DPPH (1 mL) was added to an EtOH solution (3 mL) of the tested samples at different concentrations (200, 150, 100, 50, and 25 mg/mL). An equal volume of EtOH was added in the control test. The mixture was shaken vigorously and allowed to stand at room temperature for 30 min. Then the absorbance at 517 nm was measured with a UV–Vis spectrophotometer. The percentage of inhibition (I%) of DPPH radical was calculated as follows:

Microbial strains

  Ablank  Asample I% ¼ 100, Ablank

Nine microorganisms; three Gram-positive bacteria: Bacillus subtilis (ATCC6633), Staphylococcus aureus (ATCC29737), and Enterococcus faecalis (ATCC19433); three Gram-negative bacteria: Pseudomonas aeruginosa (ATCC9027), Escherichia coli (ATCC10536), and Klebsiella pneumonia (ATCC13883), and three fungal/yeast: Aspergilus niger (ATCC16888), Candida albicans (ATCC10231) and Saccharomyces cerevisiae (ATCC7754), were used. Strains were grown on nutrient agar (NA) for bacteria and sabouraud dextrose agar (SDA) for fungal/yeast. For the activity tests, nutrient broth (NB) for bacteria and sabouraud dextrose broth (SDB) for fungal/yeast strains were used. Disk diffusion

where Ablank is the absorbance value of the control reaction (containing all reagents except the sample) and Asample is the absorbance value of the test sample. The sample concentration providing 50% inhibition (IC50 value) was calculated by plotting inhibition percentages against concentrations of the sample. All tests were carried out in triplicate and IC50 values were reported as mean ± SD of triplicates. Total phenolic content The total phenolic content (TPC) was determined according to the method described by Slinkard and Singleton (1977) with minor modifications. A sample of stock solution (1.0 mg/ mL) was diluted in MeOH to final concentrations of 1000, 800, 600, 400, and 200 mg/mL. A 0.1 mL aliquot of sample was pipetted into a test tube containing 0.9 mL of MeOH, then 0.05 mL Folin–Ciocalteu’s reagent was added, and the flask was shaken thoroughly. After 3 min, 0.5 mL of 5% Na2CO3 solution was added and the mixture was allowed to stand for 2 h with intermittent shaking. Then, 2.5 mL of MeOH was added and left to stand in the dark for 1 h. The absorbance measurements were recorded at 765 nm. The same procedure was repeated for the standard gallic acid solution. The concentration of TPC in the oils was expressed as milligram of gallic acid equivalent per gram of sample. Tests were carried out in triplicate and the gallic acid equivalent value was reported as mean ± SD of triplicates. FRAP assay The total reducing capacity was determined using FRAP assay (Benzie & Strain, 1996). The stock solutions included 300 mM acetate buffer pH 3.6, 10 mM TPTZ solution in 40 mM HCl, and 20 mM FeCl36H2O solution. The fresh working solution was prepared by mixing 25 mL acetate buffer, 2.5 mL TPTZ, and 2.5 mL FeCl36H2O. The temperature of the solution was raised to 37  C prior to use. The essential oil (150 mL) was allowed to react with 2850 mL of the FRAP solution for 30 min in the dark condition. After incubation, the absorbance was read at 593 nm using a UV– vis spectrophotometer. The results were calculated by

Antimicrobial tests were determined by the disk diffusion method described by Gulluce et al. (2004) with slight modifications as described by Salleh et al. (2011). Briefly, 400 mL suspension containing 108 CFU/mL bacteria and 106 CFU/mL fungal/yeast were spread on NA and SDA mediums, respectively. The essential oil was dissolved in DMSO (4 mg/mL). The discs (6 mm diameter) were impregnated with 10 mL essential oil and DMSO (negative control) and the placed on the inoculated agar. The inoculated plates were incubated for 24 h at 37  C (bacterial) and 48 h at 30  C (fungal/yeast). Streptomycin sulfate (10 mg/mL) and nystatin (100 IU) were used as the positive controls for bacteria and fungi/yeast, respectively. Clear inhibition zones around the discs indicated the presence of antibacterial/antifungal activity. All tests and analysis were carried out in triplicate. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) The MIC and MBC were determined by the broth microdilution method using 96-well microplates described by Gulluce et al. (2004) with modifications stated by Salleh et al. (2011). The inocula of the microbial strains were prepared from 24 h broth cultures and suspensions were adjusted to 0.5 McFarland standard turbidity. The essential oil was dissolved in DMSO to produce 1000 mg/mL stock solution. A number of wells (A–H) were reserved in each plate for positive and negative controls. Sterile broth (100 mL) was added to the wells from rows B to H. The stock solutions of samples (100 mL) were added to the wells at rows A and B. Then, the mixture of samples and sterile broth (100 mL) at row B were transferred to each well in order to obtain a two-fold serial dilution of the stock samples (concentration of 1000, 500, 250, 125, 62.5, 31.3, 15.6, and 7.8 mg/mL). The inoculum (100 mL) was added to each well. The final volume in each well was 200 mL. Streptomycin sulfate for bacteria and nystatin for fungal/yeast were used as positive controls. The plates were incubated at 37  C for 24 h. Microbial growth was indicated by the presence of turbidity and a pellet at the bottom of the well. Samples from the MIC study which did

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not show any growth of bacteria were removed from each well (10 mL) and then subculture on the surface of the freshly prepared nutrient agar in disposable Petri dishes (50 mm  15 mm). Then, the Petri dishes were inverted and incubated for 16–20 h at 37  C. After 16–20 h, the number of surviving organisms was determined.

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Antityrosinase activity Tyrosinase inhibition assay was determined according to the method reported by Masuda et al. (2005) with slight modifications. Briefly, the essential oil and kojic acid were dissolved in DMSO prepared in the amount of 1 mg/mL. The essential oil (40 mL) dissolved in DMSO with 80 mL phosphate buffer (pH 6.8), 40 mL tyrosinase enzyme, and 40 mL L-DOPA were placed in each well. Each sample was accompanied by a blank that contained all the components except L-DOPA. Kojic acid was used as a reference standard inhibitor for comparison. The reaction was carried out using a 96-well microplate and a microplate reader (Epoch MicroVolume Spectrophotometer, Bio-Tek, Winooski, VT) was used to measure the absorbance at 475 nm. The percentage of tyrosinase inhibition (I%) was calculated as follows:   Acontrol Asample I% ¼ 100 Acontrol where Acontrol is the absorbance of the control reaction and Asample is the absorbance of the essential oil/reference. Analyses were expressed as mean ± SD of triplicates. Anti-inflammatory activity The reagents were prepared according to the standard protocol (Lipoxygenase inhibitor screening assay kit, item no. 760700 Cayman Chemicals Co., Ann Arbor, MI). Stock solutions of essential oil were prepared to obtain the concentrations of 100, 50, 25, 12.5, and 6.25 mM in the respective wells. The prepared solutions were then introduced onto 96-well plates where the cells were distributed as blank 1A–2A–1D (triplicate), positive control 1B–2B (duplicate), and 100% initial activity wells 1C–2C–2D (triplicate). The remaining wells were designated for inhibitor (essential oil) solutions in duplicate. The addition of the reagents was done according to the standard protocol, in which 100 mL assay buffer was added to the blank wells, whereas 90 mL lipoxygenase (5-LOX) enzyme and 10 mL assay buffer was added to the positive control wells. About 90 mL lipoxygenase enzyme and 10 mL solvent (DMSO) were added to wells with 100% initial activity. The inhibitor (essential oil) wells were charged with 90 mL lipoxygenase enzyme and 10 mL respective stock (essential oil) solutions. The reaction was initiated by adding 10 mL of the substrate (AA) to all wells. The plate was then shaken for 5 min on an orbital shaker. Ultimately, 100 mL of chromogen solution (prepared according to the standard protocol) was added to each well to stop enzyme catalysis. The plate was incubated for 30 min and was read at 500 nm. The percentage of inhibition (I%) of the essential oil was calculated using the following equation:   Ainitial activity Ainhibitor I% ¼ 100 Ainitial activity

Pharm Biol, Early Online: 1–9

where Ainitial activity is the absorbance of 100% initial activity wells without sample and Ainhibitor is the absorbance of essential oil/reference. Analyses were expressed as mean ± SD of triplicates. Anticholinesterase activity AChE/BChE inhibitory activity of the essential oil was measured by slightly modifying the spectrophotometric method developed by Ellman et al. (1961) and Orhan et al. (2008). Briefly, 140 mL sodium phosphate buffer (pH 8.0), 20 mL DTNB, 20 mL essential oil (concentration of 1000 mg/mL), and 20 mL AChE/BChE solution were added by a multichannel automatic pipette in a 96-well microplate and incubated for 15 min at 25  C. The reaction was then initiated with the addition of 10 mL acetylthiocholine iodide/ butyrylthiocholine chloride. The hydrolysis of acetylthiocholine iodide/butyrylthiocholine chloride was monitored by the formation of yellow 5-thio-2-nitrobenzoate anion from the reaction of DTNB with thiocholine, catalyzed by enzymes at 412 nm, and utilizing a 96-well microplate reader (Epoch Micro-Volume Spectrophotometer, Bio-Tek, Winooski, VT). Galantamine was used as a reference. The percentage inhibition (I%) of AChE/BChE was determined by comparing the rates of reaction of samples relative to the blank sample (EtOH in phosphate buffer pH 8) using the following formula:   ES I% ¼ 100 E where E is the activity of enzyme without test sample and S is the activity of enzyme with test sample. Analyses were expressed as mean ± SD of triplicates. Statistical analysis Data obtained from essential oil analysis and biological activities were expressed as mean values. The statistical analyses were carried out by employing one-way ANOVA (p40.05). A statistical package (SPSS version 11.0, SPSS Inc., Chicago, IL) was used for data analysis.

Results and discussion Chemical compositions of essential oil The essential oil was obtained by hydrodistillation of air-dried aerial parts (leaf and stem bark) with a yield of 2.85 g (w/w) (0.0095%), as a pale yellow liquid. Analysis of GC and GC–MS (Figure 1) of this oil has successfully identified 42 components accounted for 93.8% of the total oil. Table 1 shows the chemical compositions of B. pulverulenta oil along with their retention indices in the GC capillary column, Ultra1 (100% polymethylsiloxsane). The identified compounds were four phenylpropanoids and 34 sesquiterpenes. Phenylpropanoid was the most dominant group in the oil of B. pulverulenta amounting to 51.1% with eugenol (45.3%) and eugenol acetate (5.6%) as the main components. Other phenylpropanoids were dihydroeugenol (0.1%) and dihydroeugenol acetate (0.1%). The sesquiterpenes components consisted of 22 sesquiterpene hydrocarbons (25.0%) and 12 oxygenated sesquiterpenes (16.0%). Many sesquiterpenes were present in a minute quantity in the oil. Those present in

Chemical composition and biological activity of B. pulverulenta

DOI: 10.3109/13880209.2015.1037003

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Abundance TIC: 1527.D

8000000

19.38

23.19

7000000 6000000 5000000

24.79 24.72

4000000 3000000 20.15

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2000000

26.76 26.23 25.90 26.17 24.59 26.42 23.48 23.62 24.91 26.36 22.52 22.7023.89 24.04 23.37 22.96

32.24 30.64 41.33

1000000 0 18.00

20.00

22.00

24.00

26.00

28.00

30.00

32.00

34.00

36.00

38.00

40.00

42.00

Time-->

Figure 1. GC-MS chromatogram of the essential oil of B. pulverulenta.

more than 2.0% were a-ylangene (3.6%), cadalene (3.2%), t-muurolol (2.5%), caryophyllene oxide (2.3%), spathulenol (2.2%), aromadendrene (2.0%), and caryophylla-4(12),8(13)dien-5b-ol (2.0%). However, the groups of monoterpenes were not detected in this oil. Eugenol has been identified as the major and most common phenylpropanoid present in some species of Lauraceae family. Among them are Cinnamomum sintoc Blume (Indonesia: stem barks 38.8%), C. illicioides A.Chev. (Vietnam: bark 41.2%), C. zeylanicum Blume (Sri Lanka: leaf 74.9%), C. verum J.Presl (Fiji: leaf 86.0%), and C. tamala Nees et Eberm (Bangladesh: leaf 77.3%) (Chowdhury et al., 1981; Giang et al., 2006; Iskandar & Supriyatna, 2008; Schmidt et al., 2006; Patel et al., 2007). Pharmacological studies show that eugenol demonstrated anesthetic, hypothermic, muscle-relaxant, anti-stress, and anticonvulsant activities (Dallmeier & Carlini, 1981). Antioxidant activity Antioxidant activity of the essential oil was determined by measuring the scavenging activity on DPPH free radicals, b-carotene-linoleic acid bleaching, FRAP, and TPC. The results are shown in Table 2. In DPPH scavenging assay, the antioxidant activity was measured by the decrease in the absorbance as the DPPH radical received an electron or hydrogen radical from an antioxidant compound to become a stable diamagnetic molecule (Juntachote & Berghofer, 2005). The essential oil revealed low activity with an IC50 value of 94.5 mg/mL compared with the standard, BHT (IC50 value 18.5 mg/mL). This finding suggests that the antioxidant activity of the essential oil is not only due to the presence of eugenol, but other constituents also have a significant effect on the activity. The b-carotene-linoleic acid bleaching assay measured the ability of an antioxidant to inhibit lipid peroxidation. In this assay, a model system made of

b-carotene and linoleic acid undergoes rapid discoloration in the absence of an antioxidant. The free linoleic acid radical formed upon the abstraction of a hydrogen atom from one of its methylene groups that attacked the b-carotene molecules, which lost the double bonds and subsequently, its characteristic (orange color) (Juntachote & Berghofer, 2005). The essential oil showed inhibition of 93.9%, which was comparable with that of BHT (95.2%). Compounds containing hydrogen atoms (eugenol) in the allylic and/or benzylic positions give better activity in this test due to relatively easy abstraction of hydrogen atom from these functional groups by peroxy radicals formed in the test circumstances (Ebrahimabadi et al., 2010). The results of phenolic content assay showed that the essential oil possessed greater antioxidative properties with the value of 660.1 mg gallic acid/g (GA/g), and this is probably due to the high percentage of eugenol as the major component in the oil. The current results are found to be correlated with previous findings that mentioned the strong antioxidant activity of eugenol (Gulcin, 2011; Ito et al., 2005). The FRAP assay measured the ability of an antioxidant to donate electron to Fe(III), resulting in the reduction of Fe3+/ferricyanide complex to Fe2+ complex. The results were expressed as mg ascorbic acid equivalent (mg AA/g). The essential oil gave 604.0 mg AA/g, which seemed to be related to the presence of high percentage of phenolic. According to previous studies, a significant correlation between FRAP values and TPC of the essential oils has been observed (Politeo et al., 2010). Eugenol, known as a common antioxidant, is widely used as a flavoring agent in cosmetic and food products, and it is also utilized as an antiseptic drug in dentistry. Structure–activity relationship studies on eugenol revealed that the side-chain structure in addition to the phenolic ring has an important role in antioxidant function (Tai et al., 2002).

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Table 1. Chemical components identified from the essential oil of B. pulverulenta.

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Components a-Cubebene Eugenol Dihydro eugenol a-Ylangene a-Copaene iso-Longifolene (Z)-Jasmone g-Gurjunene Longifolene a-Caryophyllene b-Caryophyllene Aromadendrene Dihydro aromadendrene ar-Curcumene a-Amorphene b-Selinene Cadina-1,4-diene a-Selinene a-Muurolene (E,Z)-a-Farnesene d-Cadinene Eugenol acetate cis-Calamenene Dihydro eugenol acetate a-Calacorene Germacrene B (E)-Nerolidol Palustrol Spathulenol Caryophyllene oxide Viridiflorol b-Oplopenone Caryophylla-4(12),8(13)-dien-5b-ol allo-aromadendrene epoxide t-Muurolol a-Cadinol Cadalene (5E,9E)-Farnesyl acetone Methyl hexadecanoate Phytol Hexadecanoic acid Methyl linoleate Group components Phenylpropanoids Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Others Identified components (%)

KIa 1345 1356 1367 1372 1374 1390 1395 1402 1405 1408 1415 1445 1462 1482 1483 1490 1495 1495 1502 1510 1522 1525 1528 1536 1545 1560 1562 1565 1580 1585 1595 1602 1635 1640 1646 1653 1675 1915 1924 1940 1960 2094

KIb 1345 1355 1365 1372 1374 1390 1395 1401 1407 1408 1417 1440 1460 1480 1483 1490 1495 1498 1502 1508 1520 1525 1528 1535 1545 1560 1562 1567 1578 1585 1595 1605 1639 1640 1645 1655 1676 1913 1921 1942 1959 2095

Table 2. Antioxidant activity of the essential oil of B. pulverulenta.

Percentage (%) 1.8 45.3 0.1 3.6 0.8 1.0 0.6 1.4 0.7 0.2 0.2 2.0 1.2 0.4 1.5 0.7 0.2 1.6 0.2 0.4 1.2 5.6 1.3 0.1 0.8 0.6 1.8 0.9 2.2 2.3 0.2 0.5 2.0 1.1 2.5 1.6 3.2 0.3 1.0 0.4 0.2 0.1 51.1 – – 25.0 16.0 1.7 93.8

a

Kovats index experimental. Kovats index from literature (Adams, 2001). Bold value refer to the major components.

b

Antimicrobial activity The antimicrobial activity of the investigated essential oil is shown in Table 3. The results revealed that the essential oil showed variable levels of antimicrobial activity against all tested bacterial/fungal strains. The essential oil displayed good antibacterial activity against Gram-positive bacteria, B. subtilis, S. aureus, and E. faecalis, each with MIC and MBC values of 62.5 and 125 mg/mL, respectively. Moderate activity was observed on fungal strains A. niger, C. albicans, and S. cerevisiae, each with an MIC and MBC value of 125 mg/mL. According to several authors (Kokoska et al.,

Samples Essential oil BHT AA

b-Carotene/ linoleic acid (I%)

DPPH IC50 (mg/mL)

TPC (mg GA/g)

FRAP (mg AA/g)

93.9 ± 0.2 95.2 ± 0.3 –

94.5 18.5 25.8

660.1 ± 0.2 – –

604.0 ± 0.3 – –

AA, ascorbic acid; BHT, butylated hydroxytoluene; GA, gallic acid.

2002; Yayli et al., 2005), Gram-negative bacteria appeared to be less sensitive to the action of many other plant essential oils such as from Centaurea species. This higher resistance among Gram-negative bacteria could be due to the highly hydrophilic cell membrane of this bacterial group, whereas the cell membrane of Gram-positive bacteria may facilitate penetration by hydrophobic compounds. This antimicrobial activity can be explained by the higher amounts of eugenol (45.3%) in the essential oil. The presence of eugenol present in essential oils of many plants have been proven to be active against various pathogens such as E. coli, Bacillus cereus, Helicobacter pylori, S. aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, and Streptococcus pyogenes (Ali et al., 2005; Leite et al., 2007; van Zyl et al., 2006). Synergistic effect of eugenol with other minor components may also contribute to the significant antimicrobial activity of the essential oil (Hemaiswarya & Doble, 2009). Antityrosinase activity Table 4 shows the antityrosinase, anti-inflammatory, and anticholinesterase activities of the essential oil of B. pulverulenta. The essential oil inhibited antityrosinase activity by the oxidation of L-DOPA catalyzed by mushroom tyrosinase enzyme. The enzyme activity was not suppressed but rather decreased rapidly. At a concentration of 1000 mg/ mL, the essential oil showed inhibitory activity of mushroom tyrosinase of 67.6%. The inhibition of the tyrosinase activity of kojic acid was determined to be 84.0%, which was found to be significantly more pronounced than the essential oil. Several studies have demonstrated cinnamaldehyde, anisaldehyde, benzaldehyde, geranial, and neral as potent competitive inhibitors of mushroom tyrosinase (Chang et al., 2013). The existence of these components will increase the activity. Meanwhile, eugenol has also been found to be the major component in other plant essential oils and exhibited potent tyrosinase activity as shown by the essential oils of Piper betle Linn. (Piperaceae) (an IC50 value of 126 ppm) (Row & Ho, 2009), Eugenia caryophyllata Thunb. (Myrtaceae) (an IC50 value of 9.6 mg/mL) (Watcharee et al., 2012), and Cinnamomum zeylanicum Blume (Lauraceae) (an IC50 value of 18.4 mg/mL) (Chericoni et al., 2005). Anti-inflammatory activity In anti-inflammatory activity, the essential oil showed moderate activity on lipoxygenase assay with an inhibition of 62.5%. Quercetin was used as the standard reference with an inhibition of 81.9%. The inhibition activity may be attributed to the presence of eugenol, which is known as the LOX inhibitor. Leem et al. (2011) showed that the essential

Chemical composition and biological activity of B. pulverulenta

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Table 3. Antimicrobial activity of the essential oil of B. pulverulenta. EO Samples/microbes Gram-positive Bacillus subtilis Staphylococcus aureus Enterococcus faecalis Gram-negative Pseudomonas aeruginosa Escherichia coli Klebsiella pneumoniae Fungal/yeast Aspergillus niger Candida albicans Saccharomyces cerevisiae

SS

NYS

DD

MIC

MBC

DD

MIC

MBC

DD

MIC

MBC

10.5 10.2 10.0

62.5 62.5 62.5

125 125 125

15.3 15.5 15.5

7.8 7.8 7.8

7.8 7.8 7.8

– – –

– – –

– – –

9.5 9.7 9.8

250 250 250

250 250 500

15.2 15.8 15.4

7.8 7.8 7.8

7.8 7.8 7.8

– – –

– – –

– – –

9.6 9.2 9.5

125 125 125

125 125 125

– – –

– – –

– – –

15.8 15.5 15.2

7.8 7.8 7.8

7.8 7.8 7.8

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DD, disk diffusion (zone of inhibition including the diameter of disc: 6 mm); MIC, minimum inhibitory concentration (mg/mL); MBC, minimum bactericidal concentration (mg/mL); EO, essential oil; SS, streptomycin sulfate; NYS, nystatin.

Table 4. Antityrosinase, anti-inflammatory, and anticholinesterase activities of the essential oil of B. pulverulenta. Anticholinesterase (I%)

Samples Essential oil Kojic acid Quercetin Galantamine

AntiAntityrosinase inflammatory (I%) (I%) 67.6 ± 0.4 84.0 ± 0.2 – –

62.5 ± 0.2 – 81.9 ± 0.2 –

AChE

BChE

56.5 ± 0.3 – – 95.9 ± 0.2

48.2 ± 0.3 – – 88.7 ± 0.2

AChE, acetylcholinesterase; BChE, butyrylcholinesterase

oil of Eugenia caryophyllata, which contains eugenol as the main constituent, exhibited strong inhibitory activity against COX-2 and 15-LOX enzymes. Moreover, eugenol derivatives have been evaluated as potential inhibitors of 15-LOX through molecular docking studies (Sadeghian et al., 2008). Previous studies have been carried out on the anti-inflammatory activity of eugenol with different methods and mechanisms, and showed a significant activity. These include croton oil which induced edema (Dohi et al., 1989), tooth pulp microsomes and homogenates, leukocyte, kidney medulla (Anamura, 1989), IL-1b-stimulated gingival fibroblast (Koh et al., 2013), and carrageenan-induced paw edema (Saeed et al., 1995). Generally, inflammation involves the formation of both prostaglandins and leukotrienes as mediators followed by the liberation of neutrophils and the production of reactive oxygen species (ROS). The inhibition of COX-II is normally used as a criterion for anti-inflammatory activity. This will lead to the dismissal of other potential lead compounds, which might serve as good inhibitors of 5-LOX and other inflammatory mediators as NFkB, ROS, and many others (Schmitz & Bacher, 2005). Anticholinesterase activity The anticholinesterase activities of the essential oil of B. pulverulenta were compared with that of galantamine, which is used as a standard drug against Alzheimer’s disease. The essential oil showed AChE and BChE inhibitory activities of 56.5 and 48.2%, respectively. These results can

be explained by the presence of eugenol in the oil, which has been reported to inhibit AChE/BChE in vitro (Dohi et al., 2009), besides the fact that the presence of phenylpropanoids in the oil may act synergistically to inhibit AChE (Savelev et al., 2003). The results clearly indicate that some structural features are important for biological activity. The presence of a conjugated double bond and a hydroxyl group is an important criterion for the activity. The anticholinesterase activity of these phenylpropanoids can be explained by hydrophobic interactions between hydrophobic active site of AChE and hydrocarbon skeleton of the compounds (Mukherjee et al., 2007).

Conclusion These studies revealed the antioxidant, antimicrobial, antityrosinase, anti-inflammatory, and anticholinesterase activities of essential oil of B. pulverulenta. The essential oil and its active constituents, such as eugenol, are promising sources of bioactivity substances, and the data presented in this study highlight the potential roles that phenylpropanoids can contribute to this field. Therefore, the essential oil could potentially be employed as effective skin-whitening agents and as antioxidants for future development of complementary and alternative medicine-based aromatherapy. To validate the above-mentioned activities, clinical trials should be carried out in order to ensure a safe use of the essential oil and their major components as therapeutic agents against various diseases.

Declaration of interest The authors report that they have no conflicts of interest. The authors would like to thank the Ministry of Science, Technology and Innovation Malaysia for the financial support under Vot Q.J130000.2526.03H93 and the Faculty of Science, Universiti Teknologi Malaysia for the research facilities.

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Chemical composition and biological activities of essential oil of Beilschmiedia pulverulenta.

The ethnopharmacological study of Beilschmiedia indicates that several species are used for the treatment of various ailments...
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