ijms International Journal of Molecular Sciences Int. J. Mol. Sci. 1422-0067 Molecular Diversity Preservation International (MDPI) 10.3390/ijms12117720 ijms-12-07720 Article Chemical Compositions, Antioxidant and Antimicrobial Activities of Essential Oils of Piper caninum Blume SallehWan Mohd Nuzul Hakimi Wan1 AhmadFarediah1* YenKhong Heng2 SiratHasnah Mohd1 Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia (UTM), Skudai, Johor 81310, Malaysia; E-Mails: nuzoule208@yahoo.com (W.M.N.H.W.S.); hasnah@kimia.fs.utm.my (H.M.S.) School of Chemistry and Environment Studies, Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM) Sarawak, Kota Samarahan, Sarawak 94300, Malaysia; E-Mail: khonghy@sarawak.uitm.edu.my Author to whom correspondence should be addressed; E-Mail: farediah@kimia.fs.utm.my; Tel.: +607-5534317; Fax: +6-075566162. 2011 08 11 2011 12 11 7720 7731 12 10 2011 28 10 2011 02 11 2011 © 2011 by the authors; licensee MDPI, Basel, Switzerland. 2011

This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

Chemical composition, antioxidant and antimicrobial activities of the fresh leaves and stems oils of Piper caninum were investigated. A total of forty eight constituents were identified in the leaves (77.9%) and stems (87.0%) oil which were characterized by high proportions of phenylpropanoid, safrole with 17.1% for leaves and 25.5% for stems oil. Antioxidant activities were evaluated by using β-carotene/linoleic acid bleaching, DPPH radical scavenging and total phenolic content. Stems oil showed the highest inhibitory activity towards lipid peroxidation (114.9 ± 0.9%), compared to BHT (95.5 ± 0.5%), while leaves oil showed significant total phenolic content (27.4 ± 0.5 mg GA/g) equivalent to gallic acid. However, the essential oils showed weak activity towards DPPH free-radical scavenging. Evaluation of antimicrobial activity revealed that both oils exhibited strong activity against all bacteria strains with MIC values in the range 62.5 to 250 μg/mL, but weak activity against fungal strains. These findings suggest that the essential oils can be used as antioxidant and antimicrobial agents for therapeutic, nutraceutical industries and food manufactures.

 essential oil Piperaceae Piper caninum antioxidant antimicrobial 1. Introduction

The genus Piper is one of the largest and the most important aromatic and medicinal plants of the Piperaceae family which comprises four genera and approximately 2000 species distributed in the tropical and subtropical regions [1,2]. Piper species are used in traditional remedies and folklore medicine all over the world for their antioxidant [3–5], antimicrobial [6–8], anti-inflammatory [9–12] and antifungal [13–15] activities.

Piper caninum Bl., locally known as “cabai hutan” or “lada hantu” in Malaysia, is found throughout the tropic mostly in South East Asia such as Thailand and Malaysia. It grows as climber on a small tree in lowland and upland rain forest. The leaves are chartaceous, undersurface glaucous, upper surface green when fresh and turn black when dry. The flowers are very small, about 0.6–0.8 mm diameter, consisting of a petal scale and stamens. The fruits are inflorescence axillary with orange color and have a smell like aniseed. The leaves are used by peoples for chewing. They are added to the betel-quid for treating hoarseness. Besides, it is also used after childbirth [16,17].

To the best of our knowledge, there is no report on the chemical compositions and bioactivity studies of the essential oil of P. caninum. So, here we would like to report the chemical composition, antioxidant and antimicrobial activities of the leaves and stems oils of P. caninum.

 2. Results and Discussion 2.1. Composition of the Essential Oils

The essential oils, pale yellow color were obtained by hydrodistillation of the leaves and stems of P. caninum are presented in Table 1. The chemical compositions of the leaves and stems oils were analyzed by GC, GC-MS and Kovats Indices.

Thirty six components (77.9%) and thirty seven components (87.0%) were identified from the leaves and stems oil, respectively. The leaves oil consists of two phenylpropanoids (18.0%), eleven monoterpenes (29.3%) and nineteen sesquiterpenes (26.7%). The main fraction in the leaves oils were sesquiterpenes hydrocarbons (21.9%) and monoterpene hydrocarbons (21.8%). The most abundant components in the leaves oil were safrole (17.1%), β-pinene (8.9%), linalool (7.0%) and β-caryophyllene (6.7%). In addition, germacrene D (4.9%), α-pinene (4.0%), (E)-nerolidol (3.9%), (Z)-β-ocimene (3.4%), β-elemene (2.1%) and 2-undecanone (2.0%) were also detected in the leaves oil. On the other hand, the stems oil contains two phenylpropanoids (27.9%), nine monoterpenes (14.7%) and twenty three sesquiterpenes (42.1%). The sesquiterpenes hydrocarbon made up a major fraction in the stems oil which constituted 35.2%. The major components in the stems oils were safrole (25.5%), β-caryophyllene (9.8%), germacrene D (7.8%), β-pinene (4.9%) and δ-elemene (4.1%). Besides, those present in more than 2% were linalool (2.9%), limonene (2.7%), eugenol (2.4%), t-muurolol (2.4%) and bicyclogermacrene (2.3%).

Most of the oil components were similar in both oils. However, camphor (0.3%), α-cubebene (0.5%), eugenol (2.4%), β-cubebene (0.3%), aromadendrene (0.8%), allo-aromadendrene (0.3%), α-bisabolene (0.4%), germacrene B (1.1%), globulol (0.3%), t-muurolol (2.4%), α-cadinol (1.0%) and farnesyl acetate (1.2%) were found to be absent in the leaves oil. In addition, myrcene (0.9%), γ-terpinene (0.1%), α-terpinolene (0.4%), 2-nonanone (1.2%), pinocarvone (0.2%), cyclosativene (0.2%), α-ylangene (0.2%), β-bourbonene (1.1%), β-gurjunene (0.2%), (E,E)-α-farnesene (0.9%), chavibetol (0.9%), elemol (0.2%) and β-eudesmol (0.9%) were absent in the stems oil.

Similar to our results, safrole has been reported to be the major constituents of Piper auritum H.B.K. (Panama: leaves oil, 70%) [18], Piper callosum Ruiz & Pav. (Brazil: leaves oil, 70%), Piper hispidinervium C. DC. (Brazil: leaves oil, 81–88%) [19], Piper betle Linn. flowers oil (Taiwan: flowers oil, 27.6%) [20], Piper mikanianum Kunth (Brazil: leaves oil, 82%) [21] and Piper xylosteoides C. DC. (Brazil: aerial parts, 47.83%) [22]. Safrole is a precursor of many products used as natural insecticides as well as in the perfumery industries [23], while β-caryophyllene is known for its anti-inflammatory and local anaesthetic activities [24]. Since the oils are rich in safrole and β-caryophyllene, leaves and stems oils of P. caninum can be used as the source of these components.

 2.2. Antioxidant Activity

The essential oils were screened for their possible antioxidant activity by β-carotene/linoleic acid, DPPH radical scavenging and total phenolic contents. The results of the antioxidant activity are shown in Table 2.

β-Carotene/linoleic acid bleaching assay was evaluated by measuring the inhibition of conjugated diene hydroperoxides starting from linoleic acid oxidation. The effectiveness of the stems oil (114.9 ± 0.9%) was higher than leaves oil (103.5 ± 0.8%), which were higher compared to BHT (95.5 ± 0.5%). Compounds containing hydrogen atoms in the allylic and/or benzylic positions give better activity in this test because of relatively easy abstraction of hydrogen atom from these functional groups by peroxy radicals formed in the test circumstances [25]. In the stems oil, safrole (25.5%) was present in high amount, which is responsible for the antioxidant activity. The effect of antioxidant on DPPH radical scavenging was thought to be due to their hydrogen donating ability. It has been reported that free radical scavenging activity is greatly influenced by the phenolic components of samples [26]. In this study, the essential oils demonstrated low antioxidant activities, compared to BHT (43.5 ± 0.25 mg/mL) due to low abundance of phenolic components in the essential oils of P. caninum.

The amounts of total phenolic in the essential oils were determined spectrometrically according to the Folin-Ciocalteu’s method and calculated as garlic acid equivalents as shown in Table 2. The total phenolic contents of the essential oils are 27.4 ± 0.54 mg GA/g and 19.8 ± 0.42 mg GA/g for leaves and stems oil, respectively. The results indicated that leaves oils gave higher total phenolic than the stems oil, which revealed that there is a relationship between the antioxidant ability and total phenolic contents. The antioxidant activity is mainly due to their redox properties and chemical structure, which can play important role in inhibiting lipoxygenase and free radical scavenging [27].

 2.3. Antimicrobial Activity

The antimicrobial activity of the essential oil against Gram-positive bacteria (B. subtilis, S. aureus), Gram-negative bacteria (P. aeruginosa, P. putida, E. coli), yeast (C. albicans) and fungi (A. niger) was assessed both qualitatively and quantitatively by disc diffusion method and MIC values. Table 3 shows the antimicrobial activity of the essential oil of P. caninum.

Both oils showed strong antimicrobial activities against all bacterial strains but moderate to weak activity against fungal strains. The inhibition zones and MIC values for microbial strains, sensitive to the essential oils, were in the range 7–12 mm and 62.5–250 μg/mL, respectively. The leaves oil showed strong MIC value, 62.5 μg/mL towards E. coli while the stems oil exhibited MIC value 125 μg/mL against S. aureus and E. coli. The presence of safrole and β-caryophyllene may contribute to the antimicrobial activity of both oils. Studies on the antimicrobial activities of single aroma compounds found that safrole [28] inhibited the Gram negative, E. coli while β-caryophyllene [29] active against the Gram positive bacterium (S. aureus and E. fecalis). The stems oil showed moderate antimicrobial activity against C. albicans and A. niger with MIC value 500 μg/mL, however the leaves oil showed weak activity in both disc diffusion and micro-well dilution methods. Our findings revealed that the sensitivity of the bacterial strains to the leaves oil decreases in the order: E. coli > P. putida > B. subtilis = S. aureus = P. aeruginosa while for the stems oil, the order is: S. aureus = E. coli > P. putida = P. aeruginosa = B. subtilis.

In general, the essential oil showed better antimicrobial activity against the Gram-positive bacteria than the Gram-negative bacteria. Gram-positive bacteria, B. subtilis and S. aureus, were the most susceptible to this oil, with inhibition zone of 7–12 mm with MIC values of 250–500 μg/mL. Gram-negative bacteria were most resistant to this oil with inhibition zones between 7–8 mm with MIC values of 125–500 μg/mL. These results are consistent to those of previous reports in the literature, indicating that Gram-negative bacteria are more resistant to essential oils than Gram-positive bacteria due to its outer membrane [30]. Some studies also concluded that the essential oils have greater antimicrobial activity due to their additive, synergistic or antagonistic effects [31].

 3. Experimental Section 3.1. Plant Materials

Samples of Piper caninum Bl. were collected from Kuala Kangsar, Perak, Malaysia, in April 2011. This species was identified by Mr. Ahmed Zainuddin Ibrahim and the voucher specimen (HTBP3389) was deposited at the Herbarium of Taman Botani, Putrajaya.

 3.2. Solvent and Chemicals

β-Carotene, linoleic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH), gallic acid and butylated hydroxytoluene (BHT) were obtained from Sigma-Aldrich Chemie (Steinheim, Germany). Analytical grade methanol, ethanol and dimethylsulfoxide (DMSO), HPLC grade chloroform, Folin-Ciocalteu’s reagent, anhydrous sodium sulfate, sodium carbonate, polyoxyethylene sorbitan monopalmitate (Tween-40) were purchased from Merck (Darmstadt, Germany).

 3.3. Extraction of Essential Oils

The fresh leaves and stems were subjected to hydrodistillation in an all glass Dean-stark apparatus for 8 hours. The oils were dried over anhydrous magnesium sulfate and stored at 4–6 °C. The oils yields (w/w) were 0.46% and 0.31% for leaves and stems, respectively, based on their fresh weight.

 3.4. Gas Chromatography (GC)

GC analysis were performed on a Hewlett Packard 6890 series II A gas chromatograph equipped with an Ultra-1 column (25 m long, 0.33 μm 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 temperature 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 μL were injected manually (split ratio 50:1). The injection was repeated three times and the peak area percents were reported as means ± SD of triplicates. Calculation of peak area percentage was carried out by using the GC HP Chemstation software (Agilent Technologies).

 3.5. Gas Chromatography-Mass Spectrometry (GC-MS)

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 μm thickness and 0.20 mm inner diameter). Helium was used as carrier gas at flow rate of 1 mL/min. Injector temperature was 250 °C. Oven temperature was programmed from 50 °C (5 min hold) to 250 °C at 10 °C/min and finally held isothermally for 15 min. For GC-MS detection, an electron ionization system, with 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 50–400 amu.

 3.6. Identification of Constituents

The constituents of the oils were identified by comparison of their mass spectra with reference spectra in the computer library (Wiley) and also by comparing their retention indices, with those of authentic compounds or data in the literature [32]. The quantitative data were obtained electronically from FID area percentage without the use of correction factor.

 3.7. Antioxidant Activity 3.7.1. β-Carotene-Linoleic acid Assay

The β-carotene-linoleic acid bleaching assay described by Miraliakbari, and Shahidi [33] was used with minor modifications. A mixture of β-carotene and linoleic acid was prepared by adding together of 0.5 mg β-carotene in 1 mL chloroform (HPLC grade), 25 μL linoleic acid and 200 mg Tween 40. The chloroform 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 (2 g/L) were in methanol and 350 μL of each sample solution were added to 2.5 mL of the above mixture in test tubes and mixed thoroughly. The test tubes were incubated in a water bath at 50 °C for 2 h together with two blanks, one contained BHT (positive control) and the other contained the same volume of methanol. The absorbance was measured at 470 nm on an ultraviolet-visible (UV–Vis) spectrometer. Antioxidant activities (inhibitions percentage, I%) of the samples were calculated using the following Equation:

[ I % = ( A β - carotene after  2   h / A initial  β - carotene ) × 100 ]

where Aβ-carotene after 2 h assay is the absorbance value of β-carotene after 2 h assay remaining in the samples and Ainitial β-carotene is the absorbance value of β-carotene at the beginning of the experiment. All tests were carried out in triplicate and inhibition percentages were reported as means ± SD of triplicates.

 3.7.2. DPPH Radical Scavenging Assay

The free radical scavenging activity was measured by the DPPH method as described by Loo, Jain, and Darah [34] with minor modification. Each sample of stock solution (1.0 mg/L was diluted to final concentration of 1000, 500, 250, 125, 62.5, 31.3, 15.63 and 7.81 μg/mL. Then, a total of 3.8 ml of 50 μM DPPH methanolic solution was added to 0.2 mL of each sample solution and allowed to react at room temperature for 30 min. The absorbance of the mixtures was measured at 517 nm. A control was prepared without sample or standard and measured immediately at 0 min. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity, and vice versa. Inhibitions of DPPH radical in percent (I%) were calculated as follow:

[ % = [ ( A blank - A sample) ) / A blank ] × 100 ]

where Ablank is the absorbance value of the control reaction (containing all reagents except the test compound) and Asample is the absorbance values of the test compounds. The sample concentration that provides 50% inhibition (IC50) was calculated by plotting inhibition percentages against concentrations of the sample. All tests were carried out in triplicate and IC50 values were reported as means ± SD of triplicates.

 3.7.3. Total Phenolic Content

Total phenolic contents of the essential oils were determined as described by Loo, Jain, and Darah [34]. Sample of stock solution (1.0 mg/mL) was diluted in methanol to final concentrations of 1000, 800, 600, 400, and 200 μg/mL. A 0.1 mL aliquot of sample was pipetted into a test tube containing 0.9 mL of methanol, then 0.05 mL Folin-Ciocalteu’s reagent was added, and the flask was thoroughly shaken. 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 methanol 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 solutions and a standard curve obtained with the following Equation:

[ y = 0.0021 x - 0.0223 , r 2 = 0.9928 ]

The concentration of total phenolic compounds in the oils was expressed as mg of gallic acid equivalent per gram of sample. Test was carried out in triplicate and gallic acid equivalent value was reported as mean ± SD of triplicate.

 3.8. Antimicrobial Activity 3.8.1. Microbial Strains

The test microorganisms, Staphylococcus aureus (ATCC29737), Bacillus subtilis (ATCC6633), Pseudomonas aeruginosa (ATCC9027), Pseudomonas putida (ATCC49128), Escherichia coli (ATCC10536), Candida albicans (ATCC10231), and Aspergillus niger (ATCC16888) were used. The strains were grown on Nutrient agar (Oxoid, Italy) for the bacteria and Potato dextrose agar (PDA) for yeasts and fungi.

 3.8.2. Disc Diffusion Assay

Antimicrobial activity of the essential oils of P. caninum was carried out by the disc diffusion method reported by Murray, Baron, Pfaller, Tenover, and Yolken [35]. The essential oils were dissolved in DMSO (4 mg/mL). Inocolumn of 400 μL suspension containing 108 CFU/mL of bacteria and 106 CFU/mL of fungi each was spread on the nutrient agar (NA) and potato dextrose agar (PDA) medium. The discs (6 mm diameter) impregnated with 10 μL of the essential oils and DMSO (negative control) were placed on the inoculated agar, and were incubated for 24 h at 37 °C (bacterial), 48 h at 30 °C (yeast) and 72 h at 30 °C (fungi). Streptomysin sulfate (10 μg/mL) and nystatin (100 IU) were used as the positive controls for bacteria and fungi, respectively. Clear inhibition zones around the discs indicated the positive antimicrobial activity. All tests and analysis were carried out in triplicates.

 3.8.3. Minimum Inhibitory Concentration (MIC)

The minimal inhibitory concentration (MIC) was determined by broth micro dilution method using 96-well microplates as described by Gulluce [36]. The inocula of the microbial strains were prepared from 24 h broth cultures and suspensions were adjusted to 0.5 McFarland standard turbidity. Essential oil (1 mg) was dissolved in DMSO (1 mL) to get 1000 μg/mL stock solution. A number of wells were reserved in each plate for positive and negative controls. Sterile broth (100 μL) was added to well from row B to H. The stock solutions of samples (100 μL) were added to wells at row A and B. Then, the mixture of samples and sterile broth (100 μL) at row B were transferred to each well in order to obtain a twofold serial dilution of the stock samples (concentration of 1000, 500, 250, 125, 62.5, 31.3, 15.63 and 7.81 μg/mL). The inoculated bacteria (100 μL) were added to each well. The final volume in each well was 200 μL. Strepyomysin sulfate and nystatin were used as positive controls for bacterial and fungal, respectively. Plates were incubated at 37 °C for 24 h. Microbial growth was indicated by the turbidity and the presence of pellet at the bottom of the well.

 3.9. Statistical Analysis

Data obtained from essential oil analysis, antioxidant and antimicrobial activity were expressed as mean values. The statistical analyses were carried out employing one way ANOVA (p < 0.05). A statistical package (SPSS version 11.0) was used for the data analysis.

 4. Conclusions

The result demonstrated that safrole, β-caryophyllene, β-pinene and germacrene D were the main components from the leaves and stems oil of P. caninum. The highest activity was observed for inhibition of lipid peroxidation in the β-carotene/linoleic acid system by the stems oil. The essential oil of P. caninum showed strong antimicrobial activity; therefore it might well be used as an antimicrobial agent, as well as in food preservatives.

 Acknowledgments

The authors would like to thank Ministry of Science, Technology and Innovation Malaysia (MOSTI) vote 79378 and GUP Universiti Teknologi Malaysia grant Q.J130000.7126.02H30 for financial supports.

 References ParmarV.S.JainS.C.BishtK.S.JainR.TanejaP.JhaA.TyagiO.D.PrasadA.K.WengelJ.OlsenC.E.Phytochemistry of the genus PiperPhytochemistry19974659767310.1016/S0031-9422(97)00328-2 KatoM.J.FurlanM.Chemistry and evolution of PiperaceaePure Appl. Chem20077952953810.1351/pac200779040529 DasguptaN.DeB.Antioxidant activity of Piper betle L. leaf extract in vitroFood Chem20048821922410.1016/j.foodchem.2004.01.036 YamaguchiL.LagoJ.H.G.TanizakiT.M.MascioP.D.KatoM.J.Antioxidant activity of prenylated hydroquinone and benzoic acid derivatives from Piper crassinervium KunthPhytochemistry2006671838184310.1016/j.phytochem.2006.03.00116624353 RegasiniL.O.CotinguibaF.MorandimA.A.KatoM.J.ScorzoniL.Mendes-GianniniM.J.BolzaniV.S.FurlanM.Radical scavenging capacity of Piper arboretum and Piper tuberculatum (Piperaceae)Lat. Am. J. Pharm200827900903 MasudaT.InazumiA.YamadaY.PadolinaW.G.KikuzakiH.NakataniN.Antimicrobial phenylpropanoids from Piper sarmentosumPhytochemistry1991303227322810.1016/0031-9422(91)83180-S CostantinM.B.SartorelliP.LimbergerR.HenriquesA.T.SteppeM.FerreiraM.J.OharaM.T.EmerencianoV.P.KatoM.J.Essential oils from Piper cernuum and Piper regnellii: Antimicrobial activities and analysis by GC/MS and 13C-NMRPlanta Med20016777177310.1055/s-2001-1836311731928 SilvaD.R.EndoE.H.FilhoB.P.D.NakamuraC.V.SvidzinskiT.I.E.SouzaA.YoungM.C.M.Ueda-NakamuraT.CortezD.A.G.Chemical composition and antimicrobial properties of Piper ovatum VahlMolecules2009141171118210.3390/molecules1403117119325517 ChoiE.M.HwangJ.K.Investigations of anti-inflammatory and antinociceptive activities of Piper cubeba, Physalis angulata and Rosa hybridJ. Ethnopharmacol20038917117510.1016/S0378-8741(03)00280-014522451 RajudinE.AhmadF.SiratH.M.JamaluddinF.MustaphaN.M.AliR.M.ArbainD.Anti-inflammatory activity of Piper magnibaccum (Piperaceae)Nat. Prod. Commun2008317191721 Rodrigues SilvaD.BaroniS.SvidzinskiA.E.Bersani-AmadoC.A.CortezD.A.Anti-inflammatory activity of the extracts, fractions and amides from the leaves of Piper ovatum Vahl. (Piperaceae)J. Ethnopharmacol200811656957310.1016/j.jep.2007.12.01818289815 ZakariaZ.A.PatahuddinH.MohamadA.S.IsrafD.A.SulaimanM.R.In vivo anti-nociceptive and anti-inflammatory activities of the aqueous extract of the leaves of Piper sarmentosumJ. Ethnopharmacol2010128424810.1016/j.jep.2009.12.02120035852 TerreauxC.GuptaM.P.HostettmannK.Antifungal benzoic acid derivatives from Piper dilatatumPhytochemistry19984946146410.1016/S0031-9422(98)00197-6 LagoJ.H.RamosC.S.CasanovaD.C.Morandim AdeA.BergamoD.C.CavalheiroA.J.Bolzani VdaS.FurlanM.GuimaraesE.F.YoungM.J.Benzoic acid derivatives from Piper species and their fungitoxic activity against Cladosporium cladosporioides and C. sphaerospermumJ. Nat. Prod2004671783178810.1021/np030530j15568762 PessiniG.L.FilhoB.P.D.NakamuraC.V.CortezD.A.G.Antifungal activity of the extracts and neoligans from Piper regnelli (Miq.) C.DC. var. pallescens (C. DC.) YunckJ. Brazil Chem. Soc20051611301133 BurkillH.I.A Dictionary of Economic Pproducts of the Malay PeninsulaMinistry of Agriculture and Co-operativeKuala Lumpur, Malaysia1966217721773 tawanC.S.IporI.B.FasihuddinB.A.SaniH.A brief account on the wild Piper (Piperaceae) of the crocker range, SabahASEAN Rev. Biodivers. Environ. Conserv2002111 GuptaM.P.AriasT.D.WilliamsN.H.BosR.TattjeD.H.E.Safrole, the main component of the essential oil from Piper auritum of PanamaJ. Nat. Prod19854833033010.1021/np50038a026 MaiaJ.G.S.SilvaM.H.L.LuzA.I.R.ZoghbiM.G.B.RamosL.S.Especies de Piper da Amazonia ricasQuim. Nova198710200204 HwangL.S.WangC.K.SheuM.J.KaoL.S.Phenolic compounds of Piper betle flower as flavoring and neuronal activity modulating agentsACS Symp. Ser1992506200213 AbreuA.M.SevegnaniL.MachicadoA.R.ZimermannD.RebeloR.A.Piper mikanianum (Kunth) Steudel from Santa Catarina, Brazil—A new souce of safroleJ. Essent. Oil Res20021436136310.1080/10412905.2002.9699884 FerrazA.BalbinoJ.M.ZiniC.A.RibeiroV.L.BordignonS.A.PoserG.Acaricidal activity and chemical composition of the essential oil from three Piper speciesParasitology Res201010724324810.1007/s00436-010-1878-y MaiaJ.G.S.AndradeE.H.A.Database of the Amazon aromatic plants and their essential oilsQuim. Nova20093259562210.1590/S0100-40422009000300006 SivasothyY.ChongW.K.HamidA.EldeenI.M.SulaimanS.F.AwangK.Essential oils of Zingiber officinale var. rubrum Theilade and their antibacterial activitiesFood Chem201112451451710.1016/j.foodchem.2010.06.062 EbrahimabadiA.H.EbrahimabadiE.H.Djafari-BidgoliZ.KashiF.J.MazoochiA.BatooliH.Composition and antioxidant and antimicrobial activity of the essential oil and extracts of Stchys inflata Benth from IranFood Chem201011945245810.1016/j.foodchem.2009.06.037 CheungL.M.CheungP.C.K.OoiV.E.C.Antioxidant activity and total phenolics of edible mushroom extractsFood Chem20038124925510.1016/S0308-8146(02)00419-3 RubertoG.BarattaM.T.Antioxidant activity of selected essential oil components in two lipid model systemsFood Chem200969167174 KhayyatS.A.Al-ZahraniS.H.Thermal, photosynthesis and antibacterial studies of bioactive safrole derivative as precursor for natural flavor and fragranceArab. J. Chem201110.1016/ j.arabjc.2011.09.014 SchmidtE.BailS.FriedlS.M.JirovetzL.BuchbauerG.WannerJ.DenkovaZ.SlavchevA.StoyanovaA.GeisslerM.Antimicrobial activities of single aroma compoundsNat. Prod. Commun201051365136820922992 HanamanthagoudaM.S.KakkalameliS.B.NaikP.M.NagellaP.SeetharamareddyH.R.MurthyH.N.Essential oils of Lavandula bipinnata and their antimicrobial activitiesFood Chem201011883683910.1016/j.foodchem.2009.05.032 RandrianariveloR.SarterS.OdouxE.BratP.LebrunM.RomestandB.MenutC.AndrianoelisoaH.S.RaherimandimbyM.DanthuP.Composition and antimicrobial activity of essential oils of Cinnamosma fragransFood Chem200911468068410.1016/j.foodchem.2008.10.007 AdamsR.P.Identification of Essential Oil by Gas Chromatography/quadrupole Mass SpectroscopyAllured Publishing CorporationCarol Stream, IL, USA2001 MiraliakbariH.ShahidiF.Antioxidant activity of minor components of tree nut oilsFood Chem200811142142710.1016/j.foodchem.2008.04.008 LooA.Y.JainK.DarahIAntioxidant activity of compounds isolated from the pyroligneus acid Rhizopora apiculataFood Chem20081071151116010.1016/j.foodchem.2007.09.044 MurrayP.R.BaronE.J.PfallerM.A.TenoverF.C.YolkenR.H.Manual of Clinical Microbiology7th ed.ASMWashington, DC, USA1999 GulluceM.OzerH.BarisO.DafereraD.SahinF.PolissiouM.Chemical composition of the essential oils of Salvia aethiopis LTurk. J. Biol.200430231233 Tables

Components identified in the leaves and stems oils of P. caninum.

Components KI b Percentage a
Leaves Stems
α-Pinene 932 4.0 ± 0.15 1.6 ± 0.08
Camphene 946 0.2 ± 0.14 0.6 ± 0.12
β-Pinene 974 8.9 ± 0.09 4.9 ± 0.11
Myrcene 988 0.9 ± 0.10 −
Limonene 1024 3.9 ± 0.18 2.7 ± 0.08
(Z)-β-Ocimene 1032 3.4 ± 0.06 0.2 ± 0.12
γ-Terpinene 1054 0.1 ± 0.09 −
α-Terpinolene 1086 0.4 ± 0.17 −
2-Nonanone 1087 1.2 ± 0.05 −
Linalool 1095 7.0 ± 0.07 2.9 ± 0.15
Camphor 1141 − 0.3 ± 0.08
Pinocarvone 1160 0.1 ± 0.15 −
Terpinen-4-ol 1174 0.2 ± 0.12 0.5 ± 0.05
α-Terpineol 1186 0.3 ± 0.10 1.0 ± 0.14
n-Decanal 1201 0.2 ± 0.15 0.8 ± 0.12
Safrole 1285 17.1 ± 0.07 25.5 ± 0.07
2-Undecanone 1293 2.0 ± 0.14 1.1 ± 0.12
δ-Elemene 1335 1.8 ± 0.11 4.1 ± 0.16
α-Cubebene 1345 − 0.5 ± 0.15
Eugenol 1356 − 2.4 ± 0.08
Cyclosativene 1369 0.2 ± 0.14 −
α-Ylangene 1373 0.2 ± 0.04 −
α-Copaene 1374 0.5 ± 0.06 0.9 ± 0.06
β-Cubebene 1387 − 0.3 ± 0.12
β-Bourbonene 1387 1.1 ± 0.08
β-Elemene 1389 2.1 ± 0.14 2.4 ± 0.14
α-Gurjunene 1409 0.3 ± 0.08 0.9 ± 0.08
β-Caryophyllene 1417 6.7 ± 0.12 9.8 ± 0.09
β-Gurjunene 1431 0.2 ± 0.04 −
Aromadendrene 1439 − 0.8 ± 0.15
α-Humulene 1452 1.0 ± 0.13 1.6 ± 0.12
allo-Aromadendrene 1458 − 0.3 ± 0.07
Germacrene D 1484 4.9 ± 0.12 7.8 ± 0.14
Zingiberene 1493 0.4 ± 0.07 0.6 ± 0.21
Bicyclogermacrene 1502 1.1 ± 0.04 2.3 ± 0.14
(E,E)-α-Farnesene 1505 0.9 ± 0.08 −
α-Bisabolene 1506 − 0.4 ± 0.08
δ-Cadinene 1522 0.4 ± 0.04 1.0 ± 0.05
Chavibetol 1524 0.9 ± 0.11 −
Elemol 1548 0.2 ± 0.14 −
Germacrene B 1559 − 1.1 ± 0.11
(E)-Nerolidol 1561 3.9 ± 0.09 1.6 ± 0.17
Caryophyllene oxide 1582 0.2 ± 0.12 0.4 ± 0.07
Globulol 1590 − 0.3 ± 0.12
t-Muurolol 1644 − 2.4 ± 0.09
β-Eudesmol 1649 0.9 ± 0.06 −
α-Cadinol 1652 − 1.0 ± 0.06
Farnesyl acetate 1845 − 1.2 ± 0.15
Group components
Phenylpropanoids 18.0 ± 0.21 27.9 ± 0.22
Monoterpene Hydrocarbons 21.8 ± 0.19 10.0 ± 0.18
Oxygenated Monoterpenes 7.5 ± 0.24 4.7 ± 0.16
Sesquiterpene Hydrocarbons 21.9 ± 0.18 35.2 ± 0.29
Oxygenated Sesquiterpenes 5.2 ± 0.21 6.9 ± 0.18
Others 3.5 ± 0.24 2.3 ± 0.12
Identified Components (%) 77.9 ± 0.24 87.0 ± 0.26

Each value is expressed as means ± SD of three injections;

Retention indices on Ultra-1 capillary column.

Antioxidant activity of the leaves and stems oils of P. caninum a.

Samples β-carotene/linoleic acid (%) DPPH IC50 (mg/mL) Total phenolic contentGallic acid equivalent (mg GA/g)
Leaves oil 103.5 ± 0.35 187.6 ± 0.45 27.4 ± 0.54
Stems oil 114.9 ± 0.42 452.4 ± 0.55 19.8 ± 0.42
BHT 95.5 ± 0.30 43.5 ± 0.25 ND

Data represent mean ± standard deviation of three independent experiments; ND—not determined.

Antimicrobial activity of the leaves and stems oils of P. caninum a.

Leaves oil Stems oil Antibiotics SS Nystatin
Test microorganisms DD b MIC c DD MIC DD MIC DD MIC
Bacillus subtilis 12.2 ± 0.4 250 10.4 ± 0.5 250 17.6 ± 0.2 7.81 ND ND
Staphylococcus aureus 7.2 ± 0.4 250 7.0 ± 0.5 125 17.8 ± 0.2 7.81 ND ND
Pseudomonas aeruginosa 8.2 ± 0.4 250 8.8 ± 0.4 250 17.2 ± 0.2 7.81 ND ND
Pseudomonas putida 7.8 ± 0.5 125 7.5 ± 0.4 250 17.3 ± 0.2 7.81 ND ND
Escherichia coli 7.2 ± 0.4 62.5 7.8 ± 0.5 125 17.5 ± 0.2 7.81 ND ND
Candida albicans 7.0 ± 0.3 >1000 7.8 ± 0.3 500 ND ND 15.2 ± 0.2 7.81
Aspergillus niger 7.5 ± 0.3 >1000 8.0 ± 0.3 500 ND ND 15.3 ± 0.2 7.81

Data represent mean ± standard deviation of three independent experiments;

DD—disc diffusion method (including the diameter of disc 6 mm);

MIC—minimum inhibitory concentration (μg/mL);

SS—streptomysin sulfate; ND—not determined.