Chemical contents and antibacterial activity of volatile oils from Paederia pilifera

Abstract Volatile oils from aerial parts, leaves and flowers of Paederia pilifera were extracted by steam distillation. The volatile constituents and chemical compositions were investigated by gas chromatography-mass spectrometry (GC-MS). A total of 28, 18 and 26 volatile compounds were identified in the oils obtained from the aerial parts, leaves and flowers, respectively. Interestingly, hexadecanoic acid (25) was the highest component, accounting for 56.78% of the volatile oil derived from the aerial parts and 60.37% from the leaves. The most abundant compounds in the volatile oil from flowers were diisooctyl sebacate (42, 27.03%), followed by bis(2-ethylhexyl) phthalate (37, 9.03%) and docosane (34, 6.82%). The volatile oils of Paederia pilifera were evaluated for antibacterial activity and the bioassay results revealed that the oils displayed antibacterial activity against strains of Bacillus cereus B4ac-DSM4384, Staphylococcus aureus TISTR746 and Escherichia coli JM107 with values of MIC ranging from 0.278 to 35.63 mg/mL. The flower oil was found to be the most active agent against Escherichia coli with MIC value of 0.278 mg/mL. The volatile oil obtained from the aerial parts exhibited antimicrobial activity against all three strains of Bacillus cereus, Staphylococcus aureus and Escherichia coli with MIC values of 33.63, 5.11 and 0.531 mg/mL, respectively. The leaf oil possessed antibacterial activity against the strains of Bacillus cereus and Escherichia coli with MIC values of 31.64 and 0.494 mg/mL, respectively and a similar pattern was observed in the flower oil with MIC values of 35.63 and 0.278 mg/mL, respectively.


Introduction
Plants in the genus Paederia, comprised of about 20-30 species, are widely distributed throughout the tropics of Asia.Paederia pilifera Hook.f. is in the genus Paederia and belongs to the family Rubiaceae 1 .It is native to Thailand, India, Japan, Philippines, China and other countries in Southeast Asia 1 .It is characterized as a climbing plant and normally grows at elevations of 400-800 m on trees in deciduous forests or on the margins of evergreen and secondary forests.The plant blooms and fruits early in the year during the months of January and February 1,2 .Paederia pilifera grows locally in Thailand.The fresh or steamed plant is consumed in Thai households 3 .For example, the fresh whole plant can be squeezed and used as an ingredient in Thai desserts, while the chopped fresh leaves have been served on the rice of a dish, named "Kao Yum".The roots have also been boiled in water and drank as an antiflatulent 3,4 .In addition, it has emerged as a good source of traditional medicine.The entire plant can be used in treating diarrhea, flatulence, toothaches, chest pains, piles, inflammation of the spleen, emetic food poisoning, rheumatic arthritis and dysentery 1,2 .
The chemical constituents and the plant extracts obtained from Peadaria genus exhibit many beneficial pharmacological activities, including antimicrobial 5 , insecticidal 14 , antioxidant 3,5,15,16 , antibacterial 17 , antitumor 18 , antidiarrhoeal 19 , anti-inflammatory 20 , antidiabetic 21 and antinociceptive 22,23 activities.These constituents have also been found to have a beneficial effect on the digestive system 3 .Among these isolated compounds, iridoid glycosides are considered to be the major bioactive components 13 .Accordingly, iridoid glycosides separated from Paederia scandens; namely asperuloside, paederoside and scanderosiderevealed exhibited certain antiinflammatory and immunomodulatory effects 20 while paederosidic acid methyl ester (PAME) showed strong anti-nociceptive activity 23 .Also, some iridoids; namely paederoside, genipin and kutkoside, along with methanolic extracts obtained from Paederia scandens displayed inhibitory effect on Epstein-Barr virus 18 .Most recently, iridoid glycosides obtained from the aerial parts of Paederia scandens displayed significant levels of inhibition for nitric oxide production, which would indicate that a potentially promising anti-inflammatory agent could be obtained from this natural material 24 .
Accordingly, some of the relevant available scientific references on the investigation of volatile oils from Paederia genus has been reviewed.Volatile oils obtained from Paedaria foetida, including those from the stems, leaves and flowers, were isolated by steam distillation and their chemical compositions were analysed by Gas Chromatography-Mass Spectrometry (GC-MS) 13,25 .Notably, monoterpenoids including oxygen were the main groups of compounds found in all parts of this plant.Among the oxygen-containing monoterpenes, linalol was found in the highest amounts at 21.5%, 38.8% and 25.2% in the stems, leaves and flowers, respectively.In this regard, linalol was expected to be a vital aroma component 13,25 .Additionally, the presence of thiol-containing compounds was observed, especially for dimethyl disulphide, which was most abundant in the leaf oil 25 .
Similarly, the volatile oil obtained from Paederia scandens was extracted and the relevant chemical compositions were also determined 13,14,26,27 .
Accordingly, β-pinene (24.77%), α-pinene (8.22%) and 1,8-cineol (8.01%) were reported as the major constituents in the volatile oil obtained from aerial parts of this plant and the oil was also found to exhibit an insecticidal activity 14 .Furthermore, the main components of the volatile oils obtained from the dried aerial parts or leaves of this plant were identified as isopentyl acetate (20.2%), phenylmethyl acetate (8.0%), 2-phenylethyl acetate (8.0%), pentadecanoic acid ethyl ester (6.8%) and hexadecanoic acid (6.8%) 13,26 .However, some significant differences were observed in the volatile oil obtained from the fresh plants.Acetic acid (31.1%), linalool oxide (8.5%), furfural (7.5%) and fenchyl alcohol (7.3%) were dominant in this oil 13,27 .The volatile oil obtained from Paederia scandens was declared to possess crucial antiviral activity during in vitro studies in the way of anti-HBV activity 13,28 while it displayed anti-oxidative activity against non-alcoholic fatty liver disease in chickens by inhibiting the regulating protein HSP7C 29 .
A limited amount of data, acquired from the phytochemical studies of Paederia pilifera, has been reported in published literature.For example, an ethanolic extract obtained from the roots of this plant revealed gastroprotective effects on ethanol-induce gastric ulcers in mice.
Notably, it also exhibited antioxidant activities with high total phenolic contents and free radical scavenging activity 3 .Phenolic components isolated from the leaves of the plant displayed antioxidant activity 15 .Recently, phenolic acids and flavonoids were identified from the crude extracts obtained from leaves of this plant.The extracts showed antibacterial activity against Staphylococcus aureus DMST 8840, Staphylococcus epidermidis DMST 15505 and Methicillin-resistant Staphylococcus aureus (MRSA) DMST 20651 strains 17 .However, the chemical constituents isolated from this plant have not been fully characterized up to now.Consequently, this research work aimed to extract volatile oils from the aerial parts, leaves and flowers of Paederia pilifera and to further investigate their chemical compositions and bioactivity.To the best of our knowledge, this is the first report on chemical constituents and chemical compositions of volatile oils obtained from three different parts of the Paederia pilifera and their antibacterial activity.

Plant material
Paederia pilifera was collected from Chiang Mai Province (18.7295°N, 99.1324°E, north Thailand) in January of 2016.Plant material was identified and a voucher specimen (CMUB39846) was deposited at the Herbarium of Department of Biology, Faculty of Science, Chiang Mai University.

Volatile oil extraction
Fresh Paederia pilifera was cleaned and separated into three parts (aerial parts, leaves and flowers).Each fresh part of the plant was chopped into small pieces and air-dried in the shade at 30-35°C for a period of one week.The dried plant was ground into powder using a grinding mill (Sharp EM-14, 50 Hz, 350 W) and stored at room temperature in an incubator.Dried aerial parts of the plant in the amount of 200 g, as well as 200 g of leaves and 100 g of flowers were subjected for volatile oil extraction.The plant oil was then extracted by steam distillation at 100°C using 500 mL of distilled water over a period of 4 hours by employing a steam distillation apparatus.The sample-to-solvent ratio was 1: 2.5 (w/v) for the aerial parts and the leaves while the ratio for flowers was 1: 5 (w/v).The collected volatile oil was then transferred to a glass vial and stored at a temperature of -5°C for further analysis.

GC-MS analysis
The volatile oils were directly used for chromatographic analysis.The chemical compositions of the volatile oils derived from different parts of Paederia pilifera were investigated using the gas chromatography-mass spectrometry (GC-MS) technique.Accordingly, a gas chromatograph (7890A Agilent) was coupled to a mass spectrometer (5975C (EI) Agilent) with an HP-5MS capillary fused silica column (30 m, 0.25 mm, 1D, 0.25 μm film thickness).The injector and detector temperatures were 250°C and 280°C, respectively, with an oven temperature program starting at 60°C and increasing at a rate of 3°C/min to 240°C.Helium was used as carrier gas at 1 ml/min.The injection volume of the diluted oil was 1 μl.The electron ionization mass spectra, within a range of m/z 30 to 500 amu, were recorded at 70 eV ionization energy.The Kovát retention indices (RI) were determined in relation to a homologous series of n-alkanes (C 8 -C 40 ) as standards under the same chromatographic conditions.Identification of the chemical composition was performed through comparisons of the spectral data and retention indices with those of the NIST98 and WILEY7n databases and those reported in previously published literature 30 .Quantitative analysis of the volatile components was expressed as relative area percentages which were calculated using peak area normalization measurements.

Antibacterial activity assays
Antibacterial activity of all samples was evaluated by determining the minimal inhibitory concentration (MIC) according to Reller et al. 31 .Tetracycline was used as a positive control.Accordingly, 3-5 fresh colonies of Bacillus cereus B4ac-DSM4384, Staphylococcus aureus TISTR746 and Escherichia coli JM107 were inoculated in Mueller-Hinton broth (MHB) (Difco) and incubated at 37°C for 18 h.The bacterial suspensions were adjusted to approximately 10 8 cfu/ml, using McFarland turbidity standard No. 5. To determine the inhibitory effects of the volatile oils, 50 μl of Mueller-Hinton broth (MHB) was added to each well in 96-well microtiter plate and 50 μl of sample was then added to the first well.This was followed by a step involving two-fold serial dilutions in order to obtain a final concentration.A 50 μl portion of each bacterial suspension (10 8 cfu/ml) was then added into each well.The 96well microtiter plates were incubated at 37°C for 18 h.After incubation, the first well that visually displayed no growth of bacteria was selected for determination of the minimal inhibitory concentration (MIC) values.The experiment was run in triplicate and at least two duplicates of the same reading were selected and have been reported as the value of MIC.

Results and Discussion
In this study, Paederia pilifera was separated into three parts which were the aerial parts, leaves and flowers.The air-dried samples were extracted by steam distillation and the volatile oils were obtained as pale yellowish oils.The extraction yields of the oils obtained from the aerial parts, leaves and flowers were 0.02, 0.01 and 0.0025% (w/w) in relation to the dry weight of the plant, respectively.Previously published literature reported that steam distillation of the aerial parts of Paederia scandens provided the volatile oil with a yield of 0.02% 14 .The isolation of the volatile oils from the leaves, aerial parts and flowers of Paederia foetida yielded small amounts of oils at 0.0008, 0.001 and 0.003%, respectively 25 .In a comparison of our results with those of different Paedaria species, the volatile oil extracted from the aerial parts of Paederia pilifera in this study was obtained in the same amounts (0.02%) as the volatile oil isolated from the aerial parts of Paederia scandens 14 but was in much higher yields than the oil obtained from the aerial parts of Paederia foetida (0.001%) 25 .Furthermore, the leaf oil of Paederia pilifera (0.01%) in this work was present in higher quantity when compared with the leaf oil from Paederia foetida (0.0008%) 25 .However, the amount of flower oil (0.0025%) obtained in this study was a little less than the amount of flower oil derived from Paederia foetida (0.003%) 25 .
Quantitative analysis of volatile components was expressed as relative area percentages that were calculated using peak area normalization measurements.The retention index (RI) and the relative contents of the individual chemical constituents obtained from different parts of the plant are listed in Table 1 in order of their experimental retention times.
The results indicated the presence of 28, 18 and 26 identified compounds, accounting for 93.46%, 96.94% and 86.73% of the total volatile oils from the aerial parts, leaves and flowers, respectively.The unknown constituents were unidentified due to their relatively low abundance and a lack of any reference data.As indicated in Table 1 and Table 2, a total of 43 compounds were identified in all of the volatile oils from the three different parts of the plant and were divided into ten groups including monoterpene hydrocarbon, oxygenated sesquiterpene, diterpene hydrocarbon, oxygenated diterpene, fatty acid, aliphatic carbonyl compound, alkane, aromatic carbonyl compound, fatty ester and alcohol.The highest percentage of hexadecanoic acid (25)  was noticeable in the volatile oils obtained from the aerial parts (56.78%) and leaves (60.37%), indicating the presence of high amounts of fatty acids.However, only 1.88% of 25 was detected in the flower oil.The most abundant volatile compounds present in the aerial part oils were characterized as hexadecanoic acid (25, 56.78%), phytol (29, 5.72%) and pentadecanoic acid (16, 4.81%).In addition, the major components of the leaf oils were hexadecanoic acid (25, 60.37%), hexahydrofarnesyl acetone (15, 9.39%) and oleic acid (31, 4.21%).Diisooctyl sebacate (42, 27.03%) appeared to be a major chemical constituent in the flower oil along with bis(2ethylhexyl)phthalate (37, 9.03%) and docosane (34, 6.82%).The types of compounds obtained from the flower oils were clearly distinguishable from the volatile oils derived from the aerial parts and leaves.From the flower oil, higher amounts of alkanes (35.78%) were detected as a major group, followed by aliphatic carbonyl compounds (28.92%) and aromatic carbonyl compounds (14.98%).In contrast, most of the components of the volatile oils from the aerial parts and leaves belonged to fatty acids, while the presence of alkanes, aliphatic carbonyl compounds and aromatic carbonyl compounds were present in relatively low amounts (at only around 12-16% of the overall contents).Among the volatile samples examined in this study, four types of terpenoids were found in the samples including monoterpene hydrocarbon, oxygenated sesquiterpene, diterpene hydrocarbon and oxygenated diterpene.However, other classes of terpenoids were not observed.A comparison of the chemical compositions of the volatile oils obtained from Paederia pilifera with those obtained from other species was made and the results will be described herein.According to our findings, hexadecanoic acid (25), a fatty acid, was a major component present in the oils obtained from the aerial parts and the leaves while diisooctyl sebacate (42), an aliphatic carbonyl compound, was present in the highest level in the flower oil.According to the results of previous studies, hexadecanoic acid (25, 6.8%) was found in the volatile oils obtained from the dried aerial parts and leaves of Paederia scandens 13,26 .Additionally, hexadecane (7) was a mutual compound that was reported in the volatile oils obtained from the fresh whole plant of Paederia scandens 13,27 .Accordingly, alkanes and aliphatic carbonyl compounds were found as the major groups of components in the flower oils obtained from this plant.Likewise, an aliphatic carbonyl compound, isopentyl acetate (20.2%), and a carboxylic acid, acetic acid (31.1%), were found to be enriched in the volatile oils obtained from the dried and fresh plant samples of Paederia scanden, respectively 13,27 .A monoterpenoid containing oxygen, linalol, was predominant in the volatile oils obtained from the aerial parts, leaves and flowers of Paederia foetida 13,25 .However, monoterpenes were detected at a low level in our volatile oils.Significant differences were observed in the volatile oils obtained from the paedaria genus, which may have resulted from certain variation factors related to the biodiversity of breed, the geographical area of cultivation, climate, collection time, extraction methods and the experimental conditions.
The plant extracts and the volatile oils of the Paedaria genus exhibited significant inhibitory effects on various biological activities 3,5,[14][15][16][17][18][19][20][21][22][23] .The methanolic extract obtained from the leaves of Paederia pilifera exhibited antibacterial activity against three strains of Staphylococcus aureus DMST 8840, Staphylococcus epidermidis DMST 15505 and Methicillin-resistant Staphylococcus aureus (MRSA) DMST 20651 with the same MIC value of 25.60 mg/mL 17 .Furthermore, the volatile oil derived from Paederia scandens was reported to display significant anti-hepatitis B activity 28 and anti-oxidative effect against non-alcoholic fatty liver disease (NAFLD) in chickens 29 , as well as to exhibit an insecticidal activity 14 .In this present study, three distinct volatile oils obtained from the aerial parts, leaves and flowers of the Paederia pilifera plant were evaluated for antibacterial activity against strains of Bacillus cereus B4ac-DSM4384, Staphylococcus aureus TISTR746 and Escherichia coli JM107.Antimicrobial assay was achieved by determining the MIC.Tetracycline was used as a positive control.The MIC values of all tested samples are listed in Table 3.The bioassay results revealed that all volatile oils displayed antibacterial activity against strains of Bacillus cereus, Staphylococcus aureus and Escherichia coli with values of MIC ranging from 0.278 to 35.63 mg/mL.Interestingly, the flower oil was determined to be the most active against Escherichia coli with MIC value of 0.278 mg/mL.However, none of the isolated volatile oils reached the inhibitory activity level of tetracycline (MIC values of 0.0039-0.0078mg/mL), which was used as a reference drug against the microbes.Notably, the volatile oils obtained from the aerial parts possessed antimicrobial activity against all three strains of Bacillus cereus, Staphylococcus aureus and Escherichia coli with MIC values of 33.63, 5.11 and 0.531 mg/mL, respectively.However, the volatile oils obtained from the leaves and the flowers showed positive results to Bacillus cereus and Escherichia coli and a negative result to Staphylococcus aureus.The leaf oil displayed antibacterial activity against the strains of Bacillus cereus and Escherichia coli with MIC values of 31.64 and 0.494 mg/mL, respectively, and a similar pattern was observed in the flower oil with MIC values of 35.63 and 0.278 mg/mL, respectively.By comparing the inhibitory effect on all three strains, it was detected that all oils obtained from this plant were more bioactive against the strains of Escherichia coli (MIC values within a range of 0.278-0.531mg/mL) than the other tested microbes (MIC values within a range of 5.11-35.63mg/mL).In other words, the bioactive potentials of the oils obtained from the aerial parts, leaves and flowers against the strains of Escherichia coli were higher than those of Bacillus cereus by about 63, 64 and 128 times, respectively.In addition, the antibacterial results of the volatile oil obtained from aerial parts against the strains of Escherichia coli were also found to be higher than that of Staphylococcus aureus by about 9.6 times.From our literature review, a limited amount of data was available that pertained to these biologically active volatile oils obtained from the Paederia genus 13,[27][28][29] .It is particularly noteworthy that this is the first time that results are being reported that pertain to the chemical compositions and bioactivity of the volatile oils obtained from Paederia pilifera.

Conclusion
Steam distillation of the aerial parts, leaves and flowers of Paederia pilifera led to volatile oils obtained in the amounts of 0.02, 0.01 and 0.0025% (w/w), respectively.Furthermore, there were 28, 18 and 26 constituents identified from the volatile oils obtained from the aerial parts, leaves and flowers, respectively.Accordingly, their compositions were determined using GC-MS.The volatile oils obtained from the aerial parts and leaves were composed of hexadecanoic acid (25) in maximum quantities.Moreover, the major component present in the volatile oils obtained from the flowers was diisooctyl sebacate (42).According to the results of the bioassay, the volatile oils derived from three plant parts displayed antibacterial activity against strains of Bacillus cereus B4ac-DSM4384, Staphylococcus aureus TISTR746 and Escherichia coli JM107.

Table 1 .
Volatile components and relative contents of Paederia pilifera oilsRetention index from the experiment on HP-5MS column; b Retention index according to the NIST database and literature 30 ; c Relative area percentage a

Table 1 cont . Table 2 .
Type of identified compounds