Phyllanthus wightianus Müll. Arg.: a potential source for natural antimicrobial agents.

Phyllanthus wightianus belongs to Euphorbiaceae family having ethnobotanical importance. The present study deals with validating the antimicrobial potential of solvent leaf extracts of P. wightianus. 11 human bacterial pathogens (Bacillus subtilis, Streptococcus pneumoniae, Staphylococcus epidermidis, Proteus vulgaris, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhimurium, Escherichia coli, Shigella flexneri, Proteus vulgaris, and Serratia marcescens) and 4 fungal pathogens (Candida albicans, Cryptococcus neoformans, Mucor racemosus, and Aspergillus niger) were also challenged with solvent leaf extracts usingagar well and disc diffusion methods. Further, identification of the active component present in the bioactive extract was done using GC-MS analysis. Results show that all extracts exhibited broad spectrum (6-29 mm) of antibacterial activity on most of the tested organisms. The results highlight the fact that the well in agar method was more effective than disc diffusion method. Significant antimicrobial activity was detected in methanol extract against S. pneumoniae (29 mm) with MIC and MBC values of 15.62 μg/mL. GC-MS analysis revealed that 29 bioactive constituents were present in methanolic extract of P. wightianus, of which 9,12-octadecaenioic acid (peak area 22.82%; RT-23.97) and N-hexadecanoic acid (peak area 21.55% RT-21.796) are the major compounds. The findings of this study show that P. wightianus extracts may be used as an anti-infective agent in folklore medicine.

1. Introduction

Plants play an important role in human life, primarily, as a source of food and medicine. Man is continuously faced with several lethal infectious diseases caused by pathogenic microorganisms [1]. In recent years, several pathogenic microorganisms have gained resistance to currently available synthetic antimicrobial agents and also caused many health problems [2]. Hence, there is an urgent need to discover an alternative new, broad spectrum, more active, and safer antimicrobial agent. Plant materials remain an important resource to combat serious diseases in the world. Especially, the plants from Pinaceae, Cupressaceae, Apiaceae, Burseraceae, Anacardiaceae, Palmaceae, Euphorbiaceae, Dracenaceae, and Fabaceae families are rich source for antimicrobial agents [3, 4]. Plants have an exceptional ability to synthesize de novo antimicrobial agents, in response to microbial attack for its protection [5]. Plant derived natural compounds (such as flavonoids, terpenoids, and steroids) have received considerable attention due to their diverse pharmacological properties including antibacterial and antifungal activities [6].

Antimicrobial components from plants which are mainly secondary metabolites act as inhibitors of bacterial growth, bacterial adherence, exopolysaccharide synthesis, DNA gyrase, cytoplasmic membrane function, and energy metabolism [7, 8]. Berberine, an isoquinoline alkaloid, which is present in roots and stem-bark of Berberis species, shows antimicrobial potential against bacteria, fungi, protozoa, and viruses [9]. Diterpene alkaloids, commonly isolated from the plants of the Ranunculaceae family, have antimicrobial properties [10]. Several phenolic compounds such as, caffeic acid, catechol, and pyrogallol are effective antimicrobial agents. The antibacterial activity of some monoterpenes, diterpenoids, sesquiterpenes, triterpenoids, and their derivatives isolated from plants was recently reported [11]. Nowadays, search for plants with antimicrobial activity has evolved [12]. Importance of plants in drug discovery is growing due to vast diversity of the secondary metabolites which possess varied biological activities and act as main source of molecule leading the discovery of new, effective, and safer drugs [13]. Recent attention has been paid to extraction and isolation of biologically active compounds from plant species which are used in herbal medicines [14]. Pharmacognostic investigations of plants or plant extracts were needed to ascertain their biological activities which lead to the discovery of novel drugs or templates for the development of new therapeutic agents [15].

Phyllanthus wightianus (Euphorbiaceae) is a monoecious glabrous shrub which grows up to 4.5 m high and is found in the hills (750 to 1000 m) of peninsular India, on the floor and border of shoals, low altitudes in sandveld, hot dry deciduous, mopane woodlands, along banks of seasonal streams, and rivers [16]. It exhibits various biological properties, such as antimicrobial [17-19], larvicidal [20, 21], analgesic [22], wound healing [23], and antioxidant properties [24]. On the basis of the above information, the present investigation was focused on antimicrobial properties of different solvent leaf extracts and GC-MS analysis of bioactive extract of P. wightianus Müll. Arg.

2. Materials and Methods

2.1. Plant Material

Fresh, matured, uninfected leaves of P. wightianus were collected from higher altitudes (900–1100 m) of Kolli Hills (latitude 10° 12′–11° 7′ N, longitude 76°–77° 17′ E), Namakkal district, Tamil Nadu, India. The plant material was authenticated by Botanical Survey of India (BSI) (reference number: BSI/SRC/5/23/2013-14/Tech/2081) Coimbatore, Tamil Nadu, India. The voucher specimen (specimen number: PU/BT/NDRL/2010/05) has been deposited in the Natural Drug Research Laboratory, Department of Biotechnology, Periyar University, Salem, Tamil Nadu, India.

2.2. Preparation of Extracts

Collected leaves were washed and shade-dried for three weeks and then powdered. The powdered plant material (500 g) was extracted in increasing polarity order (successively) with hexane, chloroform, acetone, ethyl acetate, and methanol in a Soxhlet apparatus for 72 hours. The extractives were filtered through Whatman number 1 filter paper and evaporated under vacuum at 40°C to yield crude extracts.

2.3. Used Microorganisms

Three gram positive bacteria, namely, B. subtilis (MTCC 441), S. pneumoniae (MTCC 655), S. epidermidis (MTCC 435), and eight gram negative bacterial strains, such as P. vulgaris (MTCC 426), P. aeruginosa (MTCC 741), K. pneumoniae (MTCC 109), S. typhimurium (MTCC 98), E. coli (MTCC 739), and S. flexneri (MTCC 1457), with two clinical isolates (P. vulgaris and S. marcescens) and 4 fungal pathogens (C. albicans, C. neoformans, M. racemosus, and A. niger) were used in this investigation. The fungal strains were obtained from clinical laboratories of Salem District, Tamil Nadu. Each test organism was prepared by inoculating a loop-full of mother culture in a 5 mL of broth (Muller-Hinton broth for bacteria and Sabourd Dextrose broth for fungal cultures) and incubated at appropriate temperature and time for bacterial pathogens (37°C for 16 hours) and fungal strains (room temperature (28°C) for 72 hours). The culture turbidity was adjusted to 0.5 McFarland equivalence (1.5 × 108 CUF) prior to use.

2.4. Agar Well Diffusion Method

The agar well diffusion method was employed to determine antibacterial activity of extracts as per the modified method of Natarajan et al. [19]. The standardized test cultures (50 μL) were swabbed on the per-molten Müeller Hinton Agar (MHA) for bacteria and Sabourd Dextrose Agar (SDA) for fungus using aseptic cotton swab. Six wells were made in the seeded plates using sterile cork borer (5 mm diameter). Then, each extract (50 μL = 50 μg) was separately introduced into wells and allowed to diffuse at room temperature. Equal volume of DMSO was served as negative control. About 25 μL of standard antibiotics like fluconazole (fungus) and ciprofloxacin (bacteria) was used as positive control (each 1 μg/μL). The bacterial and fungal plates were incubated at 37°C for 24 hours and at room temperature for 72 hours, respectively. After the incubation period, the zone of growth inhibition was measured (in mm).

2.5. Disc Diffusion Method

The disc diffusion test was performed by the method of NCCLS [25] with minor modifications. Test microbial suspension culture (50 μL) was spread on the MHA for bacteria and SDA for fungus by a sterile cotton swab. Sterile discs (5 mm diameter) were loaded with each extract (50 μL) and allowed to dry at room temperature. The dried discs were placed aseptically on the seeded plates. Standard antibiotic discs were used as positive control (gentamicin, vancomycin, and ampicillin for bacteria and fluconazole for fungus (10 mcg/disc)). The plates were incubated as the conditions mentioned in the well diffusion methods and the diameter (in mm) of clear zone of growth inhibition was recorded.

2.6. Minimum Inhibitory Concentration (MIC)

The MIC was determined by broth microdilution bioassay method using the modified method of Eloff et al. [26]. MIC was carried out on the basis of antimicrobial results, the extracts which exhibited considerable antimicrobial activity against tested organisms. 100 μL of different concentrations (1–1000 μg/mL) of extracts was introduced into 96-well microplates containing 200 μL of Muller-Hinton broth and 20 μL bacterial cultures were added to each well. The microplate was closed with lid and incubated for 24 h at 37°C. After incubation period, 40 μL of p-iodonitrotetrazolium violet (INT) (0.2 mg/mL) was added to the wells to serve as an indicator of bacterial growth and incubated at 37°C for 1 hour. The minimum inhibitory concentration (MIC) was taken as the lowest concentration of the extract that completely inhibited bacterial growth.

2.7. Minimum Bactericidal Concentration (MBC)

The MBC was determined as per method of Khan et al. [27]. MIC test dilutions (5 μl) which showed no color change was subcultured on freshly prepared Mueller Hinton Agar plates and incubated at 37°C for 24 h. The lowest concentration in which no bacterial growth occurred was taken as the Minimum Bactericidal Concentration.

2.8. GC-MS Analysis

GC-MS analysis of bioactive extract was carried out on GC Clarus 500 Perkin Elmer system comprising AOC-20i auto sampler. The spectrums of unknown components present in the bioactive extract were identified by compared with known components spectrum which stored in the NIST and WILEY libraries. The name, molecular weight and structure of the components present in the bioactive extract were ascertained. The GC-MS analysis was carried out at Sophisticated Analytical Instrument Facility (SAIF), Indian Institutes of Technology, Chennai, India.

2.9. Statistical Analysis

All determinations were done at least in triplicate and averaged. Values were expressed as mean ± standard deviations. Statistical analyses were conducted using SPPS software (16.0 Version). Analysis of variance (ANOVA) in a completely randomized design and Tukey's multiple range tests were used to compare any significant differences between samples. The confident limits used in this study were based on 95% (P < 0.05).

3. Results and Discussion

The color of extracts and extractive yield of the plant material are presented in Table 1. The results of antimicrobial activity of P. wightianus were given in Table 2. All the extracts of P. wightianus show broad spectrum antibacterial activity in the range between 6 and 29 mm. The results of agar well diffusion method show that methanolic extract has significant activity against S. pneumoniae (29 mm) followed by S. epidermidis (17 mm). The considerable amount of antibacterial activity was observed in acetone extract against S. pneumoniae (28 mm) followed by S. flexneri (12 mm). In disc diffusion method, methanol extract exhibits good antimicrobial activity and the maximum growth inhibition was observed in S. pneumoniae (18 mm) followed by S. epidermidis (17 mm). Acetone extract having well to moderate antimicrobial activity and maximum activity was detected in S. epidermidis (18 mm) followed by S. pneumoniae (14 mm). Most of the tested clinical fungal pathogenic strains exhibit no sensitivity on the tested extracts in both agar well and disc diffusion methods. However, high antifungal activity was recorded in methanol extract against C. neoformans (10 mm) followed by hexane extract against M. racemosus (10 mm) in well diffusion method (Table 2). The overall results highlight that methanol extract exhibits significant activity against most of the tested pathogens compared with other extracts.

Table 1

Color and extractive yield of extracts from different solvents of P.  wightianus.

Name of extract	Color	Yield (%)
Hexane	Yellowish green	6.24
Chloroform	Dark green	0.67
Acetone	Pale green	1.33
Ethyl acetate	Pale brown	2.49
Methanol	Brown	4.61

Table 2

Antimicrobial activity of different solvent extracts of P. wightianus.

S. number	Organisms	Method	Diameter of zone of inhibition (in mm)#
			Methanol	Acetone	Ethyl acetate	Chloroform	Hexane	Control*
1	Bacillus subtilis  (MTCC 441)	Well	09.42 ± 0.52b	07.96 ± 0.08b	6.66 ± 0.58b	00.00 ± 0.00a	00.00 ± 0.00a	29.74 ± 0.44c
		Disc	08.71 ± 0.51c	06.53 ± 0.50b	00.00 ± 0.00a	8.33 ± 0.58c	08.33 ± 0.58c	15.94 ± 0.10d
2	Escherichia coli  (MTCC 739)	Well	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	27.56 ± 0.51b
		Disc	07.71 ± 0.51b	07.67 ± 0.58b	07.62 ± 0.65b	08.67 ± 0.58b	08.70 ± 0.61b	00.00 ± 0.00a
3	Klebsiella  pneumoniae  (MTCC 109)	Well	09.52 ± 0.50b	10.72 ± 0.63c	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	29.54 ± 0.51d
		Disc	09.60 ± 0.36c	08.00 ± 0.08b	09.83 ± 0.48c	08.67 ± 0.58b,c	08.72 ± 0.63b,c	00.00 ± 0.00a
4	Proteus vulgaris  (MTCC 426)	Well	09.56 ± 0.45b	10.96 ± 0.08c	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	24.65 ± 0.36d
		Disc	09.82 ± 0.32d	08.74 ± 0.65c,d	07.67 ± 0.58b,c	07.05 ± 0.93b	07.51 ± 0.50b,c	00.00 ± 0.00a
5	Pseudomonas aeruginosa  (MTCC 741)	Well	12.75 ± 0.66b	11.65 ± 0.56b	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	22.67 ± 0.58c
		Disc	11.75 ± 0.44d	09.83 ± 0.30c	6.88 ± 0.43b	00.00 ± 0.00a	09.64 ± 0.40c	00.00 ± 0.00a
6	Salmonella  typhimurium  (MTCC 98)	Well	09.59 ± 0.66c	11.77 ± 0.68d	08.47 ± 0.50b	00.00 ± 0.00a	08.47 ± 0.50b	29.96 ± 0.08e
		Disc	09.67 ± 0.58c	08.70 ± 0.61b,c	08.59 ± 0.71b,c	07.27 ± 1.10b	07.87 ± 0.23b	00.00 ± 0.00a
7	Shigella  flexneri  (MTCC 1457)	Well	13.80 ± 0.35d	12.77 ± 0.68d	07.81 ± 0.33c	06.51 ± 0.50b	00.00 ± 0.00a	15.67 ± 0.58e
		Disc	13.84 ± 0.28d	11.77 ± 0.68c	07.82 ± 0.45b	07.78 ± 0.47b	08.85 ± 0.25b	00.00 ± 0.00a
8	Streptococcus  pneumoniae  (MTCC 655)	Well	29.71 ± 0.51c	28.55 ± 0.51c	10.73 ± 0.64b	08.67 ± 0.58a	08.00 ± 1.00a	34.67 ± 0.58d
		Disc	18.85 ± 0.25d	14.54 ± 0.50c	08.13 ± 0.13a	08.67 ± 0.58a,b	09.50 ± 0.50b	19.52 ± 0.50d
9	Staphylococcus epidermidis  (MTCC 435)	Well	17.83 ± 0.29b	10.73 ± 0.64a	17.00 ± 1.00b	10.67 ± 0.58a	11.84 ± 0.77a	29.83 ± 0.29c
		Disc	17.93 ± 0.12b	18.33 ± 0.58b	07.67 ± 0.58a	07.87 ± 0.23a	08.67 ± 0.58a	17.80 ± 0.72b
10	Proteus vulgaris	Well	00.00 ± 0.00a	07.77 ± 0.39b	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	21.84 ± 0.27c
		Disc	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	10.75 ± 0.67b
11	Serratia marcescens	Well	00.00 ± 0.00a	00.00 ± 0.00a	08.33 ± 0.32b	00.00 ± 0.00a	00.00 ± 0.00a	24.67 ± 0.58c
		Disc	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	12.42 ± 1.01b
Fungal cultures
1	C. albicans	Well	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	00.000 ± 0.0	00.00 ± 0.00a
		Disc	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a
2	C. neoformans	Well	10.41 ± 0.52d	07.56 ± 0.39c	09.78 ± 0.39d	06.51 ± 0.50b	00.00 ± 0.00a	00.00 ± 0.00a
		Disc	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	00.00 ± 0.00a	27.29 ± 10.3b
3	M. racemosus	Well	07.67 ± 0.58b	09.07 ± 0.40b,c	09.67 ± 0.58b,c	09.73 ± 0.46b,c	10.67 ± 0.58c	00.00 ± 0.00a
		Disc	00.00 ± 0.00a	00.00 ± 0.00a	07.93 ± 0.12c	06.80 ± 0.35b	09.07 ± 0.12c	00.00 ± 0.00a
4	A. niger	Well	09.67 ± 0.58d	07.93 ± 0.12b,c	09.77 ± 0.40c,d	07.77 ± 0.25b	09.57 ± 0.51d	00.00 ± 0.00a
		Disc	07.80 ± 0.35b,c	06.67 ± 0.58b	07.67 ± 0.58b,c	00.00 ± 0.00a	08.67 ± 0.58d	00.00 ± 0.00a

*Standard antibiotics (in well diffusion: ciprofloxacin (1 mg mL−1) for bacteria and fluconazole (1 mg mL−1) for fungus; in disc diffusion: gentamicin, vancomycin, and ampicillin (10 mcg/disc) for bacteria and fluconazole (10 mcg/disc) for fungus). #The values are mean of triplicates with standard deviations (mean ± S.D; n = 3). Different superscript letters (a–e) in rows indicate significant differences (at P < 0.05) when subject to Tukey's multiple comparison test.

The MIC and MBC results (Tables 3 and 4) indicate that the crude extracts of P. wightianus inhibited the growth of selected bacterial species. The lowest MIC and MBC value were detected in methanolic extract against S. pneumoniae (15.62 μg/mL) and S. epidermidis (31.25 μg/mL). The rest of extracts showed moderate to high MIC and MBC values (500–>1000a μg/mL) against most of the tested bacterial pathogens. Plant extracts are considered as having a good inhibitory activity, if they present MICs ≤ 100 μg/mL, a moderate inhibitory activity, if they present MICs ranging from 100 to 500 μg/mL, a weak inhibitory activity, if they present MICs ranging from 500 to 1000 μg/mL, and no inhibitory activity, if they present MICs > 1000a μg/mL [28, 29]. While considering these reports, the MIC and MBC values recorded from antibacterial activity of present investigation might be good to moderate.

Table 3

MIC (μg/mL) for antibacterial activity of P. wightianus against some pathogens.

Organisms	Methanol	Acetone	Ethyl acetate	Chloroform	Hexane	Ciprofloxacin
Bacillus subtilis  (MTCC 441)	>1000a	>1000a	>1000a	>1000a	>1000a	3.9b
Escherichia coli  (MTCC 739)	>1000a	>1000a	>1000a	>1000a	>1000a	15.62b
Klebsiella pneumoniae  (MTCC 109)	500b	>1000a	>1000a	>1000a	>1000a	7.81c
Proteus vulgaris  (MTCC 426)	>1000a	>1000a	>1000a	>1000a	>1000a	15.62b
Pseudomonas aeruginosa  (MTCC 741)	250c	250c	500b	>1000a	>1000a	31.25d
Salmonella typhimurium  (MTCC 98)	500	>1000a	>1000a	>1000a	>1000a	15.62
Shigella  flexneri  (MTCC 1457)	125d	250c	500b	>1000a	>1000a	31.25e
Streptococcus pneumoniae  (MTCC 655)	15.62d	31.25c	250b	>1000a	>1000a	3.9e
Staphylococcus epidermidis  (MTCC 435)	31.25d	62.5c	500b	>1000a	>1000a	3.9c

The values are mean of triplicates. Different superscript letters (a–e) in rows indicate significant differences (at P < 0.05) when subject to Tukey's multiple comparison test.

Table 4

MBC (μg/mL) for antibacterial activity of P. wightianus against some pathogens.

Organisms	Methanol	Acetone	Ethyl acetate	Chloroform	Hexane	Ciprofloxacin
Bacillus subtilis  (MTCC 441)	>1000a	>1000a	>1000a	>1000a	>1000a	15.62b
Escherichia coli  (MTCC 739)	>1000a	>1000a	>1000a	>1000a	>1000a	31.25b
Klebsiella pneumoniae  (MTCC 109)	>1000a	>1000a	>1000a	>1000a	>1000a	15.62b
Proteus vulgaris  (MTCC 426)	>1000a	>1000a	>1000a	>1000a	>1000a	31.25b
Pseudomonas aeruginosa  (MTCC 741)	500b	500b	500b	>1000a	>1000a	31.25c
Salmonella typhimurium  (MTCC 98)	500b	>1000a	>1000a	>1000a	>1000a	31.25c
Shigella  flexneri  (MTCC 1457)	250c	250c	500b	>1000a	>1000a	62.5d
Streptococcus pneumoniae  (MTCC 655)	15.62d	62.5c	500b	>1000a	>1000a	3.9e
Staphylococcus epidermidis  (MTCC 435)	31.25c	125b	1000a	>1000a	>1000a	3.9d

The values are mean of triplicates. Different superscript letters (a–e) in rows indicate significant differences (at P < 0.05) when subject to Tukey's multiple comparison test.

The results highlights that significant antibacterial activity was observed in methanol extract against S. flexneri, S. pneumoniae, and S. epidermidis. Similar observations were made with P. wightianus methanolic extract [17]. Our previous findings, report that acetone extract expresses significant activity against most of the clinical bacterial pathogens compared to methanol extract [19]. Whereas, the present results show MTCC bacterial strains to be more sensitive to the methanol extract than acetone extract of P. wightianus. This difference in observation may be due to the high concentration (5 mg/well/disc) of extracts being used in the earlier study, whereas, in the present findings, low concentrations of extracts did not inhibit bacterial growth.

Studies have shown that methanolic extracts of many Phyllanthus species, such as P. acidus [30], P. muellerianus [31], P. amarus, P. maderaspatensis [32], P. debilis [33], P. amarus, P. emblica [34], and P. niruri [35], harbor promising antimicrobial activity which strengthens the present findings. Several reports stated that methanol is potent solvent for extracting variety of important phytoconstituents, like alkaloids, phenols, tannins, fatty acids, and flavonoids, which harbor antimicrobial potential which support the findings of present investigation [36, 37].

GC-MS analysis shows the presence of 29 compounds which were identified based on their retention time (RT), molecular formula, molecular weight (MW), and concentration (%) (Figure 1 and Table 5). Methnolic extract of P. wightianus has 9,12-octadecadienoic acid (with the peak area 22.82% and retention time 23.970) and N-hexadecanoic acid (with the peak area 21.55% and retention time 21.796) acting as major components. A variety of compounds, such as aliphatic ether, aliphatic carboxylic acid, aliphatic ester, alkene, and phenolic compounds were identified. Some of the compounds were present only in low quantities (ranging from 0.6 to 4%).

Figure 1

GC-MS chromatogram of methanol leaf extract of P. wightianus.

Table 5

Compounds detected in methanol leaf extract of P. wightianus using GC-MS analysis.

Peak	R. time	Peak area (%)	Molecular formula	Molecular weight	Compound name
1	4.726	0.94	C6H12O2	116	2-Pentanone, 4-hydroxy-4-methyl-3 2-hydroxy-2-methyl-4-pentanone 2-
2	9.358	0.28	C6H6O3	126	Levoglucosenone 6,8-ioxabicyclooct-2-en-4-one
3	10.907	0.67	C8H8O	120	4-Vinylphenol
4	11.198	0.26	C8H18O2	174	T-Butyldimethylsilyl acetate
5	12.387	0.08	C13H16O2	204	1-(4-Methoxyphenyl)-5-hexen-1-one
6	13.436	1.60	C6H6O3	126	1,2,3-Benzenetriol (pyrogallol)
7	15.093	6.07	C15H24O2	236	Butanoic acid, 3-methyl-, 3,7 dimethyl-2,4,6-octatrienyl ester
8	15.241	3.56	C15H20Br2O2	390	3,8-Dioxabicyclononane, 6-bromo-4-(1-bromopropyl)-2-[2-penten-4-ynyl]
9	15.525	1.04	C14H22O	206	Phenol, 2,4-bis(1,1-dimethylethyl)-2,4-ditert-butylphenol 1-hydroxy-2, 4-di-tert-butylbenze
10	16.037	2.46	C11H16O2	180	2-Benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl- (2,6,6-trimethyl-2-hydroxycyclohexylidene)acetic acid lactone 4,5
11	16.586	0.55	C12H14O4	222	1,2-Benzenedicarboxylic acid, diethyl ester phthalic acid
12	17.658	0.77	C21H32O3	332	β-Dodecahydro
13	19.062	0.71	C11H16O3	196	2(4h)-Benzofuranone, 5,6,7,7a-tetrahydro-6-hydroxy-4,4,7a-trimethyl-, (6s-cis)-(—)-loliolide
14	19.716	3.03	C13H20O2	208	Pluchidiol
15	20.264	10.65	C20H38	278	2,6,10-Trimethyl,14-ethylene-14-pentadecne
16	20.347	1.19	C18H36O	268	2-Pentadecanone, 6,10,14-trimethyl- hexahydrofarnesyl acetone 6,10,14
17	20.591	3.16	C20H40	296	3,7,11,15-Tetramethyl-2-hexadecen-1-ol (2e)-3,7,11,15-tetramethyl
18	20.831	5.85	C20H40	296	3,7,11,15-Tetramethyl-2-hexadecen-1-ol (2e)-3,7,11,15-tetramethyl
19	20.925	0.36	C24H34O2	446	Butanal
20	21.674	0.58	C10H20	156	6-Octen-1-ol, 3,7-dimethyl-
21	21.796	21.55	C16H32O2	256	N-Hexadecanoic acid, palmitic acid, pentadecanecarboxylic acid
22	23.300	0.53	C18H38O	270	Bis-(3,5,5-trimethylhexyl) ether
23	23.808	0.40	C20H38	278	2,6,10-Trimethyl,14-ethylene-14-pentadecne neophytadiene
24	23.881	3.81	C11H22O2	186	Nonanoic acid, 7-methyl ester methyl 7-methylnonanoate
25	23.970	22.82	C18H32O2	280	9,12-Octadecadienoic acid-linoleic acid grape seed oil linoleic linole
26	24.199	5.93	C18H30O2	278	9,12,15-Octadecatrienoic acid, linolenic acids alpha-linolenic acid
27	24.308	0.68	C18H36O2	284	Octadecanoic acid (stearic acid)
28	26.055	0.46	C8H14O	126	(3z)-6-methyl-3,6-heptadien-1-ol

N-Hexadecanoic acid and 9,12-octadecadienoic acid are common secondary metabolites present in several plants [38-43] and are reported as having many biological properties, like antimicrobial, anti-inflammatory, hypocholesterolemic, cancer preventive, hepatoprotective, and antioxidant [44, 45]. 9,12-Octadecadienoic acid was also stated as a model compound of unsaturated fatty acids, which selectively inhibits FabI enzyme in S. aureus and E. coli, catalyzing the final and rate-limiting step of the chain elongation process of the type II fatty acid synthesis (FAS-II) in bacteria [46].

Several fatty acids and phenolic compounds were identified in GC-MS analysis of methanol extract which may be the responsible for the antimicrobial activity. The mechanisms of antimicrobial action of fatty acids are nonspecific modes of action [47]. However, antimicrobial effects of fatty acids were observed to form mostly either by a complete inhibition of oxygen uptake or stimulating uptake of amino acids into the cells, which occurs in a dose dependent manner [48]. Fatty acids intercalate in the phospholipid bilayer of microbes due to their lipophilicity, which increases the permeability of the cell membrane, dissipation of the proton-motive force, and leakage of inorganic ions, leading to cell death [49, 50].

Studies have shown that phenolic compounds have bactericidal action by interfering with bacterial cell adhesins, enzymes, cell envelope, and transport proteins [51]. They also increase the free radical concentration within the bacterial protoplasm and irreversibly complex with nucleophilic amino acids in microbial proteins determining loss of their function [52]. As a result, this causes bacterial cell lysis [53]. The antibacterial activity of methanol extract is not only caused by their major compounds, but it could be due to a synergism among their other components present in it. Hence, the presence of these components in higher quantity in methanol extract of P. wightianus may be responsible for better bioactivity.

4. Conclusions

The findings of present investigation show that agar well diffusion method is ideal for determining the antimicrobial activity of P. wightianus extracts. Methanolic extract of P. wightianus contributed significant activity against most of the tested bacterial pathogens with least MIC and MBC values. Hence, methanol is the best solvent system for extracting the bioactive principles from leaves of P. wightianus which possesses promising antimicrobial principles which may be used in the treatment of infectious diseases caused by pathogenic microbes. The antimicrobial principles from the bioactive extracts may need further purifications to have its synthetic analogues which will be carried out in the future.