Phyllanthus wightianus Müll. Arg.: A Potential Source for Natural Antimicrobial Agents

D Natarajan; R Srinivasan; M S Shivakumar

doi:10.1155/2014/135082

. 2014 May 5;2014:135082. doi: 10.1155/2014/135082

Phyllanthus wightianus Müll. Arg.: A Potential Source for Natural Antimicrobial Agents

D Natarajan ^1,^*, R Srinivasan ¹, M S Shivakumar ²

PMCID: PMC4026884 PMID: 24883301

Abstract

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

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Table 2.

Antimicrobial activity of different solvent extracts of P. wightianus.

S. number	Organisms	Method	Diameter of zone of inhibition (in mm)^#
S. number	Organisms	Method	Methanol	Acetone	Ethyl acetate	Chloroform	Hexane	Control*
1	Bacillus subtilis (MTCC 441)	Well	09.42 ± 0.52^b	07.96 ± 0.08^b	6.66 ± 0.58^b	00.00 ± 0.00^a	00.00 ± 0.00^a	29.74 ± 0.44^c
1	Bacillus subtilis (MTCC 441)	Disc	08.71 ± 0.51^c	06.53 ± 0.50^b	00.00 ± 0.00^a	8.33 ± 0.58^c	08.33 ± 0.58^c	15.94 ± 0.10^d

2	Escherichia coli (MTCC 739)	Well	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	27.56 ± 0.51^b
2	Escherichia coli (MTCC 739)	Disc	07.71 ± 0.51^b	07.67 ± 0.58^b	07.62 ± 0.65^b	08.67 ± 0.58^b	08.70 ± 0.61^b	00.00 ± 0.00^a

3	Klebsiella pneumoniae (MTCC 109)	Well	09.52 ± 0.50^b	10.72 ± 0.63^c	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	29.54 ± 0.51^d
3	Klebsiella pneumoniae (MTCC 109)	Disc	09.60 ± 0.36^c	08.00 ± 0.08^b	09.83 ± 0.48^c	08.67 ± 0.58^b,c	08.72 ± 0.63^b,c	00.00 ± 0.00^a

4	Proteus vulgaris (MTCC 426)	Well	09.56 ± 0.45^b	10.96 ± 0.08^c	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	24.65 ± 0.36^d
4	Proteus vulgaris (MTCC 426)	Disc	09.82 ± 0.32^d	08.74 ± 0.65^c,d	07.67 ± 0.58^b,c	07.05 ± 0.93^b	07.51 ± 0.50^b,c	00.00 ± 0.00^a

5	Pseudomonas aeruginosa (MTCC 741)	Well	12.75 ± 0.66^b	11.65 ± 0.56^b	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	22.67 ± 0.58^c
5	Pseudomonas aeruginosa (MTCC 741)	Disc	11.75 ± 0.44^d	09.83 ± 0.30^c	6.88 ± 0.43^b	00.00 ± 0.00^a	09.64 ± 0.40^c	00.00 ± 0.00^a

6	Salmonella typhimurium (MTCC 98)	Well	09.59 ± 0.66^c	11.77 ± 0.68^d	08.47 ± 0.50^b	00.00 ± 0.00^a	08.47 ± 0.50^b	29.96 ± 0.08^e
6	Salmonella typhimurium (MTCC 98)	Disc	09.67 ± 0.58^c	08.70 ± 0.61^b,c	08.59 ± 0.71^b,c	07.27 ± 1.10^b	07.87 ± 0.23^b	00.00 ± 0.00^a

7	Shigella flexneri (MTCC 1457)	Well	13.80 ± 0.35^d	12.77 ± 0.68^d	07.81 ± 0.33^c	06.51 ± 0.50^b	00.00 ± 0.00^a	15.67 ± 0.58^e
7	Shigella flexneri (MTCC 1457)	Disc	13.84 ± 0.28^d	11.77 ± 0.68^c	07.82 ± 0.45^b	07.78 ± 0.47^b	08.85 ± 0.25^b	00.00 ± 0.00^a

8	Streptococcus pneumoniae (MTCC 655)	Well	29.71 ± 0.51^c	28.55 ± 0.51^c	10.73 ± 0.64^b	08.67 ± 0.58^a	08.00 ± 1.00^a	34.67 ± 0.58^d
8	Streptococcus pneumoniae (MTCC 655)	Disc	18.85 ± 0.25^d	14.54 ± 0.50^c	08.13 ± 0.13^a	08.67 ± 0.58^a,b	09.50 ± 0.50^b	19.52 ± 0.50^d

9	Staphylococcus epidermidis (MTCC 435)	Well	17.83 ± 0.29^b	10.73 ± 0.64^a	17.00 ± 1.00^b	10.67 ± 0.58^a	11.84 ± 0.77^a	29.83 ± 0.29^c
9	Staphylococcus epidermidis (MTCC 435)	Disc	17.93 ± 0.12^b	18.33 ± 0.58^b	07.67 ± 0.58^a	07.87 ± 0.23^a	08.67 ± 0.58^a	17.80 ± 0.72^b

10	Proteus vulgaris	Well	00.00 ± 0.00^a	07.77 ± 0.39^b	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	21.84 ± 0.27^c
10	Proteus vulgaris	Disc	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	10.75 ± 0.67^b

11	Serratia marcescens	Well	00.00 ± 0.00^a	00.00 ± 0.00^a	08.33 ± 0.32^b	00.00 ± 0.00^a	00.00 ± 0.00^a	24.67 ± 0.58^c
11	Serratia marcescens	Disc	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	12.42 ± 1.01^b

Fungal cultures
1	C. albicans	Well	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	00.000 ± 0.0	00.00 ± 0.00^a
1	C. albicans	Disc	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a

2	C. neoformans	Well	10.41 ± 0.52^d	07.56 ± 0.39^c	09.78 ± 0.39^d	06.51 ± 0.50^b	00.00 ± 0.00^a	00.00 ± 0.00^a
2	C. neoformans	Disc	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	00.00 ± 0.00^a	27.29 ± 10.3^b

3	M. racemosus	Well	07.67 ± 0.58^b	09.07 ± 0.40^b,c	09.67 ± 0.58^b,c	09.73 ± 0.46^b,c	10.67 ± 0.58^c	00.00 ± 0.00^a
3	M. racemosus	Disc	00.00 ± 0.00^a	00.00 ± 0.00^a	07.93 ± 0.12^c	06.80 ± 0.35^b	09.07 ± 0.12^c	00.00 ± 0.00^a

4	A. niger	Well	09.67 ± 0.58^d	07.93 ± 0.12^b,c	09.77 ± 0.40^c,d	07.77 ± 0.25^b	09.57 ± 0.51^d	00.00 ± 0.00^a
4	A. niger	Disc	07.80 ± 0.35^b,c	06.67 ± 0.58^b	07.67 ± 0.58^b,c	00.00 ± 0.00^a	08.67 ± 0.58^d	00.00 ± 0.00^a

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*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)	>1000^a	>1000^a	>1000^a	>1000^a	>1000^a	3.9^b

Escherichia coli (MTCC 739)	>1000^a	>1000^a	>1000^a	>1000^a	>1000^a	15.62^b

Klebsiella pneumoniae (MTCC 109)	500^b	>1000^a	>1000^a	>1000^a	>1000^a	7.81^c

Proteus vulgaris (MTCC 426)	>1000^a	>1000^a	>1000^a	>1000^a	>1000^a	15.62^b

Pseudomonas aeruginosa (MTCC 741)	250^c	250^c	500^b	>1000^a	>1000^a	31.25^d

Salmonella typhimurium (MTCC 98)	500	>1000^a	>1000^a	>1000^a	>1000^a	15.62

Shigella flexneri (MTCC 1457)	125^d	250^c	500^b	>1000^a	>1000^a	31.25^e

Streptococcus pneumoniae (MTCC 655)	15.62^d	31.25^c	250^b	>1000^a	>1000^a	3.9^e

Staphylococcus epidermidis (MTCC 435)	31.25^d	62.5^c	500^b	>1000^a	>1000^a	3.9^c

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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)	>1000^a	>1000^a	>1000^a	>1000^a	>1000^a	15.62^b

Escherichia coli (MTCC 739)	>1000^a	>1000^a	>1000^a	>1000^a	>1000^a	31.25^b

Klebsiella pneumoniae (MTCC 109)	>1000^a	>1000^a	>1000^a	>1000^a	>1000^a	15.62^b

Proteus vulgaris (MTCC 426)	>1000^a	>1000^a	>1000^a	>1000^a	>1000^a	31.25^b

Pseudomonas aeruginosa (MTCC 741)	500^b	500^b	500^b	>1000^a	>1000^a	31.25^c

Salmonella typhimurium (MTCC 98)	500^b	>1000^a	>1000^a	>1000^a	>1000^a	31.25^c

Shigella flexneri (MTCC 1457)	250^c	250^c	500^b	>1000^a	>1000^a	62.5^d

Streptococcus pneumoniae (MTCC 655)	15.62^d	62.5^c	500^b	>1000^a	>1000^a	3.9^e

Staphylococcus epidermidis (MTCC 435)	31.25^c	125^b	1000^a	>1000^a	>1000^a	3.9^d

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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%).

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

Open in a new tab

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.

Acknowledgments

The authors are gratefully thankful to the University Grants Commission (UGC), New Delhi (reference no. 37-296/2009 (SR)) for providing financial assistance and the Department of Biotechnology, Periyar University, Salem, for the necessary facilities.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

References

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[B1] 1.Das S, Sarkar A, Seth A, Gupta N, Agrawal RC. Evaluation of in-vitro antibacterial potential of ripe fruits of Aegle marmelos . International Journal of Pharmacy and Pharmaceutical Sciences. 2012;4(3):179–181. [Google Scholar]

[B2] 2.Doudach L, Meddah B, Alnamer R, Chibani F, Cherrah Y. In vitro antibacterial activity of the methanolic and aqueous extracts of Anacyclus pyrethrum used in Moroccan traditional medicine. International Journal of Pharmacy and Pharmaceutical Sciences. 2012;4(3):402–405. [Google Scholar]

[B3] 3.Termentzi A, Fokialakis N, Skaltsounis AL. Natural resins and bioactive natural products thereof as potential anitimicrobial agents. Current Pharmaceutical Design. 2011;17(13):1267–1290. doi: 10.2174/138161211795703807. [DOI] [PubMed] [Google Scholar]

[B4] 4.Paraschos S, Mitakou S, Skaltsounis AL. Chios gum mastic: a review of its biological activities. Current Medicinal Chemistry. 2012;19(14):2292–2302. doi: 10.2174/092986712800229014. [DOI] [PubMed] [Google Scholar]

[B5] 5.Gibbons S. Plants as a source of bacterial resistance modulators and anti-infective agents. Phytochemistry Reviews. 2005;4(1):63–78. [Google Scholar]

[B6] 6.Singha PK, Roy S, Dey S. Antimicrobial activity of Andrographis paniculata . Fitoterapia. 2003;74(7-8):692–694. doi: 10.1016/s0367-326x(03)00159-x. [DOI] [PubMed] [Google Scholar]

[B7] 7.Bakkali F, Averbeck S, Averbeck D, Idaomar M. Biological effects of essential oils—a review. Food and Chemical Toxicology. 2008;46(2):446–475. doi: 10.1016/j.fct.2007.09.106. [DOI] [PubMed] [Google Scholar]

[B8] 8.Jeon J-G, Rosalen PL, Falsetta ML, Koo H. Natural products in caries research: current (limited) knowledge, challenges and future perspective. Caries Research. 2011;45(3):243–263. doi: 10.1159/000327250. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9.Kim SH, Lee SJ, Lee JH, Sun WS, Kim JH. Antimicrobial activity of 9-O-acyl- and 9-O-alkylberberrubine derivatives. Planta Medica. 2002;68(3):277–281. doi: 10.1055/s-2002-23128. [DOI] [PubMed] [Google Scholar]

[B10] 10.Attar-ur-Rahman, Choudhary MI. Diterpenoid and steroidal alkaloids. Natural Product Reports. 1995;12(4):361–379. doi: 10.1039/np9951200361. [DOI] [PubMed] [Google Scholar]

[B11] 11.Kurek A, Grudniak AM, Kraczkiewicz-Dowjat A, Wolska KI. New antibacterial therapeutics and strategies. Polish Journal of Microbiology. 2011;60(1):3–12. [PubMed] [Google Scholar]

[B12] 12.Vermani K, Garg S. Herbal medicines for sexually transmitted diseases and AIDS. Journal of Ethnopharmacology. 2002;80(1):49–66. doi: 10.1016/s0378-8741(02)00009-0. [DOI] [PubMed] [Google Scholar]

[B13] 13.Ahmad I, Mehmood Z, Mohammad F. Screening of some Indian medicinal plants for their antimicrobial properties. Journal of Ethnopharmacology. 1998;62(2):183–193. doi: 10.1016/s0378-8741(98)00055-5. [DOI] [PubMed] [Google Scholar]

[B14] 14.Essawi T, Srour M. Screening of some Palestinian medicinal plants for antibacterial activity. Journal of Ethnopharmacology. 2000;70(3):343–349. doi: 10.1016/s0378-8741(99)00187-7. [DOI] [PubMed] [Google Scholar]

[B15] 15.Natarajan D, Shivakumar MS, Srinivasan R. Antibacterial activity of leaf extracts of Biophytum sensitivum (L.) DC. Journal of Pharmaceutical Sciences and Research. 2010;2(11):717–720. [Google Scholar]

[B16] 16.Radcliffe-Smith A. Euphorbiaceae. In: Pope GV, editor. Flora Zambesiaca. 9 (4) Kew, UK: Royal Botanic Gardens; 1999. p. p. 334. [Google Scholar]

[B17] 17.Mohanasundari C, Natarajan D, Srinivasan K, Anbuganapathi G, Gowrishankar J, Perumal G. Antibacterial efficacy of leaf extracts of Phyllanthus wightianus Müll. Arg. Journal of Phytological Research. 2005;18(2):171–173. [Google Scholar]

[B18] 18.Sengottuvel R, Srinivasan K, Mohanasundari C, Natarajan D, Perumal G. Screening of antimicrobial properties of leaf extracts of Smilax zeylanica and Phyllanthus wightianus . Advances in Plant Sciences. 2007;20(1):273–275. [Google Scholar]

[B19] 19.Natarajan D, Srinivasan R, Shivakumar MS. Phyllanthus wightianus Müll. Arg.: a potential source for antibacterial and phytochemical analysis. International Journal of Pharmacy and Pharmaceutical Sciences. 2012;4(5):241–245. [Google Scholar]

[B20] 20.Srinivasan R, Shivakumar MS, Natarajan D. Larvicidal efficacy of Phyllanthus wightianus Müll. Arg. on Aedes aegypti . In: Tyagi BK, editor. Proceedings of the 5th Conference of Medical Arthropodology; 2012; Madurai, India. pp. 37–43. [Google Scholar]

[B21] 21.Shivakumar MS, Srinivasan R, Natarajan D. Larvicidal potential of some Indian medicinal plant extracts against Aedes aegypti (L.) Asian Journal of Pharmaceutical and Clinical Research. 2013;6(3):77–80. [Google Scholar]

[B22] 22.Valarmathi R, Scnthamarai R, Akilandeswari S. Analgesic and antimicrobial activity of the dried root extracts of Reidia floribunda Wight. Hamdard Medicus. 2009;52(2):18–23. [Google Scholar]

[B23] 23.Priya OS. Wound healing activity of Phyllanthus wightianus . Proceedings of the International Herbal Conference; 2009; Bangalore, India. [Google Scholar]

[B24] 24.Priya OS, Viswanathan MBG, Balakrishna K, Venkatesan M. Chemical constituents and in vitro antioxidant activity of Phyllanthus wightianus . Natural Product Research. 2011;25(10):949–958. doi: 10.1080/14786419.2010.517203. [DOI] [PubMed] [Google Scholar]

[B25] 25.National Committee for Clinical Laboratory Standards (NCCLS) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. 7th edition. M7-A7. Wayne, Pa, USA.: National Committee on Clinical Laboratory Standards (NCCLS); 2006. [Google Scholar]

[B26] 26.Eloff JN, Katerere DR, McGaw LJ. The biological activity and chemistry of the southern African Combretaceae. Journal of Ethnopharmacology. 2008;119(3):686–699. doi: 10.1016/j.jep.2008.07.051. [DOI] [PubMed] [Google Scholar]

[B27] 27.Khan R, Islam B, Akram M, et al. Antimicrobial activity of five herbal extracts against Multi Drug Resistant (MDR) strains of bacteria and fungus of clinical origin. Molecules. 2009;14(2):586–597. doi: 10.3390/molecules14020586. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B28] 28.Tanaka JCA, da Silva CC, Filho BPD, Nakamura CV, de Carvalho JE, Foglio MA. Chemical constituents of Luehea divaricata Mart. (Tiliaceae) Quimica Nova. 2005;28(5):834–837. [Google Scholar]

[B29] 29.Dall’Agnol R, Ferraz A, Bernardi AP, et al. Antimicrobial activity of some Hypericum species. Phytomedicine. 2003;10(6):511–516. doi: 10.1078/094471103322331476. [DOI] [PubMed] [Google Scholar]

[B30] 30.Jagessar RC, Mars A, Gomes G. Selective Antimicrobial properties of Phyllanthus acidus leaf extract against Candida albicans, Escherichia coli and Staphylococcus aureus using Stokes Disc diffusion, Well diffusion, Streak plate and a dilution method. Natural Science. 2008;6(2):24–38. [Google Scholar]

[B31] 31.Onocha PA, Opegbemi AO, Kadri AO, Ajayi KM, Okorie DA. Euphorbiaceae plants 1: Phyllanthus amarus and Phyllanthus muellerianus leaf extracts. Nigerian Journal of Natural Product of Medicine. 2003;7:9–12. [Google Scholar]

[B32] 32.Ashokkumar R, Ramaswamy M. Comparative study on the antimicrobial activity of leaf extracts of four selected Indian medicinal plants against Pseudomonas aeruginosa, Pseudomonas fluorescens, Penicillium chrysogenum and Penicillium restrictum . Journal of Chemical Biology Physical Science. 2013;3(2):1279–1282. [Google Scholar]

[B33] 33.Chandrashekar KS, Satyanarayana, Prasanna KS. Antimicrobial activity of Phyllanthus debilis . International Research Journal of Pharmacy. 2011;2(6):75–76. [Google Scholar]

[B34] 34.de Britto J, Gracelin DHS, Sebastian SR. Antibacterial activity of a few medicinal plants against Xanthomonas campestris and Aeromonas hydrophila . Journal of Biopesticides. 2011;4(1):57–60. [Google Scholar]

[B35] 35.Mathur M, Sharma R, Sharma J, Pareek R, Kamal R. Phytochemical screening and antimicrobial activity of Phyllanthus niruri Linn. Applied Botany. 2012;46:8487–8489. [Google Scholar]

[B36] 36.Sivaraj R, Balakrishnan A, Thenmozhi M, Venckatesh R. Preliminary phytochemical analysis of Aegle marmelos, Ruta graveolens, Opuntia dellini, Euphorbia royleana and Euphorbia antiquoru . International Journal of Pharmaceutical Science Research. 2011;2(1):146–150. [Google Scholar]

[B37] 37.Proestos C, Chorianopoulos N, Nychas E, Komaitis M. RP-HPLC analysis of the phenolic compounds of plant extracts. Investigation of their antioxidant capacity and antimicrobial activity. Journal of Agricultural and Food Chemistry. 2005;53(4):1190–1195. doi: 10.1021/jf040083t. [DOI] [PubMed] [Google Scholar]

[B38] 38.Lee J-C, Kim H-R, Kim J, Jang Y-S. Antioxidant property of an ethanol extract of the stem of Opuntia ficus-indica var. Saboten . Journal of Agricultural and Food Chemistry. 2002;50(22):6490–6496. doi: 10.1021/jf020388c. [DOI] [PubMed] [Google Scholar]

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PERMALINK

Phyllanthus wightianus Müll. Arg.: A Potential Source for Natural Antimicrobial Agents

D Natarajan

R Srinivasan

M S Shivakumar

Abstract

1. Introduction

2. Materials and Methods

2.1. Plant Material

2.2. Preparation of Extracts

2.3. Used Microorganisms

2.4. Agar Well Diffusion Method

2.5. Disc Diffusion Method

2.6. Minimum Inhibitory Concentration (MIC)

2.7. Minimum Bactericidal Concentration (MBC)

2.8. GC-MS Analysis

2.9. Statistical Analysis

3. Results and Discussion

Table 1.

Table 2.

Table 3.

Table 4.

Figure 1.

Table 5.

4. Conclusions

Acknowledgments

Conflict of Interests

References

ACTIONS

PERMALINK

RESOURCES

Cite

Add to Collections

PERMALINK

Phyllanthus wightianus Müll. Arg.: A Potential Source for Natural Antimicrobial Agents

D Natarajan

R Srinivasan

M S Shivakumar

Abstract

1. Introduction

2. Materials and Methods

2.1. Plant Material

2.2. Preparation of Extracts

2.3. Used Microorganisms

2.4. Agar Well Diffusion Method

2.5. Disc Diffusion Method

2.6. Minimum Inhibitory Concentration (MIC)

2.7. Minimum Bactericidal Concentration (MBC)

2.8. GC-MS Analysis

2.9. Statistical Analysis

3. Results and Discussion

Table 1.

Table 2.

Table 3.

Table 4.

Figure 1.

Table 5.

4. Conclusions

Acknowledgments

Conflict of Interests

References

ACTIONS

PERMALINK

RESOURCES

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