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

Authors: D. Natarajan; R. Srinivasan; M. S. Shivakumar
Journal: BioMed Research International
 (2014)

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## 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.


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## Body

## 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 synthesizede 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 ofBerberis 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 ofP. wightianus Müll. Arg.


## 2. Materials and Methods

### 2.1. Plant Material

Fresh, matured, uninfected leaves ofP. 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 asP. vulgaris (MTCC 426),P. aeruginosa (MTCC 741),K. pneumoniae (MTCC 109),S. typhimurium (MTCC 98),E. coli (MTCC 739), andS. flexneri (MTCC 1457), with two clinical isolates (P. vulgaris andS. marcescens) and 4 fungal pathogens (C. albicans, C. neoformans, M. racemosus, andA. 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).


## 2.1. Plant Material

Fresh, matured, uninfected leaves ofP. 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 asP. vulgaris (MTCC 426),P. aeruginosa (MTCC 741),K. pneumoniae (MTCC 109),S. typhimurium (MTCC 98),E. coli (MTCC 739), andS. flexneri (MTCC 1457), with two clinical isolates (P. vulgaris andS. marcescens) and 4 fungal pathogens (C. albicans, C. neoformans, M. racemosus, andA. 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 Table1. The results of antimicrobial activity ofP. wightianus were given in Table 2. All the extracts ofP. 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 againstS. pneumoniae (29 mm) followed byS. epidermidis (17 mm). The considerable amount of antibacterial activity was observed in acetone extract againstS. pneumoniae (28 mm) followed byS. flexneri (12 mm). In disc diffusion method, methanol extract exhibits good antimicrobial activity and the maximum growth inhibition was observed inS. pneumoniae (18 mm) followed byS. epidermidis (17 mm). Acetone extract having well to moderate antimicrobial activity and maximum activity was detected inS. epidermidis (18 mm) followed byS. 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 againstC. neoformans (10 mm) followed by hexane extract againstM. 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 ofP.  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
4.61Table 2
Antimicrobial activity of different solvent extracts ofP. 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 (Tables3 and 4) indicate that the crude extracts ofP. wightianus inhibited the growth of selected bacterial species. The lowest MIC and MBC value were detected in methanolic extract againstS. pneumoniae (15.62 μg/mL) andS. 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 (atP<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 (atP<0.05) when subject to Tukey’s multiple comparison test.The results highlights that significant antibacterial activity was observed in methanol extract againstS. flexneri, S. pneumoniae, andS. epidermidis. Similar observations were made withP. 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 ofP. 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 manyPhyllanthus species, such asP. acidus [30],P. muellerianus [31],P. amarus, P. maderaspatensis [32],P. debilis [33],P. amarus, P. emblica [34], andP. 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 (%) (Figure1 and Table 5). Methnolic extract ofP. 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%).Table 5
Compounds detected in methanol leaf extract ofP. 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-olFigure 1
GC-MS chromatogram of methanol leaf extract ofP. wightianus.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 inS. aureus andE. 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 ofP. 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 ofP. wightianus extracts. Methanolic extract ofP. 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 ofP. 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.



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*Source: 135082-2014-05-05.xml*