chapter 3 antibacterial study -...

Chapter 3 Antibacterial Study

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3.1. INTRODUCTION

Bacterial diseases are a type of infectious diseases caused by pathogenic bacteria

(Solanki, 2010). Diseases caused by bacteria, viruses, fungi and other parasites are major

causes of death, disability, and social and economic disruption for millions of people

(Breman et al., 2004; WHO, 2008; UNAIDS, WHO, 2008). The frequency of life-

threatening infections caused by pathogenic microorganisms has increased worldwide

and is becoming an important cause of morbidity and mortality in immunocompromised

patients in developing countries (Al-Bari et al., 2006). The increasing prevalence of

multi-drug resistant strains of bacteria and the recent appearance of strains with reduced

susceptibility to antibiotics raised the specter of ‘untreatable’ bacterial infections and

adds urgency to the search for new infection-fighting strategies (Zy et al., 2005; Rojas et

al., 2006).

In India around 20,000 medicinal plant species have been recorded recently (Dev,

1997), however 500 traditional communities use about 800 plant species for curing

different diseases (Kamboj, 2000). Currently 80 % of the world population depends on

plant-derived medicine for the first line of primary health care for human alleviation

(Veale et al., 1992). According to World Health Organization, medicinal plants would be

the best source to obtain a variety of drugs. Therefore, such plants should be investigated

to better understand their properties, safety and efficacy (Nascimento et al., 2000) and

wide application for maintenance of good health.


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As mentioned earlier, the state of Tripura is situated in one of the mega diversity

region of the world because of her location in the sub-Himalayan area and the state is

very rich in flora and fauna. Two medicinal plants of the state with known ethno

medicinal properties have been collected and systematically investigated regarding their

medicinal properties. The chapter deals with the antibacterial properties of the studied

plants.

3.2. REVIEW OF LITERATURE

The scientific literature is full of reports of antimicrobial activity of plants and

their secondary metabolites (Erdemeier et al., 1996, Hassan and Ahmed, 1996; Darokan

et al., 1999; Cutter, 2000; Babu et al., 2002) and plants have always been a source of

potential antibacterial drugs (Iyengar, 1985; Chopra et al., 1992; Behl and Arora, 1993;

D'Souza, 1993; Youvraj et al., 1995; Ibrahim and Osman, 1995). The effects of plant

extracts on bacteria have been studied by a very large number of researchers in different

parts of the world (Santos Filho et al., 1990; Nascimento et al., 1990; Anesini and Perez,

1993; Akpulu et al., 1994; Saxena et al. 1994; Perez and Anesini, 1994; Alonso-Paz et

al., 1995; Vlietinck et al., 1995; Martinez et al., 1996; Vincenzo et al. 1998; Kudi et al.,

1999; Larshini et al. 1999; Kelmanson et al., 2000; Gislene et al. 2000; Reddy et al.,

2001; Ateb and ErdoUrul, 2003; Ajaiyeoba, 2002; Janovska et al., 2003; Abu-Shanab et

al., 2004; Seher et al., 2006; Rojas et al., 2006; El-Mahmood and Ameh 2007; Mothana

et al. 2008; Sassi et al. 2008; Rahman et al., 2008; Ibrahim et al. 2009; Koshy et al.


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2009; Dogan et al., 2010; Joshi et al., 2011; Obeidat et al., 2011; Bisi-Johnson et al.,

2011).

Regarding the antibacterial assay of Indian traditional medicinal plants, Ahmed et

al., (1998) tested eighty two Indian traditional medicinal plants against pathogenic and

opportunistic microorganisms. The results indicated that fifty six out of eighty two

exhibited activity against one or more test pathogens. The great potentiality of spices was

studied by Arora and Kaur in the same year and they suggested that spices might have a

great potential to be used as antimicrobial agents. Samy and Ignacimuthu (2000) tested

30 Indian folklore medicinal plants used by traditional healers for antimicrobial activity.

Among them, the leaf extracts of Cassia occidentalis and Cassia corniculata exhibited

significant broad-spectrum antibacterial activity against Bacillus subtilis and

Staphylococcus aureus. Nair et al., (2005) observed potentiality of nine Indian medicinal

plants as antibacterial agents and found that Sapindus emarginatus, out of these nine

plants showed strong activity against the tested bacterial strains. In the year 2006,

Duraipandiyan et al., assessed antimicrobial activity of eighteen Indian plant extracts that

are used in folk medicine in the treatment of skin diseases, venereal diseases, respiratory

problems and nervous disorders against nine bacterial strains (Bacillus subtilis,

Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia

coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Ervinia sp, Proteus vulgaris) and

one fungal strain (Candida albicans). They observed that out of 18 plants, 10 plants

exhibited antimicrobial activity against one or more of the tested microorganisms at three

different concentrations of 1.25, 2.5 and 5 mg/disc. Among the plants tested, Acalypha


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fruticosa, Peltophorum pterocarpum, Toddalia asiatica,Cassia auriculata, Punica

granatum and Syzygium lineare were most active. The ethnobotanical efficacy of Indian

medicinal plants; Achyranthes aspera, Artemisia parviflora, Azadirachta indica,

Calotropis gigantean, Lawsonia inermis, Mimosa pudica, Ixora coccinea, Parthenium

hysterophorus and Chromolaena odorata were examined by Sukanya et al. in the year,

2009 against clinical bacteria (Escherichia coli and Staphylococcus aureus) and

phytopathogenic bacteria (Xanthomonas vesicatoria and Ralstonia solanacearum).

Among treatments, maximum in vitro inhibition of tested bacteria E. coli, S. aureus, X.

vesicatoria and R. solanaccearum was scored in methanol extracts of C. odorata which

offered inhibition zone of 10, 9, 12 and 12 mm respectively. Pavithra et al. (2010)

assessed the activity of five traditional Indian medicinal plants (Delonix elata,

Enicostemma axillare, Merremia tridentata, Mollugo cerviana and Solanum incanum)

used for treatment of various diseases against two gram positive (Bacillus subtilis,

Staphylococcus aureus) and three gram negative (Escherichia coli, Klebsiella

pneumoniae, Pseudomonas aeruginosa) bacterial strains. Results revealed that the

chloroform and methanol extracts of D. elata and methanol extracts of M. cerviana

exhibited significant antibacterial activity against gram-positive and gram negative

strains. In vitro antibacterial activity of crude leaf extracts of three plants, Tecoma stans,

Coleus forskohlii and Pogostemon patchouli commonly practiced medicinal plants in the

villages of Salem District, Tamilnadu (India) was investigated by Senthil Kumar et al.

(2010) against five human pathogenic bacteria Staphylococcus aureus, Staphylococcus

epidermidis, Salmonella typhi, Klebsiella pneumoniae and Vibrio parahemolyticus.


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Maximum growth inhibition and effectiveness was remarkably observed in the extracts of

Coleus forskohlii, Pogostemon patchouli and then in Tecoma stans. Abhilash et al., in

the same year evaluated seven Indian plant extracts Moringa oleifera (Murungai) , Musa

paradisiaca (Bananana), Azardiratica indica (Neem), Solanum melongena (kathirikai),

Cynodon dactylon(Grass), Alternanthera sessilis (Ponnangkani) and Anisochilus

carnosus (Karpooravalli) for activity against four different microorganisms Escherichia

coli, Bacillus subtilis, Vibrio cholerae and Klebsiella pneumoniae. The ethanolic extract

of Azardiratica indica showed considerably high activity against Escherichia coli than

other extracts. Leaf extracts of Aloe vera, Datura stromonium, Pongamia pinnata,

Lantana camara , Calotropis procera was studied by Johnson et al. in the year 2011 for

antimicrobial activity against Staphylococcus, E.coli and Aspergillus species. Results

concluded that Aloe vera had the highest and strong activity against S.aureus and E.coli.

Lanata camara does not show antibacterial and antifungal activity. Pongamia pinnata

aqueous extract showed better anti E.coli activity than its alcoholic extract. Datura

stramonium showed better activity against Staphylococcus aureus & showed little anti

Aspergillus activity. Calotropis procera showed antibacterial activity against

Staphyloccus aureus & E.coli and did not show anti-Aspergillus activity.

Ethno medicinal use of Parkia species have been reported by many authors

(Irvine, 1961; Sinha, 1996; Nacoulma, 1999; Bhardwaj and Gakhar, 2005; Majumder et

al., 2009). Antibacterial and antifungal properties of P. biglobosa and P. bicolor were

reported by Ajaiyeoba (2002). The screening was done using standard strains of

Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa,


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Aspergillus niger and Candida utilis and broad spectrum activity of the extracts was

reported.

Ethnomedicinal uses of Evolvulus species have also been reported by many

authors (Chitralekha et al., 1964; Jain, 1991; Manandhar and Manandhar, 2002; Goyal &

Singh, 2005; Saini et al., 2007). Anti helminthic activity of E. nummularius has been

reported by Dash et al., (2003). Pavithra et al., (2009), studied antibacterial properties of

E. nummularius. According to them the methanol extract was found to be active against

Escherichia coli and Bacillus subtilus and MIC values were found to be 12.50 mg/ml and

3.125 mg/ml respectively.

3.3. MATERIALS AND METHODS

3.3.1. Preparation of plant extract

The plant material, stem bark and leaves of Parkia javanica (P. javanica) and

whole plant of Evolvulus nummularius (E. nummularius) were air-dried in shade and

ground (Dilika et al., 1996; Baris et al., 2006).

5 kilo gram (kg) grinded powder of P. javanica was extracted in 15 liter (lit) of

methanol by percolation method. The mixtures were kept for 2-5days in tightly sealed

vessels at room temperature at 220C, protected from sunlight. This mixture was filtered

through muslin cloth and the residue, was adjusted to the required concentration (15 lit of

methanol for the residue of 5 kg of powdered plant material) with the extraction fluid for

further extraction. Further extraction of the residue was repeated 3-5 times until a clear


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colorless supernatant extraction liquid was obtained indicating that no more extraction

from the plant material was possible.

Similarly, 1.5 kg grinded powder of E. nummularius aerial part was extracted in

5.0 lit of methanol by percolation method. The mixtures were kept for 2-5days in tightly

sealed vessels at room temperature. This mixture was filtered and processed as mentioned

above.

3.3.2. Removal of solvent

The total methanol extract was subjected to rota-evaporation to remove the

methanol. After drying, the residues were weighed in order to know the amount of extract

of each individual plant and percentage of yield. The % yield of crude plant extract was

calculated by using the following formula:

% yield of crude plant extract = (Wt. of dry extract) × 100/ Wt. of sample powder taken

After rota evaporation 560 g of gummy residue of P. javanica was obtained giving

11.2 % yield and 25 g of gummy residue of E. nummularius was obtained giving 1.67 %

yield.

3.3.3. Partitioning of methanol extract

After solvent removal, the extract was successively partitioned between water

(H2O) and benzene (Bz), H2O and chloroform (CHCl3), and H2O and n-butanol (BuOH).

The fractions were concentrated following the same procedure using rotavapor (Natrori et

al., 1981). These fractions along with the crude methanol part were sterilized by passing

through 0.22µM - syringe filter before studying their biological activity.


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3.3.4. Preparation of stock solution

The calculated amount of each dried plant extract obtained from different solvents

were dissolved in a calculated volume of 1:1 ethanol/ DMSO solvent (used as control) in

the ratio of 500mg/ml to make stock solution and were used for determination of Zone of

inhibition against different bacteria.

3.3.5. Culture media

Culture medium used for growth of bacteria was Nutrient broth, Nutrient Agar,

Mueller Hinton Agar purchased from HiMedia..

3.3.6. Bacterial strains

Bacterial strains used during the investigation were kind gift of Dr. Prasanta Bag,

Dept. of Immunology and Microbiology, Ballygaunj Science College, Kolkata. Three

standard strains of gram-positive bacteria Bacillus subtilis, Staphylococcus aureus,

Micrococcus luteus, and three standard strains of gram negative bacteria, Pseudomonas

aeruginosa, Klebsiella aerogens and Escherichia coli were used.

3.3.7. Preparation of stock culture

The stock culture of each organism was prepared by taking two nutrient agar

slants and sub culturing each confirmed test organism aseptically. One set slant was kept

as stock culture and another as working set. The cultures of bacteria in their appropriate

agar slants were stored at 4°C and used as stock cultures. One counter glycerol stock was

also maintained at -20oC temperature.

3.3.8. The assay methodology


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Antibacterial activity of plant extracts was determined by using the agar cup-plate

diffusion and dilution method (Cruickshank et al., 1975; Sousa et al., 1991). On each

sterile Nutrient Agar plate 100 l of bacterial suspension was added by a glass spreader.

On each dry bacteria seeded plate wells of 5 mm diameter was made by a sterilized micro

tip and solution (25 μl) of the extract was added on each well and on one well only

solvent was used as a control. The plates were kept in this condition for 30 minutes so

that solutions in the wells can spread through the agar medium. Then they were incubated

at 37oC for 24 hr. After this period, the positive antibacterial activity was ascertained by

measuring the growth inhibition zone.

3.3.9. Determination of Minimum Inhibitory Concentration (MIC)

Two fold serial Dilution Technique was used to determine the Minimum

Inhibitory Concentration (MIC). This technique was recommended by WHO (1991) and

used successively by other groups (Devkota et al., 2000; Pokharel 2000 and Devkota and

Dutta 2001).

3.4. RESULTS

Antibacterial activity of two study plants- Parkia javanica and Evolvulus

nummularius, was carried out on three gram positive bacteria namely Bacillus subtilis,

Staphylococcus aureus, Micrococcus luteus and three gram negative bacteria namely

Pseudomonas aeruginosa, Klebsiella aerogens and Escherichia coli.

The extraction was first done with methanol and further fractionated with water

(H2O) and organic solvents- benzene (Bz), chloroform (CHCl3), and n-butanol (BuOH)


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according to increasing polarity. The activity of each fraction was separately determined.

The result of antibacterial activity of the crude methanol extracts of P. javanica and E.

nummularius are summarized in Table 3.1 and Table 3.2. Crude extract of P. javanica

showed activity against all the three strains of gram positive and one gram negative

bacteria (P. aeruginosa) (Table 3.1). E. nummularius crude extract showed activity

against two gram positive (Bacillus subtilis, Micrococcus luteus) and two gram negative

bacteria (Pseudomonas aeruginosa, Escherichia coli) (Table 3.2). Therefore both the

plant extracts were active against both gram positive and gram negative bacteria. The

plant extracts showed maximum activity against P. aeruginosa (more than 15 mm)

(Table 3.1, Table 3.2). Klebsiella aerogens did not show any sensitivity towards any

extract (Table 3.1, Table 3.2). In addition, E. nummularius was also inactive against

Staphylococcus aureus (Table 3.2).

Activity of all the three fractions of P. javanica crude extract i.e., benzene,

chloroform and n- butanol soluble fractions, are presented in Table 3.3, 3.4, and 3.5. P.

javanica benzene extract showed activity against B. subtilis (16 mm), S. aureus (13 mm),

M. luteus (18 mm) and P. aeruginosa (21 mm) at 250 mg/ ml. Chloroform fraction

exhibited activity against B. subtilis (18 mm), S. aureus (11 mm), M. luteus (18 mm) and

P. aeruginosa (20 mm) at 250 mg/ ml. Similarly, butanol fraction showed activity against

B. subtilis (17 mm), S. aureus (15 mm), M. luteus (18 mm) and P. aeruginosa (21 mm) at

250 mg/ ml. No activity was observed against bacteria K. aerogens and E. coli. Bacteria

P. aeruginosa showed maximum sensitivity towards all the three fractions of P. javanica.

Activity of the fractions are similar to the activity of the crude extract.


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Activity of all the solvent extracts of E. nummularius i.e., benzene, chloroform

and n- butanol soluble fractions, are presented in Table 3.6, 3.7, and 3.8. Benzene soluble

fraction of E. nummularius crude extract showed activity against B. subtilis (10 mm) and

P. aeruginosa (14 mm) at 250 mg/ ml. Chloroform soluble extract showed against B.

subtilis (11 mm) at 250 mg/ ml. The butanol soluble fraction showed little activity against

M. luteus (8 mm), P. aeruginosa (10 mm) and E. coli (8 mm) at 250 mg/ ml.

The minimal inhibitory concentration (MIC) values of crude extracts and its

fractions were determined by using two fold dilution technique as recommended by

WHO. The values are presented in Table 3.9. The lowest the MIC value means highest

the activity. The lowest MIC value in case of P. javanica extracts was found against

bacterium P. aeruginosa (5 mg/ ml), in case of E. nummularius lowest MIC value was

found against bacteria B. subtilis and P. aeruginosa (40 mg/ ml).

Experimental results of antibacterial activity of plant extracts corroborate with the

ethno medicinal uses of the medicinal plants.


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Table 3.1. Antibacterial activity of P. javanica methanol (MeOH) extract against different

bacterial strains.

Conc.

(mg/ml)

Mean value of diameter of Inhibition zone (mm) ± SD

B. subtilis S. aureus

M. luteus P.

aeruginos

a

K.

aerogens

E.

coli

1 0 0 0 0 0 0

5 0 0 0 13 ± 0.158 0 0

10 0 0 0 14 ± 0.158 0 0

20 0 10 ± 0.158 0 16 ± 0.158 0 0

40 11 ± 0.224 11 ± 0.158 0 16 ± 0.158 0 0

60 12 ± 0.158 11.5 ± 0.158 0 16 ± 0.224 0 0

80 13 ± 0.122 13 ± 0.158 14.5 ± 0 18 ± 0.158 0 0

100 14 ± 0.122 12 ± 0.158 14.5± 0.158 18.5 ±

0.158

0 0

250 17.5± .158 13.5 ± 0.418 19 ± 0.224 21 ± 0.158 0 0


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Table 3.2. Antibacterial activity of E. nummularius MeOH extract against different

bacterial strains.

Conc.

(mg/ml)

Mean Value of Diameter of Inhibition zone (mm) ± SD

B.

subtilis

S.

aureus

M.

luteus

P.

aeruginosa

K.

aerogens

E. coli

1 0 0 0 0 0 0

5 0 0 0 0 0 0

10 0 0 0 0 0 0

20 0 0 0 0 0 0

40 11 ± 0.158 0 0 12 ± 0.418 0 0

60 11 ± 0.158 0 0 12 ± 0.224 0 0

80 12 ± 0.5 0 7.5 ± 0 13 ± 0.418 0 8 ± 0.158

100 13.5 ± 0.224 0 8 ± 0.158 15 ± 0.5 0 8 ± 0.418

250 14 ± 0.418 0 8 ± 0.418 15.5 ± 0.158 0 8 ± 0.224


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Table 3.3. Antibacterial activity of P. javanica benzene (Bz) extract against different

bacterial strains.

Conc.

(mg/ml)


B. subtilis S. aureus M. luteus P.

aeruginos

a

K.

aerogens

E. coli

1 0 0 0 0 0 0

5 0 0 0 12 ± 0.224 0 0

10 0 0 0 13 ± 0.158 0 0

20 11 ± 0.418 10 ± 0.158 0 15 ± 0 0 0

40 11 ± 0 10.5 ± 0.224 0 16 ± 0.158 0 0

60 11 ± 0.158 11.5 ± 0.224 0 16 ± 0.122 0 0

80 11 ± 0.122 12 ± 0.224 0 17 ± 0.5 0 0

100 13.5 ± 0.122 11.5 ± 0.122 14.5 ± 0.122 18 ± 0.158 0 0

250 16 ± 0.158 13 ± 0.418 18 ± 0.158 21 ± 0.5 0 0


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Table 3.4. Antibacterial activity of P. javanica chloroform (CHCl3) extract against

different bacterial strains.

Conc.

(mg/ml)

Mean value of diameter of Inhibition zone (mm)± SD


aeruginosa

K.

aerogens

E.

coli

1 0 0 0 0 0 0

5 0 0 0 13 ± 0 0 0

10 0 0 0 14 ± 0.158 0 0

20 0 0 0 15 ± 0.158 0 0

40 9 ± 0 9 ± 0 0 15 ± 0.224 0 0

60 11 ± 0.122 11 ± 0.158 0 15 ± 0.122 0 0

80 12.5 ±0.224 12 ± 0.158 0 17 ± 0.418 0 0

100 14 ± 0.418 11 ± 0.158 12 ± 0.224 17 ± 0 0 0

250 18 ± 0.158 11 ± 0 18 ± 0.224 20 ± 0.122 0 0


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Table 3.5. Antibacterial activity of P. javanica n-butanol (BuOH) extract against different

bacterial strains.

Conc.

(mg/ml)



aeruginosa

K.

aerogens

E.

coli

1 0 0 0 0 0 0

5 0 0 0 14.5 ± 0 0 0

10 0 0 0 15 ± 0.224 0 0

20 0 0 0 17.5 ± 0.158 0 0

40 10 ± 0.418 11 ± 0.224 0 19 ± 0 0 0

60 12 ± 0.122 12.5 ± 0.2 0 19 ± 0 0 0

80 14 ± 0.122 13 ± 0.158 0 20 ± 0.122 0 0

100 15 ± 0 12 ± 0 13 ± 0.224 20 ± 0.158 0 0

250 17 ± 0.122 15 ± 0.224 18 ± 0.122 21 ± 0.158 0 0


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Table 3.6. Antibacterial activity of E. nummularius Benzene extract against different

bacterial strains.

Conc.

(mg/ml)


B. subtilis S. aureus M. luteus P. aeruginosa K.

aerogens

E. coli

1 0 0 0 0 0 0

5 0 0 0 0 0 0

10 0 0 0 0 0 0

20 0 0 0 0 0 0

40 0 0 0 9.5 ± 0.224 0 0

60 0 0 0 10 ± 0.418 0 0

80 9 ± 0.158 0 0 11 ± 0.5 0 0

100 10 ± 0.224 0 0 12 ± 0.224 0 0

250 10 ± 0.418 0 0 14 ± 0.158 0 0


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Table 3.7. Antibacterial activity of E. nummularius CHCl3 extract against different

bacterial strains.

Conc.

(mg/ml)


B. subtilis S. aureus M. luteus P. aeruginosa K. aerogens E. coli

1 0 0 0 0 0 0

5 0 0 0 0 0 0

10 0 0 0 0 0 0

20 0 0 0 0 0 0

40 0 0 0 0 0 0

60 0 0 0 0 0 0

80 10 ± 0.158 0 0 0 0 0

100 10 ± 0.158 0 0 0 0 0

250 11 ± 0.224 0 0 0 0 0


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Table 3.8. Antibacterial activity of E. nummularius n-BuOH extract against different

bacterial strains.

Conc.

(mg/ml)



aeruginosa

K.

aerogens

E. coli

1 0 0 0 0 0 0

5 0 0 0 0 0 0

10 0 0 0 0 0 0

20 0 0 0 0 0 0

40 0 0 0 0 0 0

60 0 0 0 0 0 0

80 0 0 8 ± 0.224 8 ± 0.418 0 7.5 ± 0.158

100 0 0 8 ± 0.158 8 ± 0.224 0 8 ± 0.418

250 0 0 8 ± 0.224 10 ± 0.224 0 8 ± 0.158


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Table 3.9. Minimal inhibitory concentration (MIC) values (mg/ml) of P. javanica

and E. nummularius crude methanol extracts and its fractions.

Plant extract B. subtilis S. aureus M. luteus P.

aeruginosa

E. coli

P. javanica

methanol extract

40 20 80 5 -

P. javanica

benzene extract

20 20 100 5 -

P. javanica

chloroform

extract

40 40 100 5 -

P. javanica

butanol extract

40 40 100 5 -

E. nummularius

methanol extract

40 - 80 40 80

E. nummularius

benzene extract

80 - - 40 -

E. nummularius

chloroform

extract

80 - - - -

E. nummularius

butanol extract

- - 80 80 80


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3.5. DISCUSSION

The presence of anti bacterial substances in the higher plants is well established

(Srinivasan, 2001). Being situated in one of the megadiversity region of the world, the

state of Tripura is bestowed with the rich resource of medicinal plants. Two medicinal

plants Parkia javanica and Evolvulus nummularius with known ethnomedicinal uses

(sections 2.1.4 and 2.2.4), had been undertaken to investigate their medicinal properties

including anti bacterial activities.

To study the antibacterial properties, stem bark and leaves of Parkia javanica (P.

javanica) and whole plant of Evolvulus nummularius (E. nummularius) were used. Plant

materials was first extracted in methanol and subsequently fractioned in different solvents

as mentioned in earlier section. Methanol crude extracts as well as solvent fractionated

parts of both the plants were tested against six bacterial strains, out of which three were

Gram positive bacteria viz. Micrococcus luteus, Staphylococcus aureus and Bacillus

subtilis and three were Gram negative bacteria namely Pseudomonas aeruginosa,

Klebsiella aerogens and Escherichia coli. Staphylococci are primarily responsible for

suppurative lesions in the human body, such as boils, pimples, carbuncle, fruncle,

pyelitis, pharyngitis, cystitis, meningitis, acute osteomyelitis, Staphylococcal septicemia

as well as other infections. One aerobic spore former, Bacillus subtilis, Gram positive,

included in the study are widely distributed in soil, dust, milk and decomposing organic

matter. Among Gram negative enteric bacteria used in the study Escherichia coli are

normal inhabitants of human intestinal tract. They are occasionally pathogenic to man.

Pseudomonas aeruginosa, the other gram negative strain included are commonly


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involved in urinary tract infections. Klebsiella aerogens, a non pathogenic Gram negative

bacteria, used for study is non pathogenic by itself but, is closely related to pathogenic

strain Klebsiella pneumoniae which causes infections that include urinary tract, lower

respiratory tract, biliary tract, and surgical wound sites.

The activity of plant extracts was determined by using agar cup- plate diffusion

method (Kavanagh, 1972) measuring the inhibition zone in mm. For the study extraction

was first done with methanol and then fractionated with different solvents such as

benzene, chloroform, n- butanol according to increasing polarity.

The crude as well as fractionated extracts of both the plants were found more

effective against Gram positive strains compared to Gram negative ones. All the Gram

positive strains, Micrococcus luteus, Staphylococcus aureus and Bacillus subtilis, were

sensitive towards the extracts. Out of three Gram negative strains, only Pseudomonas

aeruginosa, was found to be sensitive. Various workers have already shown that Gram

positive bacteria are more susceptible towards plant extracts as compared to Gram

negative bacteria (Lin et al., 1999; Parekh and Chanda, 2006). These differences may be

attributed to the differences in the composition of cell walls (Yao et al., 1995).

Interestingly, in both the cases maximum activity was observed against P. aeruginosa,

which was multidrug resistant strain.

All the solvent fractions of Parkia javanica plants showed almost similar

activities to that of the crude methanol extract. However, methanolic extract showed the

maximum activity. Phytochemical analysis (Dinda et al., 2009) of the P. javanica extract

revealed presence of β-sitosterol, ursolic acid (pentacyclic triterpene acid), iridoid


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glucosides along with other mixture of compounds. Regarding fraction distribution of the

compounds, β-sitosterol was found in the lowest polar benzene fraction and iridoid

glucosides, ursolic acid in the highly polar n-butanol fraction. All these compounds are

reported to have antimicrobial activities (Gupta et al., 1980; Davini et al., 1986; Ramesh

et al., 2001; Shokeen et al., 2005). As all the fractions were found to possess anti

bacterial principles, the antibacterial activity of all the fractions may be contributed to

principles they possessed. However, the higher activity of the crude methanolic extract

could be due to the synergistic effect of all the active principles (Bai, 1990).

Regarding E. nummularius, the results of the present study corroborate with the

findings on antibacterial activity reported by Pavithra et al., (2009). The strains, they

used were S. aureus, B. subtilis, P. aeruginosa, K. pneumoniae and E. coli. Out of five

strains, E. coli and B. subtilis were reported to be sensitive with MIC values 12.5mg/ ml

and 3.125 mg/ml respectively. In the present study also, E. coli and B. subtilis were found

sensitive however with higher MIC values, 80 mg/ml and 40 mg/ml respectively. P.

aeruginosa was found to be resistant to the extract. However, in the present study, P.

aeruginosa was found sensitive with MIC value, 40mg/ml. The differences observed

between the studies may be attributed to the differences existing in plants growing in

ecologically different areas. Indeed, Lucas and Lewis, (1994) had indicated that some

higher plants contained antibacterial principles at certain stages of their development.

Therefore, the differences can logically be explained for the simple reason that plants had

not been collected from the same area and at the same time of the plant growth.


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Phytochemical analysis of E. nummularius extract (Dinda et al., 2007) revealed

the presence of β-sitosterol and its glucoside, stigmasterol, d-mannitol, ursolic acid and

its isomer oleanolic acid (pentacyclic triterpene acid). Therefore the antimicrobial activity

showed by the E. nummularius could be contributed to the antimicrobial principles

present in the extract.

chapter 3 antibacterial study -...

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