Antimicrobial Screening and Bioactivity Guided Fractionation of Acetone Extracts of Excoecaria Agallocha

B. G. Pagare¹ and B. L. Jadhav²*

¹Siddharth College of Arts, Science and Commerce, Fort, Mumbai - 400001

²Dept of Life Sciences, University of Mumbai, Vidyanagari Campus, Santacruz (E), Mumbai-400098

ABSTRACT:

Antimicrobial and bioactivity guided fractionation of acetone extract of various parts of Excoecaria agallocha was studied. The cold and hot acetone extracts of leaves, stem and pneumatophores were prepared and tested against clinical isolates like Staphylococcus aureus, Salmonella typhi, Klebseilla pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaris and Candida albicans. The leaves extract inhibited growth of most of the test microorganisms, in which best results were found against S.aureus and S. typhi. The leaves cold extract registered 1 mg/ml and 10 mg/ml MIC against S. aurerus and S. typhi respectively. Fungi C. albicans was found to be resistant to both leaves and stem cold extracts. Similarly in the hot extracts leaves inhibited growth of most of the test organisms. The best activity was found against S. aureus, S. typhi and C. albicans with MIC up to 5 mg/ml. Bioactivity guided fractionation study was carried out for target directed isolation and purification of active principles. To do this crude drug was fractionated in to petrolieum ether, water and acetone extracts. The best activity was noted in water (21 mm) and acetone (14 mm) fractions. This has indicated that potent bioactive principle is polar in nature and can be isolated from water fraction.

KEY WORD: Acetone extracts, Antimicrobial, Bioactivity fractionation, Excoecaria agallocha, MIC.

INTRODUCTION:

Indian coast has about 65 mangroves species belonging to 39 genera¹. Among these 25 species of 20 genera grow luxuriantly along the Mumbai coast². Mangroves are traditionally used to treat various diseases worldwide³. Recently Indian mangroves have shown antiviral⁴, antibacterial and antifungal⁵, mosquito larvicidal⁶ and antioxidant⁷ properties. The phytochemical studies on mangroves have shown chemical classes like steroid, triterpenes, saponin, flavonoids, alkaloids and tannin and polyphenols^8,9,10.

About 16 mangrove species of Mumbai coast have been thoroughly screened for antimicrobial properties. Among them A. alba and A. Marina¹¹, A. officinalis, E. agallocha¹², S. apetala^{13, 14}, Rhizophora apiculata,^15,16, Derrisindica and D. trifoliate¹⁷ were found to be most potent. Neuropharmacological, antibacterial and acute toxicity also reported by Nusrat et al²⁰ in the ethanol bark extract of this mangroves.

In the present study we have reported antibacterial and antifungal activity in the acetone extracts of various parts of the plant along with bioactivity guided fractionation.

MATERIAL AND METHODS:

Plant collection:

Mangrove Excoecaria agallocha was collected from the pollution free zone of Soonabai Pirojshah Godrej, Marine ecology centre, Vikhroli, Mumbai in the month of December. The plants species were identified by an expert taxonomist. All the parts were separated and cleaned thoroughly under running tap water. Plant parts, mainly leaves, stem and tender fruits were used for the extract preparation and studied its bioactivity.

Methods of extraction:

All cold and hot extracts of both the plant species were prepared 50%w/v in acetone as follows.

Cold extracts:

The fresh plant parts were used to prepare cold extracts. 10 gm of leaves, stem and pneumatophores were homogenized individually in a mortar and pestle using 10 ml of different solvents and kept aside for 45 min for extraction. It was then filtered through Whitman’s filter paper no. 1. To the residue again 6-8 ml of solvent was added and filtered similarly after 15min. The final volume of extract was made to 20ml and stored in airtight Borosil glass bottles at 4ºc.

Hot extracts:

The individual parts were oven dried at 40ºC, powdered and sieved through the muslin cloth. 10gms of these powdered materials were used to prepare hot extracts by Soxhlet method²¹. To prepare the hot extract, 200 ml of solvent was taken into the round bottom flask and the temperature of the heating mantle was adjusted just above the boiling point of the solvent. The vapours of the solvent pass through the extraction tube to the condenser and percolate back in to the thimble. This process continues to the level at which it flows back to the flask through the capillary attached to the side of extraction tube. This completes one cycle. This cycle continues till the solvent overflowing from the tube becomes colourless. The extract was then evaporated on water bath to reduce the volume to 20 ml (50%w/v) stored in air tight bottles and kept at 4ºc. Before bioassay all the extracts were brought to room temperature.

Test microorganisms and media:

Pathogenic strains of bacteria Staphylococcus aureus, Salmonella typhi, Klebseilla pneumoniae, Pseudomonas aeruginosa, Escherichia coli and Proteus vulgaris as well as one fungi Candida albicans used for the bioassay were clinical isolates obtained from KEM Hospital, Parel, Mumbai, India. All the bacterial strains were maintained on nutrient agar while fungi maintained on MGYP media. All the strains were sub culture from time to time.

Bioassay (In-vitro):

Primary screening and minimum inhibitory concentration (MIC) were carried out by Agar cup method²² to test the activity of the extracts against test microbes. On each plate four wells were made with the help of cork borer (8mm). In one of the wells 80μl of solvent was added as negative control while similar quantities of extracts were poured in three wells. The potent extracts were evaporated and 0.5, 1, 5 and 10 mg/ml concentration of residues were made to study MIC. On each plate four wells of 8mm diameter were made using cork borer and different concentrations of extracts were tested against pathogens. The results were observed as clear zones of inhibition around the wells after 24 and 48 hrs.

Activity guided fractionation:

The leaves extracts exhibited MIC up to 1 mg/ml were selected to study the activity guided fractionation. The activity guided fractions were prepared as follows. Acetone extract was evaporated to dryness and a residue was collected. The equal quantity of petroleum ether was added to dissolve the residue and centrifuged at 3000 rpm for 20 minutes. Thereafter the residue and filtrate were separated. The residue was collected and petroleum ether filtrate (Fraction ‘a’) used for bioassay. Further, residue was dissolved in equal quantity of water and centrifuged as per previous steps and filtrate was collected and used for bioassay as water extract (fraction ‘b’) filtrate. Finally, remained residue was dissolved in acetone designated as acetone extract (fraction ‘c’). All the filtrates were stored at 4⁰C in refrigerator.

RESULTS:

1. Cold extracts:

Cold acetone extract of leaves, and stem of E. agallocha species were prepared and screened for antimicrobial activities and results obtained are displayed in Table-I.

Table – I: Effects of cold acetone extracts against test microorganisms (Zone of inhibition in mm)

Microorganisms	Leaves (A1)	Stem (A2)	Pneumatophore (A3)
Escherichia coli	15 (D)	-	X
Staphylococcus aureus	16	-	X
Salmonella typhi	16	-	X
Pseudommonas aeruginosa	15	-	X
Proteus vulgaris	20(D)	-	X
Klebsiella pneumoniae	13	-	X
Candida albicans	-	-	X

Key: A1 – Cold acetone extract of leaves

A2 – Cold acetone extract of stem

A3 – Cold acetone extract of pneumatophores

(D) – Diffused zone

- - Negative Zone

X – Not available

Table- II: MIC results of cold acetone extracts on test microorganisms (zones of inhibition in mm)

Leaves (A1) mg/ml

Stem (A2) mg/ml

Pneumatophore (A3) mg/ml

Microorganisms

0.5

Escherichia coli

Staphylococcus aureus

Salmonella typhi

Pseudomonas aeruginosa

Proteus vulgaris

Klebsiella pneumoniae

Candida albicans

Key: A1 – Cold acetone extract of leaves

A2 – Cold acetone extract of stem

A3 – Cold acetone extract of pneumatophores

XX – Not tested

- - Negative Results

The leaves extract inhibited growth of most test microorganisms, in which best results were found against S.aureus and S. typhi, with zone size of 16 mm. This extract was also found potent against Ps. aeruginosa (15 mm) and Kl. pneumoniae (13 mm), while diffused zones were noted against E. coli (15 mm) and P. Vulgaris (20 mm). Further leaves extract have registered minimum inhibitory concentrations (MIC) of 1 mg/ml and 10 mg/ml against S. aurerus and S. typhi (Table-II) respectively. Fungi species C. albicans was found to be resistant to both leaves and stem extract.

2. Hot extracts:

Hot acetone extract of leaves, stem and pneumatophores of E.agallocha were prepared and screened for their antimicrobial properties and results obtained for bioassay are displayed in Table-III. Among all parts, leaves extracts have administrated inhibitory effect on most of the microorganism for which highest zones of 18 mm were noted against S. aureus and C. albicans. The second best activity was observed against S. typhi with inihibition zone of 15 mm, and a minor inhibition zones against Ps. aeruginosa. Diffused zones were seen against P. vulgaris (20 mm) and E.coli (13 mm) in this extracts. The leaves recorded best MIC of 1 mg/ml against S.aureus, while C. albicans inhibited at 5 mg/ml concentration (Table-IV).

Stem and pneumatophores produced a moderate activity compared to leaves extracts. Both of these parts of plants have registered minor zones of inhibition against E.coli. A significant activity was also noticed against S. aureus and C. albicans by stem and pneumatophores respectively, wherein both the microorganisms exhibited zone size of 12 mm.

Activity Guided Fractionation:

Hot acetone leaves extract exerted best activity against S. aureus, and therefore it was selected for activity guided fractionation. Among bioactivity guided fractions, water fraction (21mm) and acetone fraction (14mm) recorded better results against S. aureus (Table – V). Petroleum ether fraction did not show any activity.

Table – III: Effects of Hot acetone extracts against test microorganisms (Zone of inhibition in mm)

Microorganisms	Leaves (A1)	Stem (A2)	Pneumatophore (A3)
Escherichia coli	13 (D)	9	10
Staphylococcus aureus	18	12	10
Salmonella typhi	15	-	-
Pseudommonas aeruginosa	9	-	-
Proteus vulgaris	20 (D)	-	-
Klebsiella pneumoniae	-	-	-
Candida albicans	18	-	12

Key: A1 – Hot acetone extract of leaves

A2 – Hot acetone extract of stem

A3 – Hot acetone extract of pneumatophores

(D) – Diffused zone

- - Negative Zone

DISCUSSION:

In the present study, E. agallocha leaf gave fabulous results against S. Aureus, S.typhi, Ps. Aeruginosa and Kl. Pneumonia, while E. coli and P. Vulgaris noted diffused zones. S. Aureus and C. albicans found to be highly susceptible to the hot leaf extract showing highest zones (18 mm) of inhibition. Other parts of this species showed minute activity in hot extracts against E.coli, S. aureus and C. albicans where zones ranged between 9 mm and 12 mm. Thus acetone leaf extract proved to be highly antimicrobial with wide spectrum of activity over other parts of this species. This results are in accordance with the several reports of other plant species^23,24,25. These results are also in agreement with the findings of Suraiya and Jadhav¹³ for this species. However, in contrast these authors have noted positive results against E. coli and S. aureus in cold stem extract in E. agallocha as against negative results in present study. Besides this, slightly positive results were obtained against these microorganisms where hot crude extracts of stem and pneumatophores were used. The reasons for such contrasting results could be due to the clinical isolates used in present study and site of plant collection. Further, on heating, acetone may have extracted more bioactive principles from stem of the plant as commonly seen in many studies, responsible for the increased activity^11,26

Table IV: MIC results of hot acetone extracts on test microorganisms (zones of inhibition in mm)

Leaves (A1) mg/ml

Stem (A2) mg/ml

Pneumatophore (A3) mg/ml

Microorganisms

0.5

Escherichia coli

Staphylococcus aureus

Salmonella typhi

Pseudomonas aeruginosa

Proteus vulgaris

Klebsiella pneumoniae

Candida albicans

Key: A1 – Hot acetone extract of leaves

A2 – Hot acetone extract of stem

A3 – Hot acetone extract of pneumatophores

XX – Not tested

- - Negative Results

Hot acetone extracts of leaf, stem and pneumatophores showed better activity over cold extracts. Striking feature of hot extract was inhibition zone against C. albicans, which was not found in cold extracts against this microorganism. This indicates that this plant contains thermo stable antifungal principles, which are extracted only at higher temperature.

MIC test have revealed almost same potency (1 mg/ml) for cold and hot leaf extract against S. aureus. Beside this only cold leaf extracts registered activity against S. typhi at 10 mg/ml, while hot extract of this part inhibited C. albicans at 5 mg/ml. This shows leaf contains thermo labile antibacterial principles against S typhi and thermo stable antifungal principles of moderate potency.

In the bioactivity guided fractionation best activity was noted in water (21 mm) and acetone (14 mm) fractions against S. aureus. This indicates presence of water and acetone soluble bioactive principles in the acetone leaf extract of the plant. This further indicates more than one active principle in the E. agallocha leaf. The present results are in accordance with various studies^24,25,27. Therefore it is suggested that the activity in acetone crude leaf extract is synergistic effect of several polar antimicrobials. Overall E. agallocha proved to contain potent bioactive principles in the leaf acetone extract which can be further isolated and purified useful in health management.

Table-V: Effects of hot leaf acetone fractions on test microorganisms (zones of inhibition in mm)

Microorganisms	Fraction ‘a’ (petroleum ether)	Fraction ‘b’ (water acetone)	Fraction ‘c’ (acetone)
S.aureus	-	21mm	14mm

Key: Fraction ‘a’ – acetone extract residue soluble in petroleum ether

Fraction ‘b’ – petroleum ether residue soluble in water

Fraction ‘c’ – Final residue soluble in acetone

- - Negative result

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Received on 25.08.2013

Modified on 10.09.2013

Accepted on 15.09.2013

Research Journal of Pharmacognosy and Phytochemistry. 5(5): September – October 2013, 215-219