Preliminary in vitro screening of antibacterial and antifungal compounds of mangrove plant extracts

Annals of the Sri Lanka Department of Agriculture. 2008.10:303-309.

PRELIMINARY IN VITRO SCREENING OF ANTIBACTERIAL AND ANTIFUNGAL COMPOUNDS OF MANGROVE PLANT EXTRACTS D.M. WITHANAWASAM1 and P.D. ABEYSINGHE2 1 Rice Research Station, Ambalantota, 2 Department of Botany, University of Ruhuna

ABSTRACT The phytochemical analysis of plant extracts has revealed the presence of compounds which are cytotoxic to some microorganisms. However, extraction and screening for their antimicrobial property is an untouched area of study. Aqueous and ethanolic mangrove plant extracts obtained from mature leaves, tender leaves, bark and shoot of Avicennia marina, Bruguiera gymnorhiza and Rhizophora mucronata were screened for antimicrobial activity against clinical isolates of Shigella sp., Pseudomonas sp., Escherechia coli, Staphylococcus sp., Klebsiella sp., Acinetobactor sp. and two pathogenic soil bacterial strains isolated from chili rhizosphere and Pythium spp. and Fusarium spp. isolated from damping-off Cucumis sativus seedlings. Ethanolic extracts were found to be more effective than aqueous extracts on certain clinical strains. Both ethanolic and aqueous extracts of A. marina exhibited the highest antibacterial activity. Moreover, sequential extraction of ground materials of A. marina in ethanol and ethyl acetate showed the highest growth inhibition in Shigella sp. and Pseudomonas sp. Strains of Acinetobactor, Klebsiella and two soil bacterial strains were found to be the least sensitive for all the extracts. The growth of fungal isolates was not inhibited by any of the plant extracts. Mature leaf extracts of A. marina was separated into fractions by Thin Layer Chromatography (TLC) and each separated fraction was screened for antibacterial activity and different degrees of growth inhibition was evident. However, crude extracts of mature leaves of A. marina showed more effective antibacterial activity than the separated fractions alone. It can be concluded that the extracts of leaf, bark and shoot of three mangrove plants have the ability to inhibit bacterial growth in different degrees. Therefore, there is a pharmaceutical value in the extracts of A. marina, Bruguiera gymnorhiza and Rhizophora mucronata. KEYWORDS: Antimicrobial activity, Bio assay, Clinical isolates, Mangrove, Thin Layer Chromatography.

INTRODUCTION Traditional medicine plays a significant role in the health care systems of developing countries, especially in Asia including Sri Lanka. Phytochemical analysis of plant extracts has revealed the presence of many biologically active compounds such as tannins, flavanoids, alkaloids and phenolics (Bandaranayake, 1998). The medicinal value of plant materials typically result from the combinations of secondary metabolites (Donald, 2000). Secondary products involve in the plant defense mechanism through cytotoxicity against microbial pathogens. This provides useful antimicrobial medicines in human (Donald, 2000). Higher plants produce hundreds to thousands of diverse chemical compounds with different biological activities

48 WITHANAWASAM AND ABEYSINGHE

(Hamburger, 1991). The first compound with antimicrobial activity was found in the 1930s (Goodman, 1991). Since that period, the development and use of these substances have increased because of the appearance of resistant strains (Zinhener, 1972). There have been many investigations on the antifungal, antibacterial (Elgayyar, 2001), and antiviral (Vander, 1991) preparations and individual compounds isolated from natural sources. Mangrove plants are a rich source of steroids, triterpines, flavanoids, alkaloids and tannins (Bandaranayake, 1995). The extraction of novel chemical compounds from mangrove is in its infancy. However, crude extracts of mangrove are used in folkoric medicine for diseases such as rheumatism, small pox ulcers and various other infections (Bandaranayake, 1998). It is evident that some hospital-acquired infections are resistant to present day antibiotics and the cost of treating these infections have been a burden to national health services. Therefore, proper measures must be taken to deal with this problem. Our aim of this project was to explore the folkloric knowledge by screening some mangrove plant extracts for antibacterial and antifungal activity using pathogens from various sources. MATERIALS AND METHODS Preparation of plant extracts Avicennia marina, Bruguiera gymnorhiza and Rhizophora mucronata were used as tested mangrove plants. The specimens were identified using the “A Field Guide to the Common Trees and Shrubs of Sri Lanka” (Ashton, 1997). Plant extracts were obtained from three species by grinding method. Amounts of 0.3 g of mature leaves, tender leaves, shoot and bark was crushed with 1 ml sterilized distilled water and 95% ethanol separately using mortar and pestle. Crushed material was centrifuged at 10,000 rpm for 2 minutes. Bio assay Crude extracts were screened in vitro against bacterial strains obtained from general hospital, Matara; Escherichia coli (infected blood), Staphylococcus sp. (infected urine), Pseudomonas sp. (from wound), E. coli (ATCC 25922), Shigella sp., Acinetobactor sp. and Klebsiella sp. from the Faculty of Medicine, University of Ruhuna, Galle. In addition to above clinical bacterial strains, two pathogenic soil bacterial strains, CHA 49 and CHA 5 and two pathogenic fungi Pythium spp. and Fusarim spp. isolated from Cucumis sativus seedlings which showed symptoms of damping-off were tested. Bio assay was conducted using agar well diffusion technique. Half strength Nutrient Agar (NA) medium seeded with each bacterial strain (pour plate method) was inoculated with 50 µl of each crude extract. Antifungal activity was tested using actively growing cultures of Pythium and Fusarium

SCREENING OF ANTIBACTERIAL AND ANTIFUNGAL COMPOUNDS 49

in Potato Sucrose Agar (PSA) medium using agar well diffusion technique. Antibacterial activity was visualized as a clear zone on petri plates. Growth inhibition was measured quantitatively by measuring the diameter (mm) of the clear zone. Sequential Soxhlet extraction Large scale extraction was done using mature leaves, tender leaves and bark (50 g each) of A. marina which showed highest inhibition in the preliminary bio assay into petroleum ether, chloroform, ethyl acetate, 95% ethanol and water by Soxhlet extractor (Harborne, 1998). Plant extracts were concentrated in a rotary evaporator. The bio assay was repeated for these extracts. Thin Layer Chromatography (TLC) and Preparative TLC (PTLC) Plant extracts were separated into component(s) by analytical TLC method using different combinations and ratios of ethyl acetate, diethyl ether and ethanol. TLC plates were visualized under UV illumination (Harborne, 1998). Fractions were further separated by PTLC and each separated band on PTLC plate was scraped and resuspended in chloroform, ethyl acetate and ethanol (Hostettmann, 1986, Marston, 1991). Each resuspended fraction was screened for antibacterial activity against tested bacterial strains and the degree of inhibition before and after fractionation was compared. RESULTS AND DISCUSSION Some ethanolic and aqueous extracts of leaves, shoot and bark of A. marina, B. gymnorhiza and R. mucronata exhibited antibacterial activity against certain clinical bacterial strains under study. No growth inhibition was observed compared to control plates against soil bacterial strains and isolates of Pythium spp. and Fusarium spp. used in this experiment. Further more, it showed that the degree of antibacterial activity of these plant extracts were not similar. Almost all crude ethanolic extracts showed better inhibition than the aqueous extracts (Table 1). None of the aqueous extracts (extracted into sterilized distilled water) showed any inhibitory effect on E. coli (Table 1). Mature leaf extracts were found to be more effective against all the tested clinical bacteria in comparison to the other extracts. Comparatively A. marina was found to have the highest activity for all clinical bacterial strains tested followed by B. gymnorhiza and R. mucronata. However, bark extracts of B. gymnorhiza and R. mucronata exhibited higher inhibition than all other extracts of same two species.

50 WITHANAWASAM AND ABEYSINGHE

Considering the overall performance of plant extracts of the species, only A. marina was selected for further analysis. Previous studies also reveal that they have been used in traditional medicine against microbial infections ; A.marina spp. has been used for skin diseases, boils and wounds, blood purifier, Bruguiera spp. in eye diseases while Rhizophora spp. in diarrhea and tuberculosis (Bandaranayake, 1998). Both water and ethanol are polar solvents. Only polar compounds in plant materials are extracted into water and ethanol. When ethanol and water were tested alone, no inhibitory activity on bacteria was evident. The highest inhibitory activity was given for Shigella sp. tested with the ethyl acetate and ethanolic extracts from leaves and bark of A. marina. The compounds extracted into petroleum ether from the leaves and bark of the same plant did not show considerable amount of inhibition. Soxhlet extracts of mature leaves of A. marina into chloroform, ethyl acetate, and ethanol exhibited the highest growth inhibition in Shigella sp. The second most efficient antibacterial activity was shown against Pseudomonas sp. Neither Acinetobactor sp. nor Klebsiella sp. was inhibited by any of the tested plant extract (Table 2). Therefore, these two bacterial strains are the least sensitive to all types of extracts under study, while Shigella sp. was the most sensitive. Both petroleum ether and water extracts have given the lowest inhibition on bacterial growth. Out of five solvents, petroleum ether is the least polar solvent while water is the highest. Moderately polar chloroform, ethyl acetate and ethanol showed the more pronounced inhibition. It proves that effective chemical compounds were extracted into moderately polar solvents. All plant extracts did not show any inhibitory effect on either Pythium spp. or Fusarium spp. However, positive result against both the fungi associated with plants can be obtained at higher concentration of the plant extract. In Analytical TLC a number of combinations of ethyl acetate, diethyl ether, and ethanol were used to prepare solvent systems. Few of them only were able to give better separation of fractions. The polarity of the three solvents differed from each other. Therefore, a series of eluents in which solvents were mixed in different ratios were prepared. The degree of polarity in the final mixture differed according to the ratio of mixing. PTLC revealed that mature leaf extracts of A. marina separated into several fractions. Components of the extract separate according to the polarity of solvents and molecular weight of components. When the degree of bacterial growth inhibition is compared before and after fractionation, the inhibitory activity

SCREENING OF ANTIBACTERIAL AND ANTIFUNGAL COMPOUNDS 51

was reduced after separation into fractions (Fig. 1). It implies that when compounds are together they can exhibit good antibacterial activity. Table 1. Diameter of the inhibition/clear zone around the wells filled with plant extracts measured in millimeters (mm). Bacterial strain

Plant material A.marina

B. gymnorhiza

R. mucronata

Inhibition zone/Clear zone (mm) E.coli Shigell Pseudomo a sp. nas sp. w et w et w eth h h -5 4 8 3 6

CHA 5

CHA 49

eth

Pythium spp. w et h ---

b -4 -3 -2 -5 -----tl -6 -7 2 4 2 5 -----s -3 1 5 2 2 -3 -----ml -3 -3 2 2 -------b -4 -4 2 4 -4 -----tl ----- -2 -------s -1 -1 2 3 -------ml -1 -1 1 2 -------b -1 2 4 2 5 -3 -----tl ----- ---------s -1 -1 1 --------(ml- mature leaves, b- bark, tl- tender leaves, s- shoot, w- water and eth- ethanol. The numbers are means of 3 experiments by measuring the inhibition zone).

Table 2.

Fusariu m spp. w eth --

------------

w 3 -

E. coli (ATCC25922) pe c ea et w - 3 3 - 4 - 4 -

Diameter of the inhibition/clear zone around the wells.

Bacterial strain

Inhibition zone/Clear zone (mm)

Plant material

Staphylococcus sp. pe c ea et 4 3 6 1 1 4 -

ml tl b

Staphylococc us sp. w eth

w -

Pseudomonas sp. pe c ea et 4 6 7 2 - 3 3 2 4 3

w 2

E. coli pe c 2

ea 3 4 6

et 5 4 3

w -

Shigella sp. pe c ea 5 13 2 8 6 14

et 13 8 8

Wells filled with mature leaves (ml), tender leaves (tl) and bark (b) extracts of A. marina in pe- petroleum ether, c- chloroform, ea- ethyl acetate, et- ethanol and w- water measured in millimeters (mm).

3 M E

2 M E

M E 1

M E

B E A

M C 2

M C 1

M C

inhibition(m m ) ) m

g r o w th

18 16 14 12 10 8 6 4 2 0

i n h i b i ti o n (m

degree of growth inhibition

Degree of growth (mm)

52 WITHANAWASAM AND ABEYSINGHE

e x tra ct

S h ig e lla

1 8 1 6 1 4 1 2 1 0 8 6 4 2 0

d eg ree

o f

Figure 1. Comparison degrees of growth inhibition of Shigella sp. and Pseudomonas MC of MC1 MC2 BEA sp. BEA1 BEA2 ME sp. ME1 ME2 ME3 Shigella Pseudomonas sp. by crude Soxhlet extracts of mature leaves in chloroform (MC), mature leaves in ethanol (ME) and barkExcract in ethyl acetate (BEa) and separated components of the same extracts of A. marina. (1, 2, and 3 represent separated PTLC). x t ra c t

sp .

P s

e u d o m

o n a s

sp .

ACKNOWLEDGEMENTS Authors wish to acknowledge Prof. R. N. Pathirana, Senior Lecturer, Department of Chemistry, University of Ruhuna for his valuable advice during the research study. REFERENCES Ashton, Mark S., S. Gunatilleke, N. de Zoysa, M.D. Dassanayake, N. Gunatilleke and S. Wijesundera. 1997. A field guide to the common trees and shrubs of Sri Lanka. WHT publication, Sri Lanka. 108,310,311 p. Bandaranayake, W.M. 1998.Traditional and medicinal uses of mangroves. Mangroves and salt Marshes 2:133-148. Bandaranayake, W.M. 1995. Survey of mangrove plants from Northern Australia for phytochemical constituents and UV- absorbing compounds. Current Topics in Phytochemistry 14:69-78. Donald, P. Briskin. 2000. Medicinal plants and phytomedicines. Linking Plant Bio Chemistry and Physiology to Human Health. Plant Physiology 124:507-514.

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