DETERMINATION OF ANTIBACTERIAL PROPERTIES OF ANONA muricata L. (GUYABANO) LEAF EXTRACT USING DISC DIFFUSION METHOD
Rivera JE, Rivera T, Rodriguez MB, Rosario M, Salongcay R, Samson E, Santillan G, Sarabia JJ, Semblante C, Tacang A, Tan AM, Tan A, Tan KJ, Tan MKK, Tasico RC, Tauli BE, Thomas J, Tuliao R, Umag Z, Uy CV, Valencia JC, Verde JM, Villanueva VVR, Vizconde D, Wee PA, Wylengco MC, Zapanta JM Department of Pharmacology, College of Medicine, University of the Philippines \u2013 Manila, Philippine General Hospital ABSTRACT: Anona muricata L. has long been used as a folk remedy for a wide array of illnesses and the literature corroborates claims of medical properties like cytotoxic, antimicrobial and antiseptic properties. However, confirmation for the antimicrobial properties of the leaves (especially of the Philippine variety) is yet to be established. Aqueous extracts (of 200% and 100% concentration) were prepared from young and mature guyabano leaves and consequently .02 ml of each were pipetted onto paper discs. The paper discs were applied onto agar plates inoculated with one of the following bacteria (S. aureus, E. coli and P. aeruginosa ). Positive controls (Oxacillin for S. aureus and Ceftazidime for E. coli and P. aeruginosa) were also placed onto the plates. The zones of inhibition for the guyabano extract were then measured and compared to zones of inhibition of the positive controls. There were no zones of inhibition observed around the discs with the mature and young guyabano extracts (at both concentrations). Young and mature A. muricata leaf decoctions at concentrations of 100% and 200% did not exhibit antibacterial property comparable to Oxacillin against S. aureus and Ceftazidime against E. coli and P. aeruginosa as indicated by the failure to generate a zone of inhibition.
INTRODUCTION
The use of herbal medicine is an integral part of Philippine culture. Within this culture, various plants such as Allium sativum, Ananas comosus, Cocos nucifera have been popularly studied to determine their medicinal properties. In addition to this, other native plants are being investigated to bring about other alternative methods which could be as effective as the commercial products. This includes the recent trend in the investigation of Anona muricata L. Anona muricata L.
is reported to possess promising medicinal properties such as
cytotoxicity, antimicrobial and antiparasitic properties. Even though findings from other countries have shown that
Anona muricata has antimicrobial properties against common
pathogens, the Guyabano species in the Philippines could have a different set of properties comparable to their foreign counterparts. This study will then look at the possible antibacterial property of the guyabano leaf.
REVIEW OF LITERATURE Anona muricata
L. (Guyabano, Tagalog; Graviola, Sour sop, English)
Anona muricata L. is an evergreen tree of the Anonaceae family. In other countries, it is
also commonly called Brazilian pawpaw, guan\u00e1bano, guan\u00e1bana, guanaba, corossol \u00e9 huanaba, togebanreisi, durian benggala, nangka blanda, and cachiman \u00e9pineux. In the Philippines, it is popular for its edible fruit. It is an excellent source of Vitamins B and C. However, the flesh of this fruit was reported to be deficient in calcium, phosphorous and Vitamin A. The ripe fruit has antiscorbutic properties while the unripe fruit is used in treating dysentery. In Mexico, the guyabano is said to have pectoral, antiscorbutic and febrifuge properties. The seeds and green fruit are used as astringent and remedies for dysentery (Quisumbing 1951). The
leaves of guyabano have an alcoholic extract which yields a small amount of an essential oil. This essential oil possesses a strong somewhat agreeable odor. The extract, on the other hand, contains potassium chloride as well as dextrose, tannin, amorphous products and noncrystallizable alkaloid substance. This alkaloid substance in the bark was found to be poisonous when crude (Quisumbing, 1951). The different parts of the tree have been traditionally used as herbal medicine in numerous tropical countries for a variety of effects such as against head lice, worms and parasites, to increase milk production after childbirth, and as astringent for diarrhea and dysentery. Parts of the tree are also used to treat catarrh, diabetes, neuralgia, rheumatism, arthritis pain, cough, flu, asthma, difficult childbirth, hypertension and even liver problems. Anona muricata L. is also used as a sedative, antispasmodic and a heart tonic (Taylor 2005).
Numerous studies have been conducted on the plant Anona muricata L. Most of the attention poured to this species is due to its wealth of cytotoxic anti-cancer substances active against numerous cancer cell lines. Seeds of Anona muricata L. have yielded muricatetrocin A, muricatetrocin B, gigantetrocin A and gigantetrocin B, which were all selectively cytotoxic against HT-29 Human colon tumor cell line (Reiser
et al. 1993). Bioactivity-directed
fractionation of A. muricata seed extract also led to the discovery of five mono-tetrahydofuran ring acetogenins, which includes cis-annonacin. Cis-annonacin is selectively cytotoxic to HT-29 human colon adenocarcinoma cells (Reiser et al. 1996). Anona muricata leaf extracts also contain cytotoxic compounds. Murihexocins A and B, both mono-tetrahydrofuran acetogenin isolated from guyabano leaves, have deterring effects on different human cell lines and even exhibited selectivity to prostate (PC-30) and pancreatic (PACA-2) cell lines (Zeng et al. 1995). Annohexocin, another acetogenin extracted from Anona muricata leaves, was found to have inhibitory effects to some human tumor cell lines (Zeng et al. 1995). Also found in the leaves are
annomuricin C and muricatocin C, cytotoxic against A549 lung cells and MCF –7 Human breast solid tumor cell lines (Wu et al. 1995). Many more cytotoxic acetogenins and other hydrocarbonrich compounds have been isolated from Anona muricata L. Numerous studies have also yielded other important properties of Anona muricata L. Guyabano leaf alcoholic extracts have been shown to be a potent molluscicide against Biomphalaria glabrata. This can potentially be used for vector control of schistosomiasis (dos
Santso et al. 2001). Antiparasitic properties of Anona muricata L. have been also studied. Pericarp extracts have been studied against Leishmania braziliensis and Leishmania panamensis promastigotes, which are trypanosome protozoan parasites (Jaramillo et al. 2000). Acetogenins
in Anona muricata L. seed methanolic extracts have also shown activity against Molinema desetae (Bories et al. 1991). Anona muricata
L. is known to have antimicrobial properties. Guyabano possesses
antibacterial, antifungal and antiviral properties (Taylor 2005). Ethanolic extract of guyabano was demonstrated to have inhibitory activity against cytopathic effects of Herpes simplex virus-1 (Padma et al. 1998). On the other hand, methanolic extracts of guyabano have been shown to have activity against the lytic activity of Herpes simplex virus-2 (Betancur-Galvis et al. 1999). Various extracts from the stem, bark and leaf of Anona muricata have exhibited antibacterial activity against numerous microorganisms such as Plasmodium falciparum, Escherichia coli, Pseudomonas aeruginosa, Shigella flexneri, Staphylococcus aureus, Staphylococcus albus, Salmonella typhosa, Salmonella newport, and Serratia macescens
2004). Anona muricata L. is thus a broad spectrum antimicrobial.
(Raintree Nutrition, Inc.
RESEARCH QUESTION
Will Anona muracata leaf extract show antimicrobial properties comparable to that of Oxacillin and Ceftazidime using disc diffusion method against S. aureus, S. pneumoniae, E. coli, P. aeruginosa, and K. pneumoniae as exhibited by zones of inhibition?
OBJECTIVES
The general objective is to determine the antibacterial property of Anona muricata L. leaf extract against common pathogenic bacteria. Specifically, this study aims: 1. To compare the antibacterial property of Anona muricata L. leaf extract to Oxacillin against Staphylococcus aureus and Streptococcus pneumoniae using Disc diffusion method. 2. To compare the antibacterial property of Anona muricata L. extract to Ceftazidime against Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae using Disc diffusion method.
The study used guyabano leaf extracts prepared by the Pharmacology Laboratory staff of the UP College of Medicine. The laboratory staff prepared four concentrations: 100% and 200% Young leaf extracts and 100% and 200% Mature extracts. This study utilized two strains of gram positive bacteria (Staphylococcus aureus and negative bacteria strains (Escherichia coli, pneumoniae).
Streptococcus pneumoniae) and three gram Pseudomonas aeruginosa and Klebsiella
MATERIALS AND METHODS Research Design
This study utilized an Experimental Study Design. Sample Size Calculation
Two replicates for every bacteria (E. coli, P. aeruginosa and S. aureus) were done for each preparation of the guyabano leaf extract. Experiment Proper
Decoction Preparation
The process of decoction was employed in the extraction of the test substance from the A. muricata plant. Young and mature leaves were harvested separately. The classification of the
leaves was based on color and size. The young leaves were light green in color and small in size while mature leaves were dark green and large. The classification of the leaves was done by Prof_______, from the Department of Pharmacology. The leaves were air-dried for two weeks and were cut into small pieces. Forty (40) grams of each type were then boiled in 250 ml of water for 15 minutes, cooled, and then filtered using cheesecloths. The filtrates were placed in a water bath until 20 ml of each of the filtrate was left. The concentrates were centrifuged at 2000 rpm at 25° C for 10 minutes. The supernatants from both the young and mature leaves were filtered obtaining an amount of 2 ml each. One ml of the filtered supernatant were labeled “200% Y” for young leaves and “200% M” for mature leaves. One ml of each of these solutions was then diluted with 1 ml each of sterilized water. The resulting solutions were labeled “100% Y” for young leaves and “100% M” for mature leaves
Inoculum Preparation
The American Type Culture Collection (ATCC) strains used for E. coli (ATCC 25922), P. aeruginosa (ATCC 27853) and S. aureus (ATCC 25923) were prepared by Mrs. Concepcion F.
Ang of the PGH Infectious Disease Section. Inoculation of the Test Plate
The vial containing the culture was placed on a vortex equipment to ensure homogeneity. A sterile cotton swab was dipped into the culture. The swab was streaked into the surface of a Mueller Hinton Agar plate (Appendix 1). The streaking was repeated twice, rotating the plate approximately 60 degrees each time to ensure even distribution of the inoculum. As a final step, the rim of the agar was swabbed. Preparation of Dried Paper Discs for Susceptibility Assays
The discs prepared from filter paper had a diameter of 6.35 mm. Discs were placed on top of an aluminum wire mesh. For the positive control, an aliquot of 0.02 ml of the antibiotic solution (ceftazidime for
E. coli and
P. aeruginosa and oxacillin for
S. pneumoniae) of
concentration were pipetted onto the center of the disc. An aliquot of 0.02 ml of the A. muricata leaf extract of different concentrations (100% and 200%) was also pipetted onto the center of the discs. The discs were left to air dry in a chamber that prevents contamination of the disc surfaces with foreign matter. Application of Discs
For the susceptibility setup of the microorganisms ( E. coli, P. aeruginosa and S.aureus), each disc was pressed down to ensure complete contact with the agar surface. The disc was distributed evenly so that they are no closer than 24 mm from center to center (each disc was not
relocated once it came in contact with the agar surface). The plates were incubated at 35 ° C for 16 to 18 hours before measuring the zones of inhibition. Data Collection
The diameters of the zones of inhibition of the different concentrations of the guyabano leaf extract, oxacillin and ceftazidime were measured using a standard caliper. In order to standardize the measurement, only one person performed the measurements. The measurements were interpreted as susceptible or resistant based on the table of the Zone Diameter Interpretative Standards and E Minimal Inhibitory Concentration (MIC) Breakpoints (Appendix 2).
RESULTS
Table 1. The zones of inhibition for the mature guyabano (Anona muricata) extract at different concentrations and positive controls Zone of inhibition Organism Drug Susceptibility R1 (mm) R2 (mm) Mean (mm) S. aureus Oxacillin 20.5 21.0 20.75 S 200 % 0 0 0 R 100 % 0 0 0 R E. coli Ceftazedime 26.0 28.0 27.0 S 200 % 0 0 0 R 100 % 0 0 0 R P. aeruginosa Ceftazedime 26.0 27.0 26.5 S 200 % 0 0 0 R 100 % 0 0 0 R Table 1 shows the zones of inhibition for the mature A. muricata aqueous leaf extract at different concentrations and positive controls. S. aureus showed susceptibility to Oxacillin, having zones of inhibition averaging 20.75mm. However, both 100% and 200% concentrations of the mature A. muricata leaf extracts exhibited no zones of inhibition. Interpretative criteria obtained from NCCLS categorize S. aureus as resistant to the aqueous leaf extract. S. aureus with zones of inhibition measuring ≤ 10 mm is interpreted as resistant to the leaf extract. For E. coli and P. aeruginosa, Ceftazidime exhibited zones of inhibition averaging 27.0 mm and 26.5
mm, respectively. However, the discs containing 200% and 100% concentrations of the mature leaf extract did not exhibit zones of inhibition for both bacteria. Therefore, they were categorized as resistant.
Table 2. The zones of inhibition for the young guyabano (Anona muricata) extract at different concentrations and positive controls Zone of inhibition Organism Drug Susceptibility R1 (mm) R2 (mm) Mean (mm) S. aureus Oxacillin 20.0 19.5 19.75 S 200 % 0 0 0 R 100 % 0 0 0 R E. coli Ceftazedime 29.0 30.0 29.5 S 200 % 0 0 0 R 100 % 0 0 0 R P. aeruginosa Ceftazedime 27.0 27.5 27.25 S 200 % 0 0 0 R 100 % 0 0 0 R Table 2 shows the measured zones of inhibition (in mm) of the various concentrations of young A. muricata in comparison with positive controls. For all the Pathogens tested, only the positive controls yielded zones of inhibition that were measured to fall within the range of susceptibility: S. aureus, 19.75 mm; E. coli, 29.5 mm; P. aeruginosa, 27.25 mm. No zones of inhibition were observed for all the discs containing the guyabano leaf extract hence all the pathogens tested were categorized as resistant.
DISCUSSION
As could be seen from the results, there were zones of inhibition from the positive controls of each bacterial species while the experimental discs containing the guyabano extracts showed no signs of inhibition. This shows that the 100% and 200% concentrations of aqueous Guyabano leaf extracts of both young and mature leaves were not able to inhibit the growth of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. There are two possible
explanations for this observation. First, the Anona muricata L. in the Philippines do not have antimicrobial properties against S. aureus, E. coli and P. aeruginosa. Secondly, the concentration of the extracts was not sufficient to elicit any antimicrobial effects on these common pathogens.
The extracts were obtained through the method of decoction. The standard concentration usually employed in the preliminary screening of the potency of decoctions is 200%. However, as could be seen from the results, this was not enough to elicit any antimicrobial effects on the common Gram positive and Gram negative bacteria present in the environment. Consequently, the 100% concentration of the guyabano extracts would not show antibacterial effects if a higher concentration of 200% did not show any antimicrobial effect. Different types of Anona muricata L. can be found outside of the Philippines. The varying environmental elements found in the areas where Anona muricata L. thrive may contribute to the diversity in the antimicrobial properties of these plants. From previous studies worldwide, it has been shown that the aqueous guyabano extracts have antimicrobial properties against Micrococcus aureus (currently known as Staphylococcus aureus), Escherichia coli and Mycobacterium tuberculosis (Santos et al.
1981). Two of the microorganisms that were
employed in this study have been reported to be susceptible to guyabano extract. This then shows that guyabano leaf extracts possess antimicrobial properties. Other studies have shown that the guyabano extracts from stems, barks and leaves exhibit inhibitory actions towards numerous microorganisms such as Plasmodium falciparum, Escherichia coli, Pseudomonas aeruginosa, Shigella flexneri, Staphylococcus aureus, Staphylococcus albus, Salmonella typhosa , Salmonella newport, and Serratia macescens (Raintree Nutrition, Inc. 2004). These microorganisms are from
Gram positive and Gram negative groups. Thus, Anona muricata L. is thus a potential broad spectrum antimicrobial.
CONCLUSION
Young and mature A. muricata leaf decoctions at concentrations of 100% and 200% did not exhibit antibacterial property comparable to Oxacillin against S. aureus and Ceftazidime against E. coli and P. aeruginosa as indicated by the failure to generate a zone of inhibition.
RECOMMENDATIONS
For future studies, a larger variety of Guyabano leaf extract concentrations should be evaluated. Particularly, concentrations greater than 200% should be tested since the low concentrations of the extract may not have been sufficient to inhibit the growth of the bacteria. Different methods can also be used for the experiment such as the microbroth dilution method and agar dilution method. A negative control should also be prepared to support a probable positive result that may be exhibited by the Guyabano aqueous leaf extract.
