IN VITRO SCREENING OF SELECTED MEDICINAL PLANTS AGAINST Aeromonas hydrophila

by

ARREN CHRISTIAN M. DE GUIA

An Undergraduate Thesis Proposal presented to the faculty of the College of Fisheries in partial fulfillment of the requirements of the degree of

BACHELOR OF SCIENCE IN FISHERIES

COLLEGE OF FISHERIES CENTRAL LUZON STATE UNIVERSITY Science City of Muoz, Nueva Ecija 2011
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INTRODUCTION

Background of the Study Tilapia (Oreochromis spp.) is a very popular aquaculture species in the Philippines at present. It has been reared by hatcheries and grow-out operators because of its high market demand in the country (Yambot, 1998). Tilapia, which is considered as aquatic chicken, is offering economical and social benefits mainly for rural communities. It also plays vital role in terms of worldwide employment (Badillo, 2010). However, as reported by some farmers in the early 1990s, severe disease outbreak threatened the growing tilapia industry in the country, and according to Yambot and Inglis (1994), it can be attributed to Aeromonas hydrophila. Aeromonas are water-borne pathogens and they are common in almost all aquatic environments including fresh, brackish and marine water. At present, there are 16 existing species of Aeromonas (Khan et al., 2008). Aeromonas species are the cause of diseases in cultured and feral fishes in Europe (Cipriano et al., 2001). Motile Aeromonas Septicemia (MAS), Hemorrhagic Septicemia, Ulcer Disease, or Red-Sore Disease are some of the common diseases caused by A. hydrophila (White, 1995). According to Khan et al., (2008), like some other bacteria, Aeromonas has been found to produce exotoxin to survive undesirable condition. These toxins are composed of Aerolysin-like hemolysin (ALH), Aeromonas Serine Protease (ASP) and Aeromonas Metalloprotease (AMP). Study showed that ALHs primary action is to rupture its cell victims; however, until today its mechanism has been a mystery. ASP has been found to promote plasma coagulation by means of activating prothrombin. It is common and deadly consequence

to sepsis victims. On the other hand, the mode-of-action of AMP is not yet been determined by the experts (Khan et al., 2008). In making curative remedies against bacterial infections, antimicrobial agents are highly needed but because of increasing resistance against these antimicrobial agents more problems are challenging the science world (Kaskhedikar and Chhabra, 2009). In addition, commercial antibiotics for large-scale treatment are unaffordable for most of the farmers (Siri et al., 2008). Therefore, there is a need to develop alternative antibiotics for the treatment of infectious diseases from medicinal plants (Agarwal et al., 1996). In Philippines, thousands of species are known to have medicinal value and the use of different parts of several medicinal plants to cure specific ailments has been in vogue since ancient times. Antimicrobials of plant origin have enormous therapeutic potential. The beneficial medicinal effects of plant materials typically result from the combinations of secondary products present in the plant that include alkaloids, steroids, tannins, phenol compounds, flavonoids and resins fatty acids gums (Food and Agriculture Organization, 1993).

Significance of the Study Nowadays, multiple drug resistance has developed due to the indiscriminate use of commercial antimicrobial drugs commonly used in the treatment of infectious disease. In addition to this problem, commercial antibiotics are costly and sometimes associated with adverse effects on the host. This situation necessitates searching for new antimicrobial substances. Therefore, there is a need to develop alternative antimicrobial drugs for the treatment of infectious diseases from medicinal plants.

The set of herbal plants that will be used in this study are found effective against bacterial pathogens in humans. Harnessing the potentials of these plant materials against fish bacterial pathogens will be a great help to the aquaculture industry as a whole.

Objectives of the Study The main objective of the study is to screen and evaluate the antibacterial potential of the different plant crude extracts against A. hydrophila and specifically, to measure the zone of inhibition and colonization.

Scope and Limitation The screening will only be done in vitro and will be followed by test for lethal concentration. Leaves will only be used in this experiment.

Time and Place of the Study The study will be conducted at the Fish Pathology Laboratory of the College of FisheriesFreshwater Aquaculture Center, Central Luzon State University (CF-FAC, CLSU), Science City of Muoz, Nueva Ecija from __________________________.

REVIEW OF RELATED LITERATURE

Medicinal Plants with Antibacterial Potential Hundreds of medicinal plants are used in making powerful drugs nowadays. Their potentials as a drug are mainly because of their secondary metabolites constituents such as tannins, alkaloids and flavonoids (Ravikumar et al., 2010). Presented below are lists of common medicinal plants found in the country with their antimicrobial active components (www.stuartxchange.org): Garlic (Allium sativum) is found to contain saponin, tannins, sulfurous compounds, prostaglandins, alkaloids, volatile oils and allicin. The most important chemical constituents are the cysteine sulfoxides (alliin) and the non-volatile glutamylcysteine peptides which make up more than 82% of the sulfur content of garlic. It also contains allyl disulphide which is responsible against helminths. Duhat (Syszygium jambolanum) or Jambolan is claimed to contain alkaloid, jambosine, glycoside and jambolin which halts the diastatic conversion of starch into sugar. The leaf juice is effective in the treatment of dysentery. Jambolan leaves may be helpful as poultices on skin diseases. The leaves, stems, flower buds, opened blossoms, and barks have some antibiotic activity. Ginger (Zingiber officinale) is commonly used against sore throat and it is also famous as a food additive. In addition, some records showed that it has antibacterial activity against Pseudomonas aeruginosa, Staphylococcus aureus and Strep pyogenes. Its most active ingredients, gingerols and shagaol, can be found at the root. Almost all the

parts can be utilized for different purposes. Chemical content against bacteria is not yet sure. Makabuhay (Menispermum crispum) is a popular insecticide for rice crop. Its stems and leaves can be utilized against rheumatism, hay fever, allergic rhinitis and many more. It has also antibacterial property because it contains diterpenes which was recorded to be effective against P. aeruginosa and Bacillus subtilis. Noni (Morinda citrifolia) is found to increase immunity and was also proven to have antibacterial activity against Shigella and Salmonella. It is a very popular fruit

juice due to heavy marketing of some company. It was proven to contain antioxidant and it can reduce carcinogen in the body. The fruit contains phytochemicals, lignans, polysaccharides, flavonoids, iridoids, nonisides, scopoletin, catechin, epicatechin, damnacanthal and alkaloids. Papaya (Carica papaya) is another proven effective herbal medicine. The leaves, fruits, stems and roots all contain the proteolytic enzyme papain. In a study conducted in the University of Nigeria, they found out that ripe and unripe fruit and its seeds are effective against gram-positive bacteria, and higher concentrations are effective against gram-negative bacteria. Jathropa (Jathropa curcas) is a well-known piscicide and insecticide. The leaves of jathropa were found to contain phlobatannins and tannins that have antimicrobial activity against Salmonella typhi, S. aureus and P. aeruginosa. Wild sugarcane (Saccharum spontaneum) contains quinones, alkaloids, tannins, carbohydrates, protein, coumarin, phenol, steroid and glycosides. Locally, it is widely used as a diuretic. Decoction of roots also used for fever. Study revealed that it has

antimicrobial activity against gram-positive and gram-negative pathogenic bacteria and some fungi. Leaves, stems and roots are usually used in experiments. Acacia (Samanea saman) bark and seed contain saponin-like alkaloid, pithecolobin. Aside from antibacterial activity, it is also used against stomach ache. Bark, stems, leaves and seeds are found to contain alkaloids. It can also be used as antipyretic and antidermatoses. Sampaguita (Jasminum sambac) is usually used against infection. Active constituents are alkaloids, glycoside, flavonoids, terpines, tannin, resin and salicylic acid. Sampaguita has antibacterial effect against Salmonella typhi and S. aureus. Flowers, roots and leaves are usually used for different purposes. Alugbati (Basella rubra) is an edible vine. Studies revealed that the aqueous, ethanolic and petroleum ether extracts of leaves exhibited antimicrobial activity against some bacteria. The ethanolic extract showed maximum effect against Escherichia coli. Continuous study is needed to determine the active ingredient that causes its antimicrobial activity. Atsuete (Bixa orellana) seeds are used as antidote for cassava and jathropa poisoning. Poultice of leaves are diuretic and used for treatment of gonorrhea . Atsuete extract showed significant effect against Bacillus cereus. The seeds contain carotenoids pigments such as bixin, norbixin, -carotene, cryptoxanthin, lutein, zeaxanthin and methyl bixin. Guava (Psidium guajava) is a famous edible backyard fruit. It is known for its astringent, antispasmodic, antihelminthic and antiseptic properties. Leaves are commonly used against toothache and wounds. Study showed that the guava sprout is active against

diarrhea caused by E. coli or S. aureus produced toxin. And also, leaves extract showed significant effect against Shigella spp., Vibrio spp., S. aureus, E. coli, P. aeruginosa and B. subtilis. Jackfruit (Arthocarpus heterophyllus) is a famous edible fruit because of its delicious taste. Root is considered antiasthmatic and bark is considered sedative. Many medicinal effects have been noted about this plant. It has also antibacterial activity against B. subtilis, B. cereus, S. aureus and E. coli. Active ingredient against bacteria has not been found yet. Chili (Capsicum annuum) is a famous spice because of its taste. It is used against rheumatism. The active ingredients are capsaicinoids, capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homodihydrocapsaicin and homocapsaicin. Capsaicin and

dihydrocapsaicin are responsible against B. cereus, B. subtilis, Clostridium sporogenes, C. tetani and S. pyogenes activities. Pomelo (Citrus grandis) is a citrus fruit common locally. It contains phytochemicals such as naringin, hesperidine, diosmin and naringenin. Leaves and flowers can be used for nervous affections, coughs and ulcer. Naringenin, one of its active ingredients was found to be effective against P. aeruginosa. Indian Mango (Mangifera indica) is an effective agent against S. aureus, E. coli, P. aeruginosa and provides a basis for its medical use in Uganda. Active components are saponins, steroids and triterpenoids, alkaloids, coumarins, anthracenocides, flavonones, tannins and reducing sugars. Tiesa (Pouteria campechiana) showed slight effect against E. coli and P. aeruginosa, the fungi C. albicans and T. mentagrophytes. They were inactive against S.

aureus, B. subtilis, and A. niger. It is also active against seborrheic dermatitis of the scalp.

Zone of Inhibition

Zone of inhibition is usually observed during a test of antibiotic against bacteria. It is the clear region around the paper disk soaked in an antimicrobial medium on the agar surface cultured with bacteria. It is widely used as the index of the effectiveness of antimicrobial extract against bacteria.

MATERIALS AND METHOD

Collection of Samples Most of the sample collection will be done at CLSU compound. The selected plants will be composed of Acacia, Alugbati, Atsuete, Chili, Duhat, Jackfruit, Jathropa, Garlic, Guava, Ginger, Papaya, Pomelo, Noni, Makabuhay, Sampaguita and Wild sugar cane. Leaves and fruits of the above plant samples will be used for crude extraction. In the absence of fruit, stem or root will be used.

Preparation of Crude Extract Leaves and fruits of plant samples will be shredded and then macerated using mortar and pestle to obtain the extract (using 1 part plant material: 2 parts distilled water). The solution will be filtered using single layer sterile filter paper. The different extract will be separately stored in a screw capped clean bottle and will be refrigerated until ready for use (Ordanel, 2009).

Preparation of Aeromonas hydrophila Pure culture of A. hydrophila and will be obtained from the U.P. Los BaosNational Institute of Molecular Biology and Biotechnology (UPLB-BIOTECH). From the pure bacterial culture (not more than 48 hours), four or five colonies will be transferred to 5 ml Trypticase soy broth. The broth will be incubated at 30 C or at an optimum growth temperature until achieves or exceeds the turbidity of 0.5 Mc Farland standards. The turbidity of the test bacterial suspension with that of 0.5 McFarland will be compared against a white background with contrasting black line under adequate light.
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Preparation of Eradicant Test Against the Bacteria Whattman filter paper discs measuring 6 mm will be made using a paper puncher. It will be sterilized in an autoclave at 15 psi for 30 minutes. The sterilized discs will be soaked into the plant extracts for 1 hour and will be air-dried for 10 minutes in a chamber. The different plant extracts will serve as the treatments. Meanwhile, commercial antibiotic (tetracycline) will be used as positive control and distilled water as the negative control (Table 1). The positive control will be prepared by diluting 250 mg of tetracycline in 4 ml distilled water. Sterile cotton swab will be dipped into the standardized bacterial suspension. The swab containing the inoculum will be streaked in the prepared nutrient agar plates. Using sterile forceps, the impregnated discs will be placed on the surface of the inoculated plate. The discs will be positioned such that the minimum center distance is 24 mm and no closer than 10 to 15 mm from the edge of the petri dish. In inverted position, the plates will be incubated at room temperature and will be observed after 12, 18, 24 and 36 hours after incubation. Using ruler, the diameter of zone of inhibition will be measured in millimeter. For each control and treatment, six replicates will be used.

Preparation of Protectant Test Against the Bacteria Whattman filter paper discs measuring 6 mm will be made using a paper puncher. It will be sterilized in an autoclave at 15 psi for 30 minutes. The sterilized discs will be soaked into the standardized bacterial suspension for 1 hour. The impregnated discs will be placed in the Mueller Hinton Agar plates containing 0.5 ml plant extract (for treatments) and 0.5 ml tetracycline (for positive control). The plates will be incubated at

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room temperature in inverted position and will be observed after 12, 18, 24 and 36 hours after incubation. The microbial effect will be determined by measuring the zone of bacterial colonization on plates. For each control and treatment, six replicates will be used. Plant Toxicity test This toxicity test will be done in order to determine the Median Lethal Concentration (LC50) of the selected plant crude extracts. Twenty Nile tilapia

fingerlings (size 22) will be exposed to different crude extract concentrations (5, 10, 15, 20, 30, 40 and 50 ml crude extract per liter of tap water). The fish will be observed in seven days in parallel to the length of the experimental set-up to be employed in the study. The concentration at which survival falls to 50% at the seventh day will be used as the plant concentration for the study.

To determine the LC50, the formula below will be used (Reed and Muench, 1983): LC50= Antilog {A + [(B/C) D]} Where: A = log concentration below 50% mortality B = 50 - mortality below 50% C = mortality above 50% - mortality below 50% D = log concentration above 50% - log concentration below 50%

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Table 1. Description of controls and treatments that will be employed in the study. DESCRIPTION Control (+) Control (-) T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 Commercial antibiotics (Tetracycline) Distilled water Acacia Alugbati Atsuete Chili Duhat Jackfruit Jathropa Garlic Guava Ginger Papaya Pomelo Noni Makabuhay Sampaguita Wild sugar cane

Statistical Analysis Significant difference in the diameter of zone of inhibition and colonization in every time of observation among controls and treatments will be analyzed using OneWay Analysis of Variance (ANOVA) under the Statistical Package of Predictive Analytics Software (PASW) Statistics Version 18.

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LITERATURE CITED

AGARWAL, P., RAI V. and SINGH R.B. (1996) Randomized, placebo-controlled, single-blind trial of holy basil leaves in patients with noninsulin-dependent diabetes mellitus. International Journal of Clinical Pharmacology and Therapeutics, 1996, 34:406-409. BADILLO, L. J. (2006) Age growth-models for Oreochromis aureus (Perciformes, Cichlidae) of the Infiernillo reservoir, Mexico and reproductive behaviour. Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN.-0034-7744) Vol.54 (2):577-588, 1989 CIPRIANO, R.C. (2001) Aeromonas hydrophila and Motile Aeromonads Septicemias of fish. Fish Disease Leaflets 68. FOOD and AGRICULTURE ORGANIZATION (1993) Medicinal and aromatic plant in Asia. Bangkok, Thailand RAPA, Publication. KASKHEDIKAR, M. and CHHABRA, D. (2009) Multiple drug resistance in Aeromonas hydrophila isolates of fish. Veterinary World Vol.3 (2): 76-77. KHAN, R., TAKAHASHI, E., NAKURA H., ANSARUZZAMAN M., BANIK, S., RAMAMURTHY T. and OKAMOTO, K. (2008) Toxin production by Aeromonas sobria in natural environments: river water vs. seawater. Acta Med. Okayama. 2008. Vol. 62. No. 6, pp. 363-371. ORDANEL, J. (2009) In vitro screening of antibacterial properties of selected weeds against Bacillus subtilis Cohn. RAVIKUMAR, S., SELVAN, G.P. and GRACETIN, N.A.A. (2010) Antimicrobial activity of medicinal plants along Kanyakumari Coast, Tamil Nadu, India. African Journal of Basic and Applied Sciences 2 (5-6): 153-157, 2010. ISSN 2079-2034. IDOSI Publications, 2010. SIRI, S., WADBUA, P. WONGPHATHANAKUL W., KITANCHAROEN, N. and CHANTARANOTAI, P. (2008) Antibacterial and phytochemical studies of 20 Thai medicinal plants against Catfish-Infectious Bacteria, Aeromonas caviae. KKU Sci. J.36 (Supplement) 1-10. WHITE, M.R. and SWANN L. (1995) Diagnosis and treatment of Aeromonas hydrophila infection of fish. Taken from A Guide To Approved Chemicals In Fish Production And Fishery Resource Management, 1989. YAMBOT, A.V. (1998) Isolation of Aeromonas hydrophila from Oreochromis niloticus during fish disease outbreaks in the Philippines. Asian Fisheries Science 10(1998): 347-354.
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YAMBOT, A.V. and INGLIS, V. (1994) Aeromonas hydrophila isolated from Nile tilapia (Oreochromis niloticus) with eye disease. In: International congress o quality veterinary services for 21st century (eds. M.K. Vidyadaran, M.T. Aziz and H. Sharif), pp. 87-88. Kuala Lumpur. www.stuartxchange.org. Retrieved on December 9, 2011

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