|Year : 2014 | Volume
| Issue : 5 | Page : 29-33
Antibacterial and Cytotoxic Activities of Alocasia fornicata (Roxb.)
Mahmuda Haque1, Tanjheela Jahan1, Md Abdur Rashid2
1 Department of Pharmacy, Southeast University, Banani, Dhaka 1213, Bangladesh
2 Laboratory of Molecular Signaling, DICBR, NIAAA, NIH, Bethesda, Maryland 20892-9410, USA
|Date of Web Publication||19-Dec-2014|
Department of Pharmacy, Southeast University, Banani, Dhaka 1213
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objectives: The aim of the study was to investigate the antibacterial activity, with minimum inhibitory concentration (MIC) and cytotoxic activities of petroleum-ether, chloroform, and ethyl acetate extracts of leaves, stolons and ethanol extract of the roots of the plant Alocasia fornicata (Roxb.) from the family Araceae. Materials and Methods: The antimicrobial activity was evaluated using the disk diffusion method and then MIC was detected using the serial dilution technique. The various parts of the plant were also assessed for cytotoxic activity using the brine shrimp lethality bioassay. Results: All crude extracts, except petroleum ether extract of the leaf and stolon showed moderate-to-good levels of antimicrobial activity against most of the tested bacteria, with an average zone of inhibition of 8-20 mm. The MICs were detected using the serial dilution method and the results ranged from 64 μg/ml to 256 μg/ml. All crude extracts displayed considerable general toxicity toward brine shrimps. The chloroform extract of the leaf, ethyl acetate extract of the stolon, and ethanol extract showed good cytotoxic activity and the LC 50 values were found to be 13.98 μg/ml, 12.26 μg/ml, and 12.81 μg/ml, respectively. Conclusion : From the above context, it may be concluded that the plant A. fornicate may be a potential source for the development of traditional medicine, such as, a safer antibiotic and also as an anticancer agent.
Keywords: Alocasia fornicata, antimicrobial activity, cytotoxic activity
|How to cite this article:|
Haque M, Jahan T, Rashid MA. Antibacterial and Cytotoxic Activities of Alocasia fornicata (Roxb.). Int J Nutr Pharmacol Neurol Dis 2014;4, Suppl S1:29-33
|How to cite this URL:|
Haque M, Jahan T, Rashid MA. Antibacterial and Cytotoxic Activities of Alocasia fornicata (Roxb.). Int J Nutr Pharmacol Neurol Dis [serial online] 2014 [cited 2020 Jan 28];4, Suppl S1:29-33. Available from: http://www.ijnpnd.com/text.asp?2014/4/5/29/147462
| Introduction|| |
Plants have played a significant role in maintaining human health and in improving the quality of human life since a long time and have served humans well as valuable components of medicines, seasoning, beverages, cosmetics, and dyes. The popularity of herbal medicine in recent times is based on the premise that plants contain natural substances that can promote health and alleviate illness. Therefore, the focus on plant research has increased all over the world and a large body of evidence shows the immense potential of medicinal plants used in various traditional systems.  Most of these plants have been used in the indigenous system of medicine for centuries without having any proper investigation done on their therapeutic activities. Today, scientists are using these renewable resources to produce a new generation of therapeutic solutions. In recent years, attempts have been made to investigate indigenous drugs against infectious diseases, to help in developing safer antimicrobial and anticancer drugs. 
As part of the further advancement of this research arena, an attempt has been made to study on A. fornicata. The plant belongs to the family Araceae. It grows naturally in the marshy lands of the tropical areas in India, Bangladesh, China, and South Africa. It is also cultivated in India and Bangladesh. The typical characteristics of A. fornicata are: It is 2-3 ft in height, with a slightly pinkish colored petiole, and wide triangular shaped leaves, and a horizontally growing stolon. The species is commonly known as Shola kachu and is widely used in Ayurvedic medicines in India since time immemorial. Almost all parts of these plants are used as a food. It is also known as poor man's food, primarily due to their richness in starch.  The other phyto-constituents are triglochinin and calcium oxalate.  The plant has antioxidant properties. In India people of the Konda Reddis and Savaras tribes use rhizome paste to treat wounds and to kill worms in domestic animals. Kanda and Nuke Doras people also apply the rhizome paste for curing cracks in the heels and wounds.  Hence, this study aims to screening the A. fornicata plant for its antibacterial and cytotoxic activities, for evaluating its potential uses, and determining whether its use in traditional medicine is justified.
| Materials and Methods|| |
Collection and identification of the plant
The fresh plants of A. fornicata were collected during the month of January 2011 from the area of Manikganj, Bangladesh and identified by Dr. Hosne Ara, Director, Bangladesh National Herbarium.
Plant materials extraction and fractionation
The fresh leaves, stolons and roots were washed, sun dried and ground. The ground leaves (100 mg), stolons (80 mg) and roots (40 mg) were extracted with ethanol at room temperature, and this was followed by solvent - solvent partitioning of the leaf and stolon extracts with petroleum-ether, chloroform, and ethyl acetate. ,
The antibacterial assay was performed by the disk diffusion technique. , The sample solution of the materials to be tested was prepared by dissolving a definite amount of material in the appropriate solvent to attain a concentration of 50 mg/ml. Ten microliters of a such solution were applied on a sterile disk (5 mm diameter, filter paper) and allowed to dry off the solvent in an aseptic hood. Thus, such disks contain 500 μg of crude extracts. To compare the activity with standard antibiotics, Kanamycin (30 μg/disk) was used.
All extracts of the collected plant were tested against five Gram-positive and seven Gram-negative bacteria (Bacillus megaterium, Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Sarcina lutea and Salmonella paratyphi, Vibrio parahaemolyticus, Vibrio mimicus, Escherichia coli, Shigella dysenteriae, Pseudomonas aeruginosa and Shigella boydii). Briefly, in this study the test disks and standard disk were placed in a Petri dish seeded with particular bacteria and then left in a refrigerator at 4°C for 12-18 hours, in order to diffuse the material from the disks to the surround media in the Petri dishes. The Petri dishes were then incubated at 37°C overnight to allow bacterial growth. The antibacterial activities of the extracts were then determined by measuring the respective zone of inhibition in millimeters.
The Minimum Inhibitory Concentration (MIC) is the lowest concentration at which the test sample shows its highest activity against the tested microorganisms. The MICs of the extracts were also determined by the serial dilution technique against the bacteria, which showed the highest zone of inhibition in the antibacterial assay. 
The brine shrimp lethality bioassay is widely used in bioassay for bioactive compounds.  Here a simple zoological organism (Artemia salina) was used as a convenient monitor, for screening.
The eggs of the brine shrimp, A. salina, were collected from an aquarium (Dhaka, Bangladesh) and hatched in artificial seawater (3.8% NaCl solution) for 48 hours, to mature into shrimps called nauplii.  The cytotoxicity assay was performed on brine shrimp nauplii using the Mayer method. The test samples (extracts) were prepared by dissolving them in Dimethyl sulfoxide (DMSO) (not more than 50 μl in 5 ml solution) plus sea water (3.8% NaCl in water) to attain concentrations of 20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml, and 100 μg/ml. A vial containing 50 μl of DMSO diluted to 5ml was used as a control. Standard Vincristine Sulfate was used as the positive control. , Next the matured shrimps were applied to each of the experimental vials and the control vial. The number of the nauplii that died after 24 hours was counted. The findings were transformed to probit analysis for determination of the LC 50 values of the compound.
| Results|| |
The results of antibacterial screening:
All crude extracts of A. fornicata were tested for antibacterial activity against 12 (five Gram-positive and seven Gram-negative) human pathogenic bacteria. The results of the extracts displaying an antibacterial effect against different bacteria are shown in [Table 1] and [Table 2]. Although the petroleum-ether extracts showed no activity against the tested bacteria, the chloroform and ethyl acetate extracts of the leaf and stolon and the ethanol extract of the root showed moderate-to-good activity against most of the tested bacteria. The chloroform extract of the leaf showed the highest 14 mm zone of inhibition against the Gram-positive bacteria, B. subtilis and Gram- negative bacteria, S. dysenteriae, and the average zone of inhibition was 10 mm to 14 mm. The highest activity of the ethyl acetate extract of the leaf was recorded as 15 mm against S. boydii. and the lowest was 8 mm against B. megaterium. On the other hand, a 10 mm to 20 mm zone of inhibition was seen in the chloroform extract of the stolon and the highest zone of inhibition, of 20 mm was see against S. lutea, a Gram-positive bacterium. However, the ethyl acetate extract of the stolon showed activity against only B. subtilis, S. lutea, S. paratyphi, and P. aeruginosa, and the highest activity was 14 mm against B. subtilis. The ethanol extract of the root showed good activity against all the tested bacteria. Among the tested bacteria, the growth of P. aeruginosa (18 mm) was strongly inhibited by the ethanol extract of the root. The results of MICs in terms of micrograms per milliliter are presented in [Table 3]. The MIC of the ethyl acetate extract of the leaf was determined against B. subtilis and S. boydii and was found to be 64 μg/ml and 128 μg/ml, respectively, and for the chloroform extract of the stolon it was 64 μg/ml against B., m.subbtilis and 128 μg/ml against P. aeruginosa. The ethanol extract of the root demonstrated an MIC of 64 μg/ml against P. aeruginosa and 256 μg/ml against B. cereus.
|Table 1: Antibacterial activity of petroleum-ether, chloroform, ethyl acetate, extract of the leaf and ethanol extract of the root in terms of zone of inhibition in millimeters |
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|Table 2: Antibacterial activity of petroleum ether, chloroform, and ethyl acetate, extract of the stolon in term of zone of inhibition in millimeters |
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|Table 3: Minimum inhibitory concentration of ethyl acetate extract of the leaf, chloroform extract of the stolon, and ethanol extract of the root |
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The results of the cytotoxicity test
The cytotoxic activities of all crude extracts of A. fornicata were studied by the brine shrimp lethality bioassay. The petroleum-33 ether, chloroform, and ethyl acetate extracts of the leaf and ethanol extract of the root showed good cytotoxic activity with the LC 50 values of 13.98 μg/ml, 15.60 μg/ml, 15.29 μg/ml, and 12.81μg/ml, respectively, [Figure 1]. The LC 50 values of petroleum ether, chloroform and ethyl acetate extracts of the stolon were 18.69 μg/ml, 14.66 μg/ml, and 12.26 μg/ml, respectively, [Figure 2]. The LC 50 value of standard Vincristine Sulfate was found to be 9.61μg/ml [Figure 1] and [Figure 2]. No mortality was found in the control group.
|Figure 1: Determination of LC50 values for standard and petroleum-ether, chloroform, ethyl acetate, extracts of the leaf, and ethanol extract of the root of A. fornicate, from linear correlation between logarithms of concentration versus percentage of mortality|
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|Figure 2: Determination of LC50 values for standard and petroleum-ether, chloroform, and ethyl acetate extracts of the stolon of A. fornicate, from linear correlation between logarithms of concentration versus percentage of mortality|
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| Discussion|| |
Plants are an important source of potentially useful structures for the development of new chemotherapeutic agents. The first step toward this goal is the in vitro antibacterial activity assay.  Many plant species present inhibition zones of different diameters; however, the size difference of an inhibition zone depends primarily on many factors, for example, diffusion capacity of substances (present in the extracts) in the medium, antimicrobial activity of diffused substances, growth, and metabolic activity of the microorganisms in the medium.  The demonstration of antimicrobial activity against both Gram-positive and Gram-negative bacteria may be indicative of the presence of a broad spectrum of antibiotic compounds. ,
In the present study, we demonstrated for the first time that the plant A. fornicata has antibacterial and cytotoxic effects and could play an important role as a bioactive principle. The crude chloroform and ethyl acetate extracts of the leaf and stolon showed moderate-to-good antibacterial activity against both Gram-positive and Gram-negative bacteria. Although the chloroform extract of the stolon showed the highest zone of inhibition (20 mm against S. lutea), the crude chloroform and ethyl acetate extracts of the leaf were much more active against the tested bacteria than those of the stolon. The ethanol extract of the root showed much better activity against all the tested bacteria and the average zone of inhibition was 10 mm to 18 mm. The crude extracts also had significant minimum inhibitory concentration.
The Brine shrimp lethality bioassay has been used extensively in the primary screening of the crude extracts as well as isolated compounds, to evaluate the toxicity toward the brine shrimps, which could also provide an indication of the possible cytotoxic properties of the test material.  It has been established that the cytotoxic compounds generally exhibit significant activity in the brine shrimp lethality bioassay, and this assay can be recommended as a guide for the detection of antitumor and pesticidal compounds, because of its simplicity and low cost.  This bioassay also has a good correlation with human solid tumor cell lines. Therefore, the cytotoxic effects of plant the extracts indicate that it can be selected for a further cell-line assay, because there is a correlation between cytotoxicity and the activity against the brine shrimp nauplii, when using the extract.  In comparison with the standard (Vincristine Sulfate), all crude extracts have shown moderate-to-potent cytotoxic activity, ranging from 12.26 μg/ml to 18.69 μg/ml against the brine shrimp nauplii. Among the crude extracts, the ethanol extract of the root and ethyl acetate extract of the stolon are promisingly cytotoxic and they might have antitumor activity.
| Conclusion|| |
In conclusion, the present study demonstrates that the plant, A. fornicata possesses antibacterial and cytotoxic activities. Therefore, the plant may be utilized for the development of traditional medicine and further investigation is necessary for the development of the lead compound and to establish it as a safer antibiotic and also as an anticancer agent.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]