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ORIGINAL ARTICLE
Year : 2015  |  Volume : 5  |  Issue : 2  |  Page : 75-81

Antibacterial activity and cytotoxicity of stem bark of two common plants of Bangladesh


1 Department of Pharmacy, Southeast University, Banani, Dhaka, Bangladesh
2 Laboratory of Molecular Signaling, Division of Intramural Clinical and Biological Research (DICBR), National Institute on Alcohol Abuse and Alcoholism (NIAAA), National Institutes of Health (NIH), Bethesda, Maryland, USA

Date of Submission06-Dec-2014
Date of Acceptance05-Feb-2015
Date of Web Publication23-Mar-2015

Correspondence Address:
Mahmuda Haque
Department of Pharmacy, Southeast University, Banani, Dhaka - 1213, Bangladesh

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2231-0738.153797

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   Abstract 

Aim: The emergence and spread of antimicrobial resistance is a growing problem in both developing and developed countries and threatens to become a global crisis. In recent years, attempts have been made to investigate indigenous medicines used 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 the stem barks of Carica papaya Linn. (C. papaya; family: Caricaceae) and Tamarindus indica Linn. (T. indica; family: Fabaceae), two common plants of Bangladesh. The petroleum-ether, chloroform, and ethyl acetate extracts of the stem bark of both plants were investigated for their antibacterial activity and cytotoxicity. Materials and Methods: The antibacterial activity was evaluated using the disk diffusion method. Cytotoxicity was determined against brine shrimp nauplii. In addition, the minimum inhibitory concentration (MIC) was determined using the serial dilution technique to evaluate antibacterial potency. Results: All crude extracts of T. indica and the chloroform extract of C. papaya appeared very potent in terms of both zones of inhibition and spectrum of activity. However, all the extractives were also subjected to brine shrimp lethality bioassay for preliminary cytotoxicity evaluation. Here, the chloroform extract of C. papaya revealed the strongest cytotoxicity, LC 50 of 10.46 μg/mL. Conclusion: The stem barks of both C. papaya and T. indica show broad-spectrum antibacterial activity and may be potential sources of natural antimicrobial compounds and anticancer agents to be used in the treatment of various infectious diseases caused by resistant microorganisms and of cancer, respectively.

Keywords: Antibacterial activity, antimicrobial activity, Carica papaya, cytotoxicity, Tamarindus indica


How to cite this article:
Haque M, Chowdhury MG, Kabir MF, Rashid MA. Antibacterial activity and cytotoxicity of stem bark of two common plants of Bangladesh. Int J Nutr Pharmacol Neurol Dis 2015;5:75-81

How to cite this URL:
Haque M, Chowdhury MG, Kabir MF, Rashid MA. Antibacterial activity and cytotoxicity of stem bark of two common plants of Bangladesh. Int J Nutr Pharmacol Neurol Dis [serial online] 2015 [cited 2019 Oct 23];5:75-81. Available from: http://www.ijnpnd.com/text.asp?2015/5/2/75/153797


   Introduction Top


The use of different parts of different plants or their extracts for the treatment of various diseases is an age-old practice. Plant-derived antimicrobial compounds have received considerable attention in recent years due to the emergence of multidrug-resistant bacteria and the high costs and serious side effects of antibiotics. [1] Efforts are thus directed to identify those plants or their plant products that have broad-spectrum antimicrobial properties but no associated ill effects. [2],[3] It is expected that the screening and scientific development of plant extracts can yield novel compounds of therapeutic value. [4] In addition, in most of the developing countries, a large number of populations depend on traditional practitioners, who in turn are dependent on medicinal plants, to meet their primary health-care needs. Although modern medicines are available, herbal medicines have retained their status for historical and cultural reasons. [5] As in other developing countries, a large portion of the Bangladeshi population depend for their physical and psychological health needs on traditional systems of medicine. Medicinal plants have become the focus of intense study in terms of conservation and as to whether their traditional uses are supported by observable pharmacological effects or merely based on folklore. [6],[7]

Considering the given context, an attempt has been made to study C. papaya and T. indica, two common plants of Bangladesh. The plant C. papaya is a member of the Caricaceae family and is a dicotyledonous, polygamous, and diploid species. [8] It originated from Southern Mexico, Central America, and the northern part of South America. It is now cultivated in many tropical countries such as Bangladesh, India, Indonesia, Sri Lanka, the Philippines, the West Indies, and Malaysia. The papaya fruit is globally consumed either fresh or in the form of juices, jams, and crystallized dry fruit. [9] The ripe fruit is said to be a rich source of vitamins A and C and calcium. [10] There are many commercial products derived from the different parts of the C. papaya plant, the most prominent being papain, caricain, and chymopapain, which are produced from the latex of the young fruit, stem, and leaves. C. papaya leaves have been used in folk medicine for centuries. Recent studies have shown their beneficial effect as an anti-inflammatory agent, [11] for their wound-healing properties, [12] for their antitumor as well as immunomodulatory effects, [13] and as an antioxidant. [14] A toxicity study (acute, subacute, and chronic toxicity) conducted on Sprague-Dawley rats administered C. papaya leaf juice (CPLJ) of the sekaki variant revealed that it was safe for oral consumption. [15] CPLJ is also used in the treatment of dengue fever. [16],[17] T. indica belongs to the dicotyledonous family Fabaceae. [18] It is indigenous to tropical Africa but has become naturalized in North and South America from Florida to Brazil and is also cultivated in subtropical China, India, Pakistan, Bangladesh, the Philippines, Java (Indonesia), and Spain. [19] T indica is rich in phenolic compounds, polymeric tannins, and fatty acids, flavonoids, saponins, alkaloids, glycosides, and essential elements As, Ca, Cu, Fe, Mn, and Mg. [20],[21] Traditionally T indica has been used to treat asthma, bronchitis, leprosy, tuberculosis, wounds, ulcers, inflammation, gastralgia, diarrhea, dysentery, burning sensations, giddiness, vertigo, and diabetes. [22],[23] It has been reported that T. indica extracts have antiulcer, anti-asthmatic, antidiabetic, antioxidant, antibacterial, anti-inflammatory, and analgesic properties. [20],[24],[25],[26],[27],[28],[29],[30]

To the best of our knowledge, there are many reports on the leaf, fruit, and seed extracts but none regarding the stem bark of C. papaya. Accordingly, there are many reports on the leaf, fruit, fruit pulp, and seed extracts but very few reports regarding the antimicrobial activity and no report on the cytotoxicity of the stem bark of T. indica. As there is no sufficient literature on the antibacterial and cytotoxic activities of the stem barks of these plants, we undertook the present study as a primary biological investigation. We report herein the antibacterial and cytotoxic effects of the stem barks of C. papaya and T. indica for evaluation of their potential use as antimicrobial and cytotoxic agents, to advance research, and to further establish the scientific basis of the traditional uses of these plants.


   Materials and methods Top


Collection and identification of the plant

Fresh stem bark samples of the plant C. papaya were collected during the month of January, 2011 from the area of Rangpur, Bangladesh, and fresh stem bark samples of the plant T. indica were collected during the month of July, 2011 from the area of Dhaka, Bangladesh. Both the plants were identified by Dr. Hosne Ara, Director, Bangladesh National Herbarium.

Plant materials extraction and fractionation

The fresh stem barks of both plants were washed, sun-dried, and ground. The powdered stem barks of C. papaya (130 mg) and T. indica (150 mg) were extracted with ethanol at room temperature in flat-bottomed glass containers, with occasional stirring and shaking for 7 days. Next, the extracts were filtered, first through cotton and then through filter paper. The filtrates were concentrated to afford solid masses by using a rotary evaporator. [31],[32] The concentrated ethanol extracts of both plants were made slurry with water, and this was followed by solvent/solvent partitioning with petroleum-ether, chloroform, and ethyl acetate using the Kupchan partitioning method. [33]

Antibacterial test

The antibacterial assay was performed by the disk diffusion technique. [34],[35] 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. Of this concentrated solution, 10 mL was 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 mg of crude extracts. [34],[35] To compare the activity with standard antibiotics, kanamycin (30 mg/disk) was used.

All extracts of the collected plants were tested against five Gram-positive and seven Gram-negative bacteria (Bacillus megaterium, Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Sarcina lutea; and  Salmonella More Details paratyphi, Vibrio parahaemolyticus, Vibrio mimicus,  Escherichia More Details coli, Shigella dysenteriae, Pseudomonas aeruginosa, and Shigella boydii). Briefly, in this study the test disks and the standard disk were placed in a  Petri dish More Details seeded with particular bacteria and then left in a refrigerator at 4°C for 12-18 h, in order to diffuse the material from the disks to the surrounding 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 investigation was carried out in triplicate.

Minimum inhibitory concentration (MIC) determination

The MIC is the lowest concentration at which a test sample shows its highest activity against the tested microorganism(s). The MICs of the extracts were also determined by the serial dilution technique against the bacteria. [36] The plant extract (1.0 mg) was dissolved in 2 mL distilled water (two drops Tween 80 were added to facilitate dissolution) to obtain a stock solution. After preparing the suspensions of test organisms (10 7 organisms per microliter), one drop of suspension (0.02 mL) was added to each broth dilution. After 18 h incubation at 37°C, the tubes were examined for bacterial growth. The MIC of the extract was taken as the lowest concentration that showed no growth. Growth was observed in those tubes where the concentration of the extract was below the inhibitory level and the broth medium was observed to be turbid (cloudy). Distilled water with two drops of Tween 80 and kanamycin were used as negative and positive control, respectively. The investigation was carried out in triplicate.

Cytotoxicity screening

The brine shrimp lethality bioassay is widely used in bioassay for bioactive compounds. [37] 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 h; they then mature into nauplii. [38] The cytotoxicity assay was performed on brine shrimp nauplii using the Meyer method. The test samples (extracts) were prepared by dissolving them in dimethyl sulfoxide (DMSO) (not more than 50 μl in 5 mL solution) plus seawater (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. [38],[39] A vial containing 50 μL of DMSO diluted to 5 mL was used as a control. Standard vincristine sulfate was used as the positive control. Next, the matured shrimps were transferred individually to each of the experimental vials and the control vial. The number of the nauplii that died after 24 h was counted. The findings were transformed to probit analysis for determination of the median lethal concentration (LC 50 ) values of the compound. The bioassay was carried out in triplicate.

Statistical analysis

All the above assays were conducted in triplicate and repeated three times for consistency of results and statistical purpose. The zone of inhibition, MIC, and LC 50 were calculated as mean ± SE (n = 3) for the antibacterial screening, MIC determination, and brine shrimp lethality bioassay, respectively.

[TAG:2]RESULTS [/TAG:2]

The results of antibacterial screening

The extractives of C. papaya and T. indica demonstrated varying degrees of inhibition against the growth of microorganisms. All extracts of the collected plants were subjected to screening for inhibition of microbial growth against five Gram-positive and seven Gram-negative bacteria. [Table 1] and [Table 2] present the summary of the antibacterial activities of C. papaya and T. indica, respectively, with respect to each of the test organisms. The average zones of inhibition produced by the crude petroleum-ether, chloroform, and ethyl acetate extracts of C. papaya and T. indica were 10 mm, 11-17 mm, and 8-13 mm [Table 1], and 9-27 mm, 11-29 mm, and 13-20 mm [Table 2], respectively.
Table 1: Antibacterial activity of petroleum-ether, chloroform, and ethyl acetate extracts of the stem bark of C. papaya in terms of zone of inhibition in millimeters

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Table 2: Results of antibacterial activity of petroleum-ether, chloroform, and ethyl acetate extracts of the stem bark of T. indica in terms of zone of inhibition in millimeters

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The results of MIC determination

The results of MIC determination in terms of micrograms per milliliter are presented in [Table 3]. The MIC value of the chloroform extract of C. papaya was determined against B. megaterium and V. parahaemolyticus and was found to be 512 μg/mL in both cases. The MIC value for the ethyl acetate extract of T. indica against both S. aureus and P. aeruginosa was 128 μg/mL. The antibacterial potency of these two extracts against the tested bacteria expressed in MIC indicated that these plant extracts are equally effective against Gram-positive and Gram-negative bacteria. But for the chloroform extract of T. indica, MIC values indicated that the plant extract is more effective against Gram-negative at a lower concentration (lowest 128 μg/mL) than against Gram-positive bacteria (256 μg/mL).
Table 3: Minimum inhibitory concentrations of the stem barks of C. papaya and T. indica

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The results of the cytotoxicity test

The cytotoxicity of all crude extracts of C. papaya and T. indica was evaluated against A. salina [Table 4], and significant effects were attributable to the chloroform and ethyl acetate extracts of C. papaya and T. indica, with LC 50 values of 10.46 μg/mL and 12.26 μg/mL [Figure 1], and 12.52 μg/mL and 13.69 μg/mL [Figure 2], respectively. The LC 50 value of standard vincristine sulfate was found to be 9.61 μg/mL [Figure 1] and [Figure 2]. No mortality was seen in the control group.
Figure 1: Determination of LC50 values for standard and petroleumether, chloroform, and ethyl acetate extracts of the stem bark of C. papaya, from linear correlation between logarithms of concentration versus percentage of mortality

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Figure 2: Determination of LC50 values for standard and petroleumether, chloroform, and ethyl acetate extracts of the stem bark of T. indica, from linear correlation between logarithms of concentration versus percentage of mortality

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Table 4: Results of cytotoxic activity tests of standard and petroleum-ether, chloroform, and ethyl acetate extracts of the stem bark of C. papaya and T. indica

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   Discussion Top


All the crude extracts of T. indica showed moderate to strong zones of inhibition against all the tested microorganisms [Table 2], while the chloroform extract of C. papaya demonstrated mild to moderate activity against all the microbial strains [Table 1]. The chloroform extract of T. indica strongly inhibited the growth of E. coli (29 mm) and moderately inhibited the growth of V. mimicus (19 mm), P. aeruginosa (17 mm), B. subtilis (14 mm), B. megaterium (14 mm), and V. parahaemolyticus (14 mm). Again, the petroleum-ether extract of T. indica strongly inhibited the growth of E. coli (27 mm) and V. mimicus (22 mm). It also showed moderate activity against V. parahaemolyticus (19 mm), B. cereus (18 mm), and B. subtilis (17 mm). The ethyl acetate extract of T. indica showed strong inhibitory activity against the growth of S. aureus (20 mm) and P. aeruginosa (20 mm), whereas it showed moderate activity against the rest of the tested bacteria with an average zone of 13-17 mm. The present research results for T. indica agree with the earlier studies [20],[22] that showed that water, acetone, and ethanol extracts of T. indica stem bark exhibited antibacterial activity against Gram-negative E. coli, S. paratyphi, and P. aeruginosa and Gram- positive B. subtilis and S. aureus. On the other hand, the chloroform extract of C. papaya showed the greatest (17 mm) zone of inhibition against both Gram-positive B. megaterium and Gram-negative V. parahaemolyticus, and the average zone of inhibition was 11-17 mm, whereas the petroleum-ether extract showed activity against only S. aureus, S. lutea, S. paratyphi, and S. dysenteriae, the zone of inhibition measuring 10 mm for each case. The ethyl acetate extract of C. papaya moderately inhibited the growth of B. cereus (13 mm) and E. coli (13 mm). The antibacterial activities of these extractives revealed the presence of broad-spectrum antibiotic compounds. [40],[41]

Following the procedure of Meyer, [38] the lethality of each extractive to brine shrimp was determined, and the results are summarized in [Table 4]. The LC 50 values obtained from the best-fit line slope for the petroleum-ether, chloroform, and ethyl acetate extracts of C. papaya were 16.83 μg/mL, 10.46 μg/mL, and 12.26 μg/mL [Figure 1], respectively. The LC 50 values of the petroleum-ether, chloroform, and ethyl acetate extracts of T. indica were 16.69 μg/mL, 12.52 μg/mL, and 13.69 μg/mL, respectively, [Figure 2]. In comparison with the positive control (vincristine sulfate), the cytotoxicity exhibited by the chloroform and ethyl acetate extracts was significant. This clearly indicated the presence of potent bioactive principles in these extractives, which might be very useful as antiproliferative, antitumor, pesticidal, and other bioactive agents. [38],[42]


   Conclusion Top


The results obtained in the present study demonstrated that different extracts (petroleum-ether, chloroform, and ethyl acetate) of the plants C. papaya and T. indica have promising antibacterial properties against the tested microorganisms, which can be utilized in the treatment of infectious diseases caused by resistant microorganisms. The results of the present study also support the traditional usage of the studied plants. Therefore, these results are encouraging enough that the characterization in detail of these fractions in other models can be pursued. Further studies may also be conducted to isolate and purify the active constituents to evaluate their cytotoxicity in human cell line cultures.

 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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