Abstract
BackgroundAntibiotic resistances of pathogens and breast cancer warrant the search for new alternative strategies. Phytoextracts can eradicate microbe-borne diseases as well as cancer with lower side effects compared to conventional antibiotics.AimUnripe and ripe Azadirachta indica (neem) seed extracts were explored as potential antibiofilm and anticancer agents in combating multidrug-resistant infectious bacteria as well as anticancer agents against the MDR breast cancer cell lines.Methods Shed-dried neem seeds (both unripe and ripe) were pulverized and extracted using methanol. The chemical components were identified with FTIR and gas chromatography - mass spectrometry. Antibiofilm activity of neem seed extracts were assessed in terms of minimum biofilm inhibitory concentration (MBIC), minimum biofilm eradication concentration (MBEC), and fluorescence microscopic studies on Staphylococcus aureus and Vibrio cholerae. Bacterial cells were studied by fluorescence microscopy using acridine orange/ethidium bromide as the staining agents. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were evaluated to observe the antibacterial activities. Cytotoxicity of the extracts against human blood lymphocytes and the anticancer activity against drug-resistant breast cancer cell lines were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and fluorescence-activated cell sorting (FACS) studies.Results 4-Ethyl-2-hydroxy-2-cyclopentene-1-one, phthalic acid, and 2-hexyl-tetrahydro thiophane were the major compounds in unripe neem seed, whereas 3,5-dihydroxy-6-methyl-2,3-dihydro-4-H-pyran-4-one and 4-ethylbenzamide were predominant in ripe neem seed. Triazine derivatives were also common for both the extracts. MBIC values of unripe and ripe neem seed extracts for S. aureus are 75 and 100 µg/mL, respectively, and for V. cholerae, they are 100 and 300 µg/mL, respectively. MBEC values of unripe and ripe seed extracts are 500 and 300 µg/mL, respectively for S. aureus and for V. cholerae the values are 700 and 500 µg/mL, respectively. Fluorescence microscopic studies at 16 and 24 h, after bacterial culture, demonstrate enhanced antibiofilm activity for the ripe seed extract than that of the unripe seeds for both the bacteria. MTT assay reveals lower cytotoxicity of both the extracts towards normal blood lymphocytes, and anticancer activity against breast cancer cell line (MDA-MB-231) with superior activity of ripe seed extract. FACS studies further supported higher anticancer activity for ripe seed extract.ConclusionsMethanolic extract of neem seeds could substantially inhibit and eradicate biofilm along with their potent antibacterial and anticancer activities. Both the extracts showed higher antibiofilm and antibacterial activity against S. aureus (gram-positive) than V. cholerae (gram-negative). Moreover, ripe seed extract showed higher antibiofilm and anticancer activity than unripe extracts.Graphical
Highlights
Antibiotic resistances of pathogens and breast cancer warrant the search for new alternative strategies
The unripe and ripe neem seeds were separately washed with water and shed-dried for fifteen days at room temperature with little modification of method described previously [49]. 200 g dried seeds were pulverized for 2 min using a blender. 50 g of powdered seed was mixed with 150 mL of HPLC grade methanol and incubated at 37oC under shaking condition for 48 h
Antibiofilm studies of neem seed extracts against S. aureus and V. cholerae show that the methanolic extracts of neem seeds are potentially effective against the formation and eradication of bacterial biofilm
Summary
Antibiotic resistances of pathogens and breast cancer warrant the search for new alternative strategies. Remarkable escalation in the appearance of multidrug-resistant bacterial pathogens has caused a life-threatening emergent situation in the therapeutic arena and is responsible for the high rate of mortality, especially for immune-compromised individuals [1]. Due to the enhanced resistance, it becomes harder to treat common infectious diseases [2, 4] This situation is serious in medical indwelling-related hospital-acquired infections, and been happening in serious magnitude in the communities [5, 6]. Effective development of alternative treatment strategies to antibiotics, chemotherapy, in various aspects like organ transplantation, surgeries, and cancer treatment is becoming a concern of towering demand to achieve control over the high risk of multi drug-resistant events. About 80% of bacterial infections are biofilm-mediated, the ability to form biofilm make the situation graver as bacterial cells in a biofilm can undermine host immune attack and restricts the antimicrobial substances to penetrate the biofilm layer [3, 6, 7]
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