Background
Bacterial resistances to conventional antibiotics have emerged as one of the burning issues in therapeutic aspects. 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]. Development of host antibiotic resistance is caused mostly by their uncontrolled uses and this is mediated by increasing efflux pump activity, several modified enzymes which breaks the antibiotics and/or changing the target of antibiotics [
2,
3]. Due to the enhanced resistance, it becomes harder to treat common infectious diseases [
2,
4]. This situation is not only serious in medical indwelling-related hospital-acquired infections, but also been happening in serious magnitude in the communities [
5,
6]. Therefore, 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].
Infections caused by
Staphylococcus aureus, a gram-positive bacterial pathogen, have been indifferent to first-line antibiotics.
S. aureus causes severe soft tissue infections in hospital settings and in communities [
8,
9]. As per the report, methicillin-resistant
S. aureus (MRSA) causes 64% more death globally than the non-resistant counterpart [
8]. They cause acute infections like bacteraemia and skin abscesses by secreting several toxins and exo-enzymes [
10]. In contrast, chronic infections are associated with a biofilm mode of growth where it can attach and persist on host tissues, such as bone and heart valves, to cause osteomyelitis and endocarditis, respectively, or on implanted medical indwellings, such as catheters, hip prosthetic implants, etc. [
6,
10‐
12]. Among the food pathogens,
Vibrio cholerae is a facultative anaerobic, gram-negative human pathogen. It causes pandemic/endemic cholera, cholera-like diarrhoea, and extra-intestinal infections [
13,
14]. Consumption of contaminated water and/or food are the major causes of
V. cholerae infections and reports reveal that around 1.3 to 4.0 million people get infected by them every year all over the world [
15]. In inter-epidemic periods,
V. cholerae can survive long in their natural habitats by virtue of their ability to form biofilm and hence, they could be potential threats in causing widespread infection in near future by evolving as an epidemic clone. Continued researches on emerging strains of
V. cholerae have demonstrated a high frequency of emergence of multidrug-resistant strains [
15,
16].
Report reveal that more than 60% infections caused by bacteria are biofilm based [
17]. Biofilm is a cage-like structure formed by bacteria for its survival inside the host. This occurs through the creation of a matrix-like structure that covers and protects bacteria against different toxic agents or antimicrobial compounds as well as helps to access different nutrients [
18,
19]. Consequently, biofilm formation in bacteria creates a suitable environment for their persistence and enhances their virulence properties [
20,
21]. The extracellular matrix acts as a physical barrier that reduces the direct effects of antibiotics on bacterial cells [
22,
23]. This physical barrier prevents antibiotics to enter into the core zone of biofilm. As a result, bacterium wins against the killer-antibiotics [
24]. Common antibiotics normally kill planktonic cells and the majority of biofilm structures. However, the bacteria those can successfully survive even after the drug treatment, start a new cycle of biofilm development by repopulating the biofilm and disseminating into planktonic forms [
25]. This eventually enhances the ailment process caused by biofilm-forming pathogenic microorganisms.
Beside the threat of antibacterial resistance, cancer is another health burden despite the improvement of diagnosis and modern surgical processes [
26,
27]. Cancer cells are heterogeneous due to altered micro-environment and clonal evolution [
28]. Under-developed and developing countries are more susceptible to cancer as a result of poor lifestyles and “westernized” diets [
29]. It has been reported that 70% of cancer deaths occur in Africa, Asia, as well as in Central and South America [
30]. Breast cancer is one of the most common cancers among women worldwide, accounting for approximately 5.7 million deaths [
31]. Over 1.5 million women (25% of all women with cancer) are diagnosed with breast cancer every year globally [
31,
32]. Breast cancer is metastatic and can commonly transfer to distant organs such as the bone, liver, lung, and brain, which mainly accounts for its incurability. Although chemotherapy and several anticancer drugs are available, their adverse side effects on normal cells/tissue, such as bone marrow function inhibition, nausea, vomiting, and alopecia lead scientists to search for some alternative therapies, which has little or no major health concern [
33].
The aforementioned scenario has encouraged the present research group to make a target in identifying new bioactive compounds against the pathogenic bacterial infections as well as to target breast cancer. Inhibition of biofilm formation serves as a novel strategy to successfully combat bacteria bypassing the chance to generate bacterial resistance due to the minute or no pressure it exerts upon bacterial cell [
34]. On the other hand, inhibition of proliferation and induction of cancerous cell death are the vital targeting area to control as well as to eradicate the cancerous cells [
35]. Natural products have been playing important roles in exploring and processing of new drugs against bacterial infections and cancer development as their mode of action reduces the emergence of bacterial antibiotic resistance and abnormal cell progression, respectively [
35,
36].
A growing number of evidences clarified that plant extracts possess considerable antimicrobial and anticancer potentials without any significant risk of resistance development against them [
35,
37‐
39]. Besides, they are expected to have lower side effects. Plant-derived quinones, flavonoids, polyphenol, essential oils and tannins are known to complex with the cell wall synthesizing enzymes, inactivating it and thereby causes disruption of the microbial cell wall [
40]. Alkaloids get intercalated into the membranes and destabilize them [
40]. Polyphenols and tannins cause metal ion complexation and substrate deprivation to their target pathogens [
41]. One of the rich sources of natural products is
Azadirachta indica (neem) that has different beneficial properties in Indian as well as in African traditions [
42]. Flowers, leaves, seeds, and bark of this plant have extensively been used as insecticide, antimicrobial, larvicidal, antimalarial, antibacterial, antiviral, and anticancer agent [
43]. Components of neem are known to inhibit cancer progression as studied both
in-vitro and
in-vivo [
44]. About 300 components have so far been identified from neem [
45]. In spite of its tremendous potential, all the components and biological potentials are yet to be fully explored [
45]. Proper comparative evaluation of unripe and ripe seed extracts is also so far illusive. Hence, the current study endeavours to delineate the potential of the methanolic extracts of neem seeds against two potent human pathogenic bacteria, i.e.,
S. aureus and
V. cholerae, as antibiofilm agent, and also against breast cancer cells as anticancer agent.
S. aureus, the Gram-positive and
V. cholerae, the Gram-negative bacterial isolates were included in the study on the basis of their high biofilm forming abilities [
46,
47]. A wide range of phytochemicals, both polar and nonpolar, can easily be extracted using methanol and also it has low boiling point that prevents the phytochemicals from damage during evaporation [
48]. Antibiofilm activity of the extracts was assessed by spectrophotometric studies and fluorescence microscopy. MTT assessment was carried out on human blood lymphocytes to assess the biocompatibility of the extracts. Anticancer activity on MDA-MB-231 triple-negative breast cancer cell lines were carried out by combined methods of MTT and FACS analyses. Results from these analyses are expected to emphasize the future uses of neem seed against bacterial infection as the extract has lower side effects and easier availability.
Experimental section
Materials
Methanol and potassium bromide (KBr) were purchased from Merck Limited, Mumbai, India. Roswell Park Memorial Institute medium (RPMI) and dimethyl sulfoxide (DMSO), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), Luria-Bertani (LB) broth, and LB agar were procured from Hi-Media Laboratories, Mumbai, India. They were of AR grade and were used as received.
Discussions
Growing evidences of drug resistance in bacteria and several side effects of the conventional chemotherapeutic processes of cancer treatment have motivated the researchers to explore some alternative but convenient approaches for dealing of infections and cancer [
92,
93]. It is known that in bacterial pathogenesis, the ability of bacterial cells to cause infection gets potentiated by conforming biofilms, based on quorum sensing (QS) signaling action. Hence, the inhibition of biofilm-forming ability as well as QS, the cell-cell communication becomes a promising alternative as a controlling strategy for these healthcare issues [
8,
94]. Under such circumstances, the identification and establishment of alternative agents to antibiotics are warranted to combat bacterial infections [
95]. With that intention, the present study was designed to explore the effect of methanolic extract of unripe and ripe neem seeds on biofilm formation and eradication. Exploring anticancer activity of the neem seed extracts on MDR breast cancer cells was also another significant part of this study. Thiophene derivative compounds are known to have potential remedial properties to certain biofilm-related bacterial infections [
96]. In the present study, GC-MS analyses revealed the presence of 2-hexyl-tetrahydrothiophane in unripe methanolic neem seed extract, considered to be responsible for antibiofilm activity towards the target bacteria. Ripe neem seed extracts containing highest percentage of 3,5-dihydroxy-6-methyl-2,3-dihydro-4 H-pyran-4-one and 4-ethylbenzamide showed significantly higher (
P<0.05) antibiofilm activity, as revealed through the MBIC, MBEC and fluorescence studies, than the unripe neem seed extract. 3,5-dihydroxy-6-methyl-2,3-dihydro-4 H-pyran-4-one has been reported to have potent antioxidant and antimicrobial activities [
97,
98]. On the other hand, the antibacterial activity of 4-ethylbenzamide has already been described [
99]. It was also found that antibiofilm activities of both the extracts were higher in the case of
S. aureus than
V. cholerae. The differential structural properties as well as biofilm regulatory properties in gram-positive and gram-negative bacteria might be the underlying causes for such observations. Besides, it may be suggested that plant extracts may play some roles in modulating cell wall synthesizing enzymes and also QS cell-cell communication and regulation during biofilm formation to induce antibiofilm activity. Moreover, 3,5-Dihydroxy-6-methyl-2,3-dihydro-4 H-pyran-4-one, a derivative of kaempferol, is present in highest percentage in ripe neem seed extract. Kaempferol is clinically known to possess anticancer, antimicrobial and antioxidant activities [
100,
101]. Kaempferol derivatives and its synergistic action along with other compounds present in the seed extracts, is considered to be responsible for possessing significantly higher antibiofilm as well as anticancer activity in methanolic ripe neem seed extracts than unripe neem seed extract. MIC and MBC results also indicate that ripe neem seed extracts possess greater antibacterial activity than unripe extracts against both tested gram-positive (
S. aureus) and gram-negative (
V. cholerae) bacteria. In this context it may also be considered that some antibiotics exhibit antibiofilm activities, but report reveals that conventional antibiotics are not prudent enough against bacterial biofilm [
102]. Also, combination strategies, involving different antimicrobial peptides (AMPs) are being used along with different conventional antibiotics [
102]. Besides, numerous phytochemicals are being used as antimicrobial and antibiofilm agents. Hence depending on several reports, it appears that effective therapeutic outcome along with lesser side effects and lower propensity for resistance development of the phytochemicals validate their usefulness as an alternative to antibiotics and other chemotherapeutics [
103,
104].
MTT analysis showed the reduced viability of the breast cancer cell line, whereas, FACS study emphasizes that both the CD44 and CD326 populations of the breast cancer cells have been significantly decreased. CD44 and CD326 are cell surface markers. The population of these markers significantly increased during the cancer prognosis and, induces cell signalling, proliferation, differentiation and migration of the cancer cells [
105,
106]. Here, Ripe seed extract possessed significantly greater killing potency against the cancer cell line than unripe extract. Several studies on 3,5-dihydroxy-6-methyl-2,3-dihydro-4 H-pyran-4-one showed its antimicrobial and anti-proliferative activity [
98], and 4-ethylbenzamide, derivatives of 4-thiazolidinone conferred their anticancer potency [
107]. It was also observed that ripe seed extract has superior anticancer activity than that found with gemcitabine and unripe seed extract. Unripe seed extract could reduce the population of cancer cells similar to the magnitude of gemcitabine-based inhibition. It may be suggested from the present study that neem seed extracts have some role in the inhibition of cancer cell proliferation and differentiation as well as induction of apoptosis process in cancer cells. Further studies are warranted for identification and characterization of the particular phytochemical responsible for the antibiofilm and anticancer activities. It is also necessary to pinpoint the component transformed during ripening of the seeds, considered to be responsible for the enhanced antibiofilm and anticancer activities.
Though the effective concentrations of the extracts were relatively higher than the conventional antibiotics, it should also be considered that these extracts contain several components and the effect of each of the components are needed to be investigated. The next approach will be to identify the individual components responsible for such effects along with tracking the exact pathway by which they exert their action. When all these factors are taken into account, it will certainly be helpful in the development of some new potent alternatives to the conventional antibiotics. Besides, easy availability, low cost, fewer side effects and long history of use in folk medicine for curing several infections and disease, are considered to make plant extracts as good resource for investigations.
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