As mentioned above, the use of neem twigs as dental cleaning sticks is commonplace in many countries to which A. indica is a native species (Gupta et al., 2017). This traditional use has translated to a growing number of studies that have tested neem products for their ability to improve dental hygiene and to prevent or treat oral diseases. Typically, these studies aim to identify an application for neem extracts as a mouthwash alternative, root canal irrigant, toothpaste, etc. Previously, the antimicrobial activity of A. indica against common endodontic pathogens, such as Enterococcus faecalis, Staphylococcus aureus, Streptococcus mutans, and Candida albicans, has been well-established (Mistry et al., 2014; Barua et al., 2017; Singh et al., 2020). For example, a decade ago, it was shown that at 7.5%, aqueous neem leaf extract was able to inhibit the growth of E. faecalis, S. mutans, and C. albicans and that the MIC of an ethanolic neem leaf extract was 1.88%, 7.5%, and 3.75% against these three important dental pathogens, respectively (Nayak Aarati et al., 2011). More recently, another group was able to determine that a methanolic extract of A. indica showed considerable antimicrobial activity against a three-week-old polymicrobial dental biofilm grown on extracted human teeth consisting of S. mutans, E. faecalis, S. aureus and C. albicans (Mistry et al., 2015). The results from two small human studies indicate that a neem-based toothpaste or gel can reduce the levels of S. mutans in the mouth and that the gel formulation can reduce plaque and gingivitis to the same degree as a chlorhexidine gel control (Nimbulkar et al., 2020; Selvaraj et al., 2020). Additional biologically relevant studies may be able to provide even more evidence for the use of neem in dentistry in combination with, or as an alternative to, antimicrobials already used in the field. Of note, several studies have already suggested that neem extracts have similar levels of activity as chlorhexidine or hypochlorite (typical components of oral washes) against plaque, gingivitis, and pain in vivo (Jalaluddin et al., 2017; Hosny et al., 2021) and against biofilm-forming bacteria (e.g., Streptococcus viridans, Porphyromonas gingivalis, and S. aureus) and C. albicans in vitro or ex vivo (Joy Sinha et al., 2015; Anand et al., 2016; Kankariya et al., 2016; Heyman et al., 2017; Andonissamy et al., 2019; Bansal et al., 2019; Tasanarong et al., 2021). Another human pathogen that causes plaque and other biofilm-related diseases in the body, E. faecalis, is also just as susceptible to various neem extracts as it is to chlorhexidine in vitro (Chandrappa et al., 2015; Mustafa, 2016; Bhardwaj et al., 2017; Joy Sinha et al., 2017).

The relevance of neem as an antimicrobial in dentistry is, so far, the most researched area and has led to many conclusions about A. indica extracts as compared to those from other plants used in traditional medicine. In fact, some studies suggest that neem has greater antibacterial activity than Commiphora myrrha (myrrh), Acacia tree (e.g., catechu), Cinnamomum verum (cinnamon), Salvadora persica (miswak), Syzygium aromaticum (clove), Zingiber officinale (ginger), Allium sativum (garlic) and Curcuma longa (tumeric) extracts against some species of bacteria and cultured dental caries (Kanth et al., 2016; Jagannathan et al., 2020; Arora et al., 2021). This being said, it is important to note that certain bacterial species (e.g., S. mutans and E. faecalis) appear to be more susceptible to extracts from other plants in some studies (Jain et al., 2015; Dedhia et al., 2018; Kalita et al., 2019; Panchal et al., 2020). While this does not diminish the antimicrobial potential of A. indica, it does underline the importance of thoroughly taking advantage of the wide variety of antimicrobial plants and compounds that are at the disposal of modern medicine.

Originally introduced around the world as a potent pesticide and fertilizer for use in agriculture (Govindachari, 1992; Nicoletti et al., 2012; Chaudhary et al., 2017), neem has been recognized in more recent years as a safe and effective broad-spectrum antimicrobial with uses throughout the food industry that range from food production and storage to packaging and human consumption. During meat production, the presence of several species of bacteria can affect the quality and safety of the product, including Campylobacter, Lactobacillus, and Carnobacterium spp. Neem cake extract, which is a waste product from neem seed oil production, has antibacterial activities against all of these potentially pathogenic species (Del Serrone et al., 2015a). Additionally, Ravva and Korn (2015) found that neem leaf and bark supplements were able to successfully eliminate Escherichia coli O157:H7 from cultured cow manure; because this E. coli strain was isolated from an apple juice outbreak of O157:H7, these results may have broad applications on farms where crops and orchards frequently exist in close proximity to cattle (Ravva and Korn, 2015). In the production of another source of protein for human consumption, specifically in shrimp aquaculture, antibiotic-resistant Vibrio parahaemolyticus can compromise both shrimp and human health. A thorough study on the potential use of neem in this industry included in vitro and in vivo assays that showed that aqueous neem extract had a MIC against V. parahaemolyticus of 62.5 mg/ml and was able to significantly increase survival of shrimp by 76% as compared to the untreated control (Morales-Covarrubias et al., 2016).

One of the next steps in the production of pathogen-free human food is storage and/or packaging. In the last couple of years, several groups have shown that food preservation films that are made from polyethylene or sustainable materials such as seaweed can be manufactured to incorporate neem leaf extracts, neem oil, and other plant-based products (e.g., turmeric and curcumin) (Ahmed et al., 2022). The resulting composite films are shelf-stable, block ultraviolet light, and have increased antifungal and antibacterial activities against C. albicans and a wide range of Gram-negative and Gram-positive organisms, including E. coli, S. aureus, Pseudomonas aeruginosa, and Bacillus subtilis (Sunthar et al., 2020; Uthaya Kumar et al., 2020; Oyekanmi et al., 2021; Subbuvel and Kavan, 2021). Furthermore, the ability of A. indica to prevent activity of food-spoiling fungi is evident by several recent reports that outline the following: 1) the ability of neem oil to prevent the growth of the grape product-spoiling species Aspergillus carbonarius and to inhibit the production of mycotoxin by strains of this fungus (Rodrigues et al., 2019), 2) the ability of neem leaves to prevent the production of aflatoxins by Aspergillus parasiticus during long-term storage of rice, wheat, and maize (Sultana et al., 2015), 3) the ability of neem seed methanol and ethanol extracts to inhibit Aspergillus flavus and A. parasiticus by 10% in the context of maize storage (An et al., 2019), 4) the ability of multiple neem seed, bark, and leaf extracts to inhibit the growth of three major potato-spoiling fungi, Aspergillus niger, Fusarium oxyporium, and Pythium spp., by 72–100% (Ezeonu et al., 2019), and 5) the ability of aqueous neem leaf extract to inhibit growth of A. niger and A. parasiticus, as well as to detoxify aflatoxin B1 and ochratoxin A in vivo (Hamad et al., 2021). These diverse antifungal properties of neem are highlighted in Supplementary Table S1. Altogether, these food-related studies should encourage further investigation of the utility of A. indica-derived products throughout the food industry; these products may serve as a sustainable antimicrobial alternative that can potentially improve food security via improved stable long-term storage, as well as improve human health through the elimination of foodborne pathogens.

In several other areas of antibacterial discovery, A. indica has been shown to be effective against many important human pathogens. Overall, a large number of studies have been published in the last decade on this topic, especially as they relate to the ever-growing number of antibiotic-resistant organisms. In this vein, several bacterial species that cause wound infections are found on the long list of antibiotic resistant threats, including S. aureus and P. aeruginosa (CDC, 2019). Several groups have tested the antibacterial activity of neem against these species. For example, Garg et al. (2015) demonstrated that methanol and chloroform neem extracts performed better than other plant extracts and better than several different antibiotics against both S. aureus and P. aeruginosa (Garg et al., 2015). More specifically, it has also been determined that the MIC of limonoid compounds that were isolated from neem seeds are between 32 μg/ml and 128 μg/ml against P. aeruginosa and another opportunistic skin pathogen, Staphylococcus epidermidis (Lu et al., 2019). As a translational approach to this area of research, several studies have demonstrated that an aqueous neem leaf extract that was used to make alginate fibers for wound dressings (Hussain et al., 2017), a nanofibrous mat embedded with neem leaf extract (Ali et al., 2019), and a topical gel containing a methanolic neem extract all inhibit S. aureus growth (Raju and Jose, 2019). In the case of a polyesteramide synthesized from neem oil to produce a nanofibrous mat, the incorporation of A. indica into this wound treatment method resulted in increased tissue regeneration in rats, as compared to the control commercial cream (Killi et al., 2019). These results potentially have broad applications for many areas of medicine in which the risk of antibiotic resistant skin/wound infections is high.

Another large group of infectious bacteria that cause morbidity and mortality all over the world are the gastrointestinal pathogens, which include foodborne and diarrhea-causing organisms. Relatedly, some of the traditional uses of neem are antidiarrheal, antacid, and antiulcer; this has led to a large body of research that has investigated the antibacterial properties of neem products against pathogens such as Salmonella spp., Shigella spp., E. coli, Listeria monocytogenes, and Bacillus cereus, as shown in Figure 1. To summarize, the Salmonella spp. and Shigella spp. that have been tested, which includes more than a dozen multidrug-resistant isolates from patients suffering from typhoid fever complications, are susceptible to seed, bark, and leaf extracts of neem from either ethanol, methanol, or acetone extraction; in some cases, the activity of neem extract was also found to be greater than that of gentamycin, erythromycin, and other plants used in traditional medicine (Mahfuzul Hoque et al., 2007; Susmitha et al., 2013; Tesso et al., 2015; Melese et al., 2016; Al Akeel et al., 2017; Panchal et al., 2020; Essuman et al., 2021). Similarly, dried leaf, seed, and bark neem extracts in any of the three previously mentioned solvents have significant antibacterial activity against E. coli, with the methanolic extract of neem seeds demonstrating the greatest level of activity (Susmitha et al., 2013; Sharma and Nupur, 2014; Melese et al., 2016). Additionally, neem oil was as effective as ciprofloxacin against 48 tested isolates of E. coli, 14 of which were diarrheagenic strains (Del Serrone et al., 2015b). Given that there are more than a quarter of a million cases of E. coli each year in the United States alone, associated world-wide morbidity is a huge issue for this pathogen (CDCE, 2019). Indeed, resistant E. coli infections were found to be responsible for nearly a quarter of all disability-adjusted life years caused by resistant bacterial infections in the European Union in 2015 (Cassini et al., 2019). On the chronic disease spectrum of gastrointestinal illnesses, Helicobacter pylori is a pathogen that colonizes approximately 50% of the human population and causes ulcers in millions of people each year; it also causes stomach cancer in ∼1% of people who are infected (Kuipers, 1999; Moodley et al., 2012; Bray et al., 2018). Recently, two studies found that neem oil extract and a neem-associated phytochemical, nimbolide, have potent in vitro bactericidal activity against H. pylori in liquid cultures and in biofilms (Blum et al., 2019; Wylie et al., 2021). Similarly, another group determined that an ethanolic neem leaf extract has activity against this species as well (Saxena et al., 2021). Overall, studies like those described above provide strong evidence that novel plant-derived treatments, like neem oil extracts and/or the phytochemicals contained therein, may be able to reduce the burden of pervasive organisms like E. coli and H. pylori.

Finally, there have been many studies published on the antibacterial properties of neem against a variety of diverse human pathogens. The overwhelming conclusion from the majority of these investigations is that many elements of A. indica (e.g., seeds, bark, leaves, etc.) produce extracts that have moderate to significant levels of antimicrobial activity against several pathogens: S. aureus, E. coli, E. faecalis, P. aeruginosa, Salmonella typhi, Streptococcus agalactiae, Shigella boydii, B. subtilis, Klebsiella pneumoniae, and Candida tropicalis, as listed in Figure 1 (Dahiya and Purkayastha, 2012; Melese et al., 2016; Al Saiqali et al., 2018; Ibrahim and Kebede, 2020). Moreover, in side-by-side comparisons A. indica-based components often show superiority to other plants; a few exceptions include aqueous garlic extract and ethanolic green tea extract that were each shown to work better than neem extracts to kill Bacillus anthracis or S. aureus and E. coli, respectively (Zihadi et al., 2019; Kaur et al., 2021). Overall, studies support the notion that A. indica is an omnipotent plant that possesses antimicrobial activity against many bacterial pathogens. Future studies should include more standardized research approaches to test the potential of neem-derived products and individual compounds against other microbes both in vitro and in vivo; a priority should also be placed on determining the mechanisms of action of these products in order to fully understand the ideal clinical significance of A. indica.

Biofilms, or communities of bacteria composed of biofilm-associated cells and extracellular polymeric substance (EPS) components (e.g., proteins, polysaccharides, and extracellular DNA), are notably recalcitrant to outside stressors, including those from the immune system and from therapeutics (Bae and Jeon, 2013; Yonezawa et al., 2019; de Vor et al., 2020). Depending on the species of biofilm-associated bacteria, mechanisms of tolerance include slow diffusion/limited penetration of the drug, metabolic heterogeneity and decreased growth rates among the cells within the biofilm, and the formation of persister cells (Mah and O'Toole, 2001; Stewart and Costerton, 2001; Attaran et al., 2017). Backed by thorough research, plants such as A. indica may provide the tools needed to treat pervasive biofilm infections. To this end, there is evidence that natural products may be an ideal source of quorum sensing inhibitors, efflux pump inhibitors, and metal chelators, which are all potentially powerful antibiofilm agents (Borges et al., 2016). Additionally, synergistic interactions between the many constituents found within plant extracts may provide a benefit over a single isolated ingredient; this may explain the efficacy of lower doses of herbal products like neem oil as compared to individual compounds (Aiyegoro and Okoh, 2009).

Significant biofilm-associated human infections are caused by species such as S. aureus, E. faecalis, and P. aeruginosa (Vestby et al., 2020). There is already evidence that A. indica has activity against biofilm-forming strains of some of these pathogens. For example, a neem leaf ethanolic extract was found to inhibit S. aureus and methicillin-resistant S. aureus (MRSA) biofilm adherence at 62.5 and 125 μg/ml, respectively (Quelemes et al., 2015). More recently, another group found that a petroleum ether neem extract had a MIC and MBC of 125 and 500 μg/ml, respectively, against a strain of MRSA (S et al., 2020). In the same in vitro study, the addition of 1 mg/ml of the neem extract resulted in a 68.9% reduction in MRSA biofilm; 2 mg/ml of the same extract resulted in a 83.8% reduction (S et al., 2020). Additionally, Guchhait et al. found that ripe neem seed extracts had antibiofilm activity against S. aureus and Vibrio cholerae. The minimum biofilm inhibitory concentrations (MBIC) and minimum biofilm eradication concentrations (MBEC) for this extract were 100 and 300 μg/ml, respectively, against S. aureus and 300 and 500 μg/ml, respectively, against V. cholerae (Guchhait et al., 2022). Furthermore, using a mouse model of V. cholerae infection, Thakurta et al. showed that administration of methanolic neem leaf extract at 100–1800 mg/kg body weight reduced intestinal fluid secretion by 27.7%–77.9% and doses ≥300 mg/kg inhibited Vibrio-induced hemorrhage in the murine intestine without signs of toxicity (Thakurta et al., 2007). Of note, individual neem-associated phytochemicals, such as catechin, may have greater potential for biofilm eradication, persister cell damage, disruption of EPS structural components, and prevention of quorum sensing (Lahiri et al., 2021). In addition, a methanolic neem oil extract and nimbolide both killed cells within in vitro biofilms produced by the Gram-negative carcinogenic pathogen, H. pylori (Wylie et al., 2021). Overall, evidence indicates that neem has great potential to be used as a therapeutic for resistant bacterial infections. However, future research that utilizes animal models will be crucial to determine whether neem-derived products fit in with established antibiotic regimens and/or work alone to eradicate biofilms in vivo.

Human immunodeficiency virus, or HIV, is arguably one of the most devastating modern human pathogens. Since its discovery in the early 1980s, there have been over one million new HIV infections each year; hundreds of thousands of people still die from the subsequent acquired immunodeficiency syndrome (AIDS) annually (Barre-Sinoussi et al., 1983; Gallo et al., 1984; Levy et al., 1984; UNAIDS, 2022). Although antiretroviral therapy (ART) is well-established and is successful at diminishing viral load and preventing disease progression, ART drugs are expensive, and require life-long treatment that is not without side effects (CDC, 2022). To this end, natural products, such as those obtained from A. indica, that are traditionally used for HIV-associated infections (Nagata et al., 2011; Anywar et al., 2020), have been explored for their abilities to protect the CD4⁺ T cell population that is vulnerable during HIV infection, to reduce persistent immune activation during ART, and to decrease the toxicity of ART drugs. For instance, small trials have concluded that neem leaf extract given daily is safe and effective at improving CD4⁺ T cell counts in HIV patients (Udeinya et al., 2004; Mbah et al., 2007). Furthermore, when A. indica and Senna siamea leaf extracts were given in combination with ART, this HIV patient group had improved T cell numbers and fewer markers of hepatic and renal toxicity than the group that was given ART alone (Goni Hamad et al., 2021). To address the possibility of T cell exhaustion in HIV-infected patients, Olwenyi et al. (2021) performed an in vitro study with peripheral blood cells isolated from infected and uninfected individuals; following exposure to enterotoxin, the lymphocytic response indicated that A. indica extract, but not the extracts from two other plants, was able to down-regulate CD4⁺ T cell activation in a concentration-dependent manner without affecting general T cell-specific functions. Overall, these results support the idea that neem has immunomodulatory abilities that can be exploited to increase the efficacies of certain treatments and to improve the condition of chronically infected patients, such as those with HIV.

Herpes simplex viruses (HSV) most commonly cause oral and genital infections, with an estimated half a billion people living with HSV type 2 and nearly four billion people with HSV type 1 in any given year (James et al., 2020). Because there is no cure for HSV infections and they can recur many times throughout a person’s life, broad searches for novel antiviral medications are warranted in order to identify agents that may reduce the morbidity associated with these infections; these searches include phytochemicals isolated from medicinal plant such as A. indica. For example, the glycolipid sulfonoquinovosyldiacylglyceride (SQDG) that has been isolated from neem leaves was shown to have potent antiviral activity against HSV-1 and -2; the half maximal effective concentrations (EC₅₀) were 9.1 and 8.5 μg/ml, respectively. The same study also found that HSV-infected, SQDG-treated macrophages produced significantly less proinflammatory cytokines than untreated controls (Bharitkar et al., 2014). It has been suggested that the antiviral and anti-inflammatory properties of SQDG may indicate that A. indica contains other phytochemicals with therapeutic potential against viruses (Shanmugam et al., 2020). Concurrently, it was found that two polysaccharides isolated from neem leaves, along with their sulfated derivates, are able to inhibit HSV-1 nucleic acid synthesis at concentrations that were not cytotoxic (Faccin-Galhardi et al., 2019). Additionally, an aqueous neem bark extract was able to block HSV-1 glycoprotein binding to and virus entry into target cells in vitro (Tiwari et al., 2010). The preliminary data reported here indicate that products derived from A. indica can act at several steps of the viral life cycle to prevent herpes infections.

Several groups have demonstrated that computational screening methods can be successfully used to identify novel inhibitors of viruses that infect hundreds of millions of people globally each year, including vector-borne diseases such as Dengue (CDC, 2021). In the context of Dengue virus (DENV), 49 different bioflavonoids that are present in the neem tree were virtually screened for binding to the DENV serine protease, NS2B-NS3pro. Subsequent in vitro assays with promising candidates revealed that kaempferol 3-O-β-rutinoside and epicatechin were able to inhibit DENV-2 infectivity by 77.7% and 66.2%, respectively, without significant cell toxicity (Dwivedi et al., 2021a). Similarly, it was previously shown that three members of another important class of neem phytochemicals, the terpenoids, were able to bind to NS2B-NS3pro with high affinity in silico; this binding ability was subsequently confirmed in vitro (Dwivedi et al., 2016). Nimbin, one of the more common triterpenoids isolated from neem leaf extracts, was shown to be effective against the envelope protein of all four types of DENV in silico (Lavanya et al., 2015). Overall, the ability of neem-derived phytochemicals to block the activities of both the protease and envelope proteins of DENV, and potentially other viruses, further suggest that A. indica may be a novel source for antiviral drugs (Shanmugam et al., 2020).

Flu leads to an estimated 290,000-650,000 deaths annually (WHO, 2020). Due to these consistently high levels of associated morbidity and mortality around the world, influenza is one of the most intensely researched viruses. Though new vaccines for influenza are constantly in development, the burden of flu could be additionally reduced by the introduction of an antiviral that is effective at all stages of disease. Recent evidence suggests that A. indica may represent a robust source of novel drugs against viruses such as influenza. To summarize, molecular docking experiments identified a total of four neem phytochemicals that interact with conserved residues of either the nucleoprotein or the non-structural (NS1) protein of influenza. Though requiring further testing, this may indicate the ability to act as a universal drug against the flu virus (Ahmad et al., 2015; Ahmad et al., 2016).

Taking the lives of more than five million people over the last 2 years, SARS-CoV-2 and the associated disease, COVID-19, continue to be significant threats to public health for which there are few treatment options (WHO, 2021b). Consequently, many researchers have successfully screened chemical libraries for viral inhibitors that act against SARS-CoV-2 (Garg et al., 2020; Vardhan and Sahoo, 2020; Baildya et al., 2021; Borkotoky and Banerjee, 2021; Gogoi et al., 2021; Nallusamy et al., 2021; Navabshan et al., 2021; Senapati et al., 2021; Ogidigo et al., 2022; Vardhan and Sahoo, 2022); detailed reviews on the potential for medicinal plants to be used against SARS-CoV-2 are available (Thota et al., 2020; Adithya et al., 2021). Phytochemicals and plant-derived products like those from A. indica are sometimes included in these chemical libraries. For example, in silico binding simulations by Parida et al. (2020) identified nimolicinol as a compound with strong affinity for both the main protease and the spike protein of SARS-CoV-2; similar docking studies found that nimbocinol binds to the papain-like protease of SARS-CoV-2 with higher affinity than remdesivir, indicating its potential to hinder viral replication (Balkrishna et al., 2021). Although these types of studies are high throughput and can detect candidates for further investigation, virtual studies must typically be followed by in vitro experiments before an antiviral can have clinical relevance. Despite this, given the devastation associated with the ongoing pandemic, several clinical trials are in progress to test neem as a component of mouthwashes, nasal sprays, and capsules for suspected, confirmed, or hospitalized COVID-19 patients, or for prophylaxis (Khan et al., 2020; Farhan Raza Khan, 2021; Nesari et al., 2021). Additionally, one animal study has been completed in which coronavirus-infected mice were treated orally or intranasally with neem bark extract; overall, the therapy was able to prevent systemic injury and pathologic effects of the virus both in vivo and in vitro using multiple model systems (Sarkar et al., 2022). Advocating for additional trials of neem against SARS-CoV-2, Eze et al. (2022) made connections between some of the known mechanisms of A. indica products against other illnesses and the potential efficacy of neem against the virus that causes COVID-19. For additional information about the pharmacological implications for the use of medicinal plants against respiratory infections, see the recent review by Timalsina et al. (2021). All in all, the antiviral action of A. indica against a variety of viruses that cause human disease is evident (Supplementary Table S1). Moreover, outside of HIV, HSV, DENV, influenza, and SARS-CoV-2, there is a strong indication that the aforementioned phytochemical screening methods may be used to identify novel inhibitors of other viral life cycles.