1. Introduction
Urinary tract infections (UTIs) are among the most common bacterial diseases for women and the elderly. Although they may result in illnesses that are not life-threatening, affected patients can face extreme distress [
1]. UTIs are caused by a variety of organisms, including
Escherichia (E.) coli, Klebsiella (K.) pneumoniae, Enterococcus (E.) faecalis, Proteus (P.) mirabilis,
Staphylococcus (S.) saprophyticus,
Pseudomonas (P.) aeruginosa, Enterobacter species,
Streptococcus species, and
S. aureus [
2]. Initially, infections are usually caused by a single species of bacteria, such as the uropathogenic
E. coli or
E. faecalis [
3]. However, over time, many organisms, including
K. pneumoniae,
P. aeruginosa,
P. mirabilis, and
Morganella (M.) morganii, can colonize the urinary tract and form polymicrobial biofilms [
4,
5]. Both
P. mirabilis and
K. pneumoniae are Gram-negative bacteria that live in human fecal flora as harmless commensal bacteria, inhabiting the gastrointestinal tract. They are, however, known to cause a wide range of opportunistic human illnesses, particularly wound infections, respiratory tract infections, and UTIs [
6,
7].
UTIs predominantly affect women when the bacteria infect any part of the urinary system, including the kidneys, the bladder, the uterus, or the urethra. Pregnancy, a history of an earlier UTI, age, sexual behavior, and lack of hygiene are among the risk factors for UTIs. A significant prevalence of UTIs exists across all socioeconomic categories, exacerbating economic pressure for some families. UTIs are one of the most prevalent healthcare-associated infections, with an estimated 150 million people infected worldwide each year, with 13,000 associated deaths [
8]. Although many antibiotics are prescribed for the treatment of UTIs, a major cause for concern is the emergence of drug-resistant strains.
Because microbial infections tend to be more severe in immune-deficient patients, there is an urgent need for alternative therapies, including natural products, to ameliorate multidrug resistances (MDRs) that are commonly seen. The rapid, widespread emergence of resistance to newly introduced antimicrobial agents indicates that even new families of antimicrobial agents have a short life expectancy. Natural products have numerous advantages, including fewer side effects, improvements in patient tolerance, lower costs, widespread acceptance due to a long history of usage, and sustainability. Many essential drugs have been discovered and developed as a result of phytochemical and pharmacological studies of natural products [
9]. Historically, medicinal plants deliver a wide variety of components with confirmed therapeutic qualities [
10]. The renewed interest in plant-derived therapies seems to be due primarily to the widely held assumption that “green medicine is safe.”
The consumption of antiseptic and anti-adhesive herbs, including the leaf parts of
Arctostaphylos uva-ursi (Uva ursi) and
Juniperus spp. (Juniper) and the fruit part of
Vaccinium macrocarpon (cranberry), are able to excrete antimicrobial constituents, which may either kill microbes directly or interfere with their attachment to epithelial cells, thereby preventing acute as well as chronic UTIs [
11]. Berberine extracted from
Mahonia aquifolium (Oregon grape) and
Hydrastis canadensis (Goldenseal) is found to be an effective drug in combating infections that are caused by
E. coli and
Proteus species by hindering their adherence to the host cell [
12], suggesting their potent role in the treatment of UTIs.
Bacopa monnieri (L.) Wettst.
(B. monnieri), popularly known as Brahmi or water hyssop, is one of the perennial creeping plants found in various parts of the world. It is also known as “the herb of grace,” due to its various medicinal properties and memory-enhancing effects [
13,
14]. It belongs to the family Scrophulariaceae that grows optimally in wet, damp, and marshy areas, with succulent, oblong leaves and small white flowers as its descriptive features. The
Bacopa species is a native herb that grows in the wetlands of southern and eastern India, Europe, Australia, Africa, Asia, North America, and South America. In addition to being known as a nootropic herb, the bacopa plant aids in the regeneration of injured neurons, neuronal synthesis, and synaptic activity restoration, as well as in strengthening brain activity.
B. monnieri contains brahmine, nicotinine, herpestine, bacosides A and B, saponins A, B, and C, triterpenoid saponins, stigmastanol, β-sitosterol, betulinic acid, D-mannitol, α-alanine, serine, pseudojujubogenin glycoside, aspartic and glutamic acids, and other elements [
15]. For centuries, the magical herb has been used by Ayurveda medical practitioners for the treatment of various conditions, such as epilepsy, anxiety, and various stress-related disorders [
16]. It also has positive effects in boosting brain function and improving memory [
17]. Additionally, it is used to treat digestive complaints and skin disorders, as an antiepileptic, antipyretic, and analgesic [
18,
19], and as an antimicrobial agent against UTIs [
20] (
Figure 1).
The therapeutic activity of herbal medicines is attributed either to the individual activity or the synergistic activity of the phytomolecules. Synthetic drugs are expensive and may be inadequate for the treatment of some diseases; they are also often associated with adulterations and adverse effects. Likewise, while natural products possess a wide range of biological activities, some have toxicity issues. Therefore, computational techniques applied in drug designing have a vital role in predicting the toxicity of compounds and in determining their other properties [
21]. For example, in silico studies that focus on predictions of toxicity, via ProTox-II server, can decrease the need for subsequent drug testing in animals (in vivo), while molecular docking for each phytochemical can determines its therapeutic efficiency. Such studies require less testing time, and they are economical in determining the effects of drugs on animals [
22]. Hence, the aim of the present study was to explore the effects of
B. monnieri against uropathogens, such as
K. pneumoniae and
P. mirabilis, using in vitro methods, while studying their active molecules via in silico approaches.
3. Discussion
Infectious diseases are a primary source of morbidity and mortality, and the number of MDR strains is on the rise, as is the development of strains with reduced antibiotic sensitivity. Currently, traditional medical systems are still society’s most valuable assets in dealing with a variety of ailments. Plant-based traditional medicines are widely used for a range of disorders, as they are effective as therapies and have no negative effects in patients, such as the demonstrated effects of synthetic pharmaceuticals in COVID-19 patients [
23]. Pathogenic bacteria are thought to develop antibiotic resistance, making the hunt for novel antibiotics a never-ending process [
24]. For these reasons, researchers have recently begun to concentrate their efforts on herbal products in order to develop more effective drugs and therapeutic targets against lethal MDR microbial strains [
23,
25,
26,
27]. These initiatives inspired us to perform the current study, which triggered the search for new antibacterial compounds generated from the methanolic and ethanolic extracts of
B. monnieri that have been found to be effective against the bacteria that cause UTIs.
Several researchers have investigated
B. monnieri’s antibacterial properties, but the majority of these studies have focused on standard bacterial strains [
19,
28,
29]. Although clinical isolates of human pathogenic
E. coli and
K. pneumoniae have been evaluated, those strains are not MDR strains [
30]. All of the tested
B. monnieri extracts demonstrated antibacterial action against the pathogens. The methanolic extract of
B. monnieri was found to have a stronger inhibitory effect against
K. pneumoniae and
S. aureus [
31,
32], which is consistent with our findings. Furthermore, the findings indicating that methanolic extracts are more powerful than aqueous extracts against bacterial strains are consistent with the findings of other studies [
33,
34,
35].
The tested microorganisms were not inhibited by an aqueous extract at a concentration of 100 mg/mL, which could be due to the loss of some bioactive components during the sample extraction process. Furthermore, multiple studies have found that the type of solvent used for extraction plays a significant impact in defining the extract’s potential [
36,
37]. This is related to the fact that various phytochemicals have different relative solubilities in different polarities of solvents. The systemic phytoconstituent screening of plant extracts is an important technique for finding new medicinal lead compounds [
38,
39]. The antibacterial activities of phytochemicals are achieved through two basic pathways: direct bacterial death or the prevention of microbial adherence to epithelial cells [
11]. Some of the extracts can be developed into medications or used as blueprints for drug development, due to the presence of various phytochemicals and antioxidants [
40].
Antibacterial resistance has become one of the world’s most serious public health challenges in the last two decades [
41,
42]. Hence, there has been a growing interest in identifying and developing novel antimicrobial compounds from a variety of sources to tackle microbial resistance [
43]. Herbal medicines such as turmeric and its bioactive molecule curcumin, have been shown to improve the symptoms of chronic UTIs, protect renal tubular function, and reduce inflammatory responses [
44,
45]. Herbal remedies have been demonstrated to be effective in the treatment of UTIs [
46].
The present study confirmed that both the ethanolic and methanolic extracts of
B. monnieri provide inhibitory effects against the Gram-negative bacteria
K. pneumoniae and
P. mirabilis. Different studies reported that
B. monnieri extract can inhibit Gram-negative as well as Gram-positive bacteria [
47]. Hema et al. [
30] reported that
K. pneumoniae and
P. vulgaris were resistant to the ethanolic extract of
B. monnieri, a finding that is contrary to those of many others, including ours. The present work on
B. monnieri also showed that the crude methanolic extract was more effective than the ethanolic or aqueous extracts.
More investigations are required on different species of the bacteria that cause UTIs to provide a clearer indication for
B. monnieri as a potential agent against UTIs. Based on the present work, it is plausible that a contributory effect against UTIs is provided by the alkaloids, tannins, and flavonoids, acting individually or in combination. Similarly, the presence of all major phytoconstituents, such as alkaloids, flavonoids, steroids, and saponins (except tannin) was investigated in the methanolic extract of
B. monnieri. It was reported that different phytochemicals are responsible for antibacterial, cytotoxic, analgesic, and neuropharmacological activities [
48]. Among the chemical constituents, alkaloids and flavonoids may be responsible for antibacterial properties [
49].
Recently, many plants have been explored via in silico and in vitro methods to evaluate the antibacterial activities of their major bioactive molecules. For example, the bioactive molecules thymol and emodin of
Thymus serpyllum and
Rheum emodi, respectively, were found to reveal antimicrobial activity with the penicillin binding protein, together with drug likeness and toxicity prediction [
50,
51,
52,
53]. Similarly, a study carried out by Ramasamy et al. [
54] revealed that aglycones and their derivatives of
B. monnieri have better binding affinity and good central nervous system drug-like properties; they validated in silico receptor and acetylcholinesterase (AChE) models to predict the potential of bacosides and aglycones and their derivatives. The alcoholic extract was analyzed via an invivo study and was found to increase the memory of rats; the activity is attributed to saponin mixtures comprising the bacosides A and B [
55]. Emran et al. [
56] used via docking studies to show that luteolin, a phytochemical of
B. monnieri, has potential to act against
Staphylococcus aureus, as it has the highest fitness score and greater specificity toward the DNA gyrase binding site than the penicillin binding protein.
No study has been conducted to date to screen the major phytochemicals of B. monnieri against uropathogens. This is the first report that shows that the Oroxindin component, a flavone or a type of phenolic of B. monnieri, is a potent inhibitor of UTIs, establishing pathogens such as K. pneumoniae and P. mirabilis. It is yet confirmed that the Oroxindin component is safe for future use against UTIs. Further research is necessary to validate the full spectrum of efficacy of the Oroxindin antibacterial compounds from B. monnieri via in vitro or in vivo approaches.
Additionally, nanotechnology could be used to improve the pharmacokinetics and bioavailability of phytochemicals. Several issues that are commonly associated with the delivery of free phytochemicals, such as the rapid elimination of phytochemicals from the bloodstream, their limited absorption, and their bioavailability, reduce their potency and may necessitate greater dosages to achieve efficacy [
57]. Some natural compounds are linked with undesirable side effects, such as toxicity; the use of a drug carrier may provide several benefits, including shielding the compounds from degradation in the body [
58]. Nanocarriers can be altered to provide smart delivery mechanisms by using pH or temperature-sensitive materials [
59], targeting ligands to improve cell uptake and selectivity, and using cellular membranes for cloaking to escape recognition by the reticuloendothelial system (RES) [
60]. Several nanoparticles, liposomes, inorganic nanoparticles, and emulsions have been shown to be effective transporters for phytochemicals such as curcumin [
61,
62]. With respect to the future prospects of encapsulating
B. monnieri, polymeric nanoparticles serve as excellent nanocarriers, due to their ability to entrap or surface-adsorb active compounds. They are versatile and can be coupled with cell-penetrating peptides (CPPs), which can assist in molecular intake and absorption. CPPs can carry cargo into cells via endocytosis, which improves targeting and localization to the desired location [
63]. Combining the above nanotechnology with bioactive molecules from
B. monnieri could enhance antibacterial efficacy, increase bioavailability, and reduce toxicity, especially in UTIs (
Figure 8).