Vitex Genus as a Source of Antimicrobial Agents

Vitex L. is the largest genus of the Lamiaceae family, and most of its species are used in the traditional medicinal systems of different countries. A systematic review was conducted, according to the PRISMA methodology, to determine the potential of Vitex plants as sources of antimicrobial agents, resulting in 2610 scientific publications from which 141 articles were selected. Data analysis confirmed that Vitex species are used in traditional medicine for symptoms of possible infectious diseases. Conducted studies showed that these medicinal plants exhibited in vitro antimicrobial activity against Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. Vitex agnus-castus L. and Vitex negundo L. have been the most studied species, not only against bacterial strains but also against fungi such as Aspergillus niger and Candida albicans, viruses such as HIV-1, and parasites such as Plasmodium falciparum. Natural products like agnucastoside, negundol, negundoside, and vitegnoside have been identified in Vitex extracts and their antimicrobial activity against a wide range of microbial strains has been determined. Negundoside showed significant antimicrobial activity against Staphylococcus aureus (MIC 12.5 µg/mL). Our results show that Vitex species are potential sources of new natural antimicrobial agents. However, further experimental studies need to be conducted.


Introduction
Bacterial resistance to clinically available antibiotics is a global phenomenon whose impact has increased significantly in recent years.Multidrug-resistant infections are a very common problem, greatly affecting mortality and morbidity in populations around the world and leading to greatly increased economic burdens.The Organization for Economic Cooperation and Development (OECD) estimates that the increase in multidrug-resistant bacterial infections will result in a total expense of approximately USD 20 to USD 35 trillion by 2050 [1,2].Resistance mechanisms developed by bacteria to circumvent the effects of antibiotics are very diverse.Enzyme-based bacterial processes can directly inactivate antibiotics; efflux pumps can expel antibiotics from inside bacterial cells, reducing their concentration to subtoxic levels; mechanistic bacterial targets of antibiotics, such as ribosome subunits, DNA gyrase or RNA polymerase, can undergo conformational changes that prevent drugs from binding to them.Such mutations can be either spontaneous or adaptive, and some of them can undergo horizontal gene transfer, which can eventually lead to new naturally resistant bacteria.For example, Staphylococcus aureus, one of the most common etiological agents of infections in hospital and non-hospital contexts, shows a huge increase in resistance patterns to antibiotics of different classes, which means that this species can develop different resistance mechanisms to available drugs [2,3].and many articles were removed for the following reasons: repeated results, no relation to medicinal issues, and the inclusion of irrelevant or incomplete information.Finally, a total of 141 scientific publications were considered eligible to be included in this review as they were related to the use of Vitex species in traditional medicine, were abstracts or full texts written in English, and the studies conducted focused on the Vitex species and their antimicrobial activity against different microorganisms.

Traditional Uses
Obtained results concerning the traditional use of the Vitex species are summarized in Table 1 and classified according to the symptoms they were used against (Figure 2).

Traditional Uses
Obtained results concerning the traditional use of the Vitex species are summarized in Table 1 and classified according to the symptoms they were used against (Figure 2).From the recognized 230 species of Vitex, only 13 species have been reported as being used in traditional medicine, namely Vitex agnus-castus L., Vitex doniana L., Vitex gardneriana Schauer., Vitex mollis L. Vitex negundo L., Vitex obovata ssp.wilmsii (Gürke) Bredenkamp & Botha, Vitex peduncularis W., Vitex peduncularis L., Vitex pinnata L., Vitex polygama L., Vitex pseudo-negundo L., Vitex rehmannii sp., Vitex rotundifolia L., and Vitex trifolia L. These species are traditionally used for the treatment of menstrual disorders and hormonal imbalance, increasing breast milk production, and hypertension [18][19][20][21].Vitex species are also used for infectious diseases treatment such as cavity infections, dysentery, diarrhea, asthma, cholera, and malaria [22][23][24][25][26].The leaf of Vitex is the most frequently used plant part for medicinal purposes, but other parts like the bark, root, and flower are also referred to in the literature.

Species
Part Used Country Signals or Symptoms or Pathology Bib.References

In vitro Antibacterial Activity Studies
Reviewed articles were screened for information regarding plant species and corresponding origin, plant parts and solvents used for extract preparation, antimicrobial activity essay performed, bacteria species used to evaluate antimicrobial activity, and substances used as control.Results were expressed as minimum inhibitory concentrations, minimum bactericidal concentrations, and inhibition zones exhibited according to the type of essay performed.The gathered information is summarized in Table 2. Our analysis showed that different essays were used to study antimicrobial activity against a wide variety of bacterial species and strains.Bacillus subtilis, Escherichia In Figure 2, we can see the major symptoms that are treated with Vitex species, grouped according to the physiological systems impaired.Results showed that these species are mostly used by traditional medical practitioners as antimicrobial agents.Vitex plants are Figures 4 and 5, we can see that most of the studies focused on leaf plant parts and methanolic and ethanolic extracts of plant material.An analysis of Figure 6 shows the main bacterial strains that Vitex species have been tested on.These strains are widely known to be responsible for infections in humans, which makes the antibacterial activity exhibited by Vitex species an important focus of research for the development of new drugs.Collected data show that all Vitex species used in traditional medicine to treat symptoms of infectious diseases exhibit in vitro antimicrobial activity against several bacterial strains, which can justify their use in traditional medicine to treat symptoms of infectious diseases.

In Vitro Antifungal, Antiviral, and Antiprotozoal Activity
Results showed that Vitex species exhibit biological activity, such as through antifungal, antiprotozoal, and antiviral activities.This information is summarized in Tables 3 and 4. V. negundo and V. agnus-castus were the most studied plant species against a wider variety of these types of microorganisms.Methanolic extracts of leaf and root were the most frequent types of extract and plant parts used.Microbial agents tested were mostly fungal, namely C. albicans and A. niger, but viruses like HIV-1 and parasites like Plasmodium falciparum were also tested.Nevertheless, the most noteworthy significant value was observed in terms of antifungal activity against C. albicans.For example, an antifungal activity evaluation of ethanolic, methanolic, and aqueous extracts of the V. agnus-castus leaf showed that all had the ability to inhibit Candida species growth.Minimum inhibitory concentrations of studied extracts ranged from 25 µg/mL to 12.5 µg/mL against C. tropicalis, C. albicans, and C. ciferri while minimum fungicidal concentrations ranged from 100 µg/mL to 25 µg/mL [118].

Characteristic Vitex Secondary Metabolites with Antimicrobial Activity
Specific compounds have been isolated from Vitex species (Figure 7 and Table 5) and described for their antimicrobial activity.Most natural products were isolated from V. negundo.Different chemical classes such as phenolic compounds (like 5-hydroxy-7,4 ′ dimethoxy flavone) and terpenoids (like agnuside, negundoside, and vitegnoside) have been isolated from Vitex species [126,127].Concerning the isolated natural products' antimicrobial activity, negundoside is the most active one, showing significant antibacterial activity against B. subtilis, E. coli, M. pyogenes, P. aeruginosa, and S. aureus.Agnuside and vitegnoside, also isolated from V. negundo, showed a similar range of antibacterial activity against the same bacterial strains as negundoside.All these natural products were isolated from the methanolic leaf extract of the plant.

Materials and Methods
This review was performed following the criteria described in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement 2020 (http://www.prisma-statement.org/PRISMAStatement/FlowDiagram;accessed on 1 January 2022).

Search Strategy
The scientific data was collected from Web of Science and PubMed scientific publications that were published between

General Discussion
Results of our study confirm that Vitex species have been used in traditional medicinal systems to approach several disease symptoms, the most frequent ones being related to infectious diseases, inflammatory states, gastrointestinal disorders, hormonal imbalances, cold symptoms, skin conditions, and liver and cardiovascular symptoms [18].
From more than 200 different Vitex species, 13 species were referred to in this review as the most used in traditional medicine; however, only 10 of them have been studied in vitro for their antimicrobial activity.V. negundo and V. agnus-castus are, by far, the two species that most frequently have been the focus of scientific research.The observed antimicrobial activity against a wide variety of microbial strains in vitro essays contributes to useful scientific validation for the main utilization of Vitex plants in traditional medicinal systems against infectious diseases.Even though specific mechanistic pathways that lead to bacterial death or growth inhibition are still unknown, the published literature indicates that these can be related to major constituents' classes of natural products present in the corresponding tested extracts.[6].
In 2011, Kannathasan et al. conducted a study focusing on the antibacterial activity of several Vitex species.Leaves of V. altissima, V. diversifolia, V. negundo, V. peduncularis, and V. trifolia were used to prepare methanolic extracts, which were then evaluated for their antibacterial activity using the disc diffusion method.Results showed that Vitex extracts under analysis exhibited a wide range of activity against all tested microorganisms, like S. aureus, E. coli, K. pneumoniae, P. aeruginosa, and P. mirabilis.Mean zones of inhibition observed showed that the antibacterial activity of the extracts was selective for the microorganisms.Generally, V. peduncularis and V. trifolia extracts exhibited larger zones of inhibition than extracts of the other species against all microorganisms, being significantly active when compared to ciprofloxacin used as the control [137].
Research conducted by Berrani et al. on the phytochemical composition and biological activity of V. negundo showed that phenolic compounds were amongst the major constituents of the methanolic extracts analyzed.These extracts were prepared using leaf, root, stem, flower, and seed samples and tested separately against a panel of microbial agents commonly responsible for pathogenic infections in humans.Results showed that all plant extracts had selective antibacterial activity against all tested strains [74].
In a study conducted in 2010 by Nagarsekar et al., leaves of V. negundo were used to prepare different extracts.These extracts were studied for their antimicrobial activity against different microorganisms and characterized for their phytochemical composition.Different concentrations of ethanolic, petroleum ether, steam-distilled and supercritical fluid extracts were tested using the well diffusion method to evaluate their antimicrobial activity.Selective activity against S. aureus and B. subtilis and the increase in extract concentration were correlated with an increase in the exhibited antimicrobial activity [82].
Ababutain et al. studied ethanolic, methanolic, and aqueous leaf extracts of V. agnuscastus for their antifungal activity, using the agar well diffusion method, against C. tropicalis, C. albicans, and C. ciferrii.Tested extracts showed selective antifungal activity against Candida species.The aqueous extract was the most active one against all species, followed by the methanolic and ethanolic extracts.These results showed significant zones of inhibition when compared with the nystatin control [138].
Essential oils of the leaf and fruit of V. agnus-castus were characterized for their chemical composition in a work conducted by Stojkovic et al. in 2011.Obtained phytochemical profiles showed that 1,8-cineole and α-pinene were major constituents present in all tested essential oils.An evaluation of the antibacterial activity of the essential oils obtained from different plant parts was conducted using the microdilution method, testing against a panel of Gram-positive and Gram-negative bacteria.Selective activity was observed against M. flavus, B. subtilis, S. typhimurium, S. aureus, and E. coli, with all oils showing Minimum Inhibition Concentration (MIC) and Minimum Bactericidal Concentration (MBC) levels higher than the ones observed for the streptomycin control.The isolated compounds 1,8cineole and α-pinene were also tested for antimicrobial activity.Both compounds exhibited significantly lower MICs and MBCs, not only compared to whole essential oils but also when compared to the streptomycin control, against all tested strains.The same behaviors were observed for all essential oils and isolated compounds against fungal pathogens like A. alternata, A. flavus, A. niger, A. ochraceus, F. tricinctum, P. ochrochloron, P. funiculosum, and T. viride.In this work, antifungal activity was also evaluated through an in vivo model of A. niger-induced rotting in apple fruits.Results showed that increasing concentrations of 1,8-cineole effectively reduced infectious disease incidence after 3 days of treatment [78].
It is well known that plant extracts are characterized by different natural products belonging to a wide variety of chemical classes of secondary metabolites.Phenolic compounds such as flavonoids constitute one of the most common chemical classes of secondary metabolites isolated from Vitex extracts and have previously been shown to exert antimicrobial activity through different mechanisms of action.For instance, apigenin, a very common flavonoid present in several plant species, can inhibit nucleic acid synthesis by binding to bacterial DNA gyrase.It can also induce bacterial cell lysis through membrane disruption (which leads to intracellular content leakage) and cell envelope synthesis inhibition, compromising structural integrity; apigenin also can inhibit biofilm formation and quorum sensing, two bacterial mechanisms with a high impact on infection prevalence and pathogenicity.Quercetin, also a very common phenolic compound, exhibits some similar behaviors.It can inhibit nucleic acid synthesis, which compromises bacterial metabolic viability.It is an active membrane disruptor and inhibitor of cell envelope synthesis.Quercetin can prevent efflux pump activity, reverting or preventing antibiotic resistance mechanisms, and can also directly inhibit bacterial toxins and enzymes [101].
Terpenoids and terpenoid derivatives also constitute a very common class of secondary metabolites, with known antimicrobial activity.Mechanistic pathways that lead to bacterial death or growth inhibition are yet to be determined, but research has shown that terpenoid compounds can inhibit oxygen uptake and oxidative phosphorylation, two metabolic processes crucial for bacterial survival.Monoterpenes carvacrol, thymol, menthol, and geraniol have exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria.Geraniol can increase Enterococcus aerogenes susceptibility to antibiotics by inhibiting bacterial efflux pumps.Menthol and thymol are both active against E. coli and S. aureus.Carvacrol has been reported to inhibit the biofilm development of S. aureus and S. typhimurium.Oleanic acid, a triterpenoid, has shown antimicrobial activity against M. tuberculosis and a synergistic effect when administered with rifampicin, isoniazide, and ethambutol, significantly decreasing the MICs of these antibiotics [118].
The fact that most secondary metabolites isolated from Vitex plants are phenolic compounds or terpenoids falls in line with published literature focusing on the biological activity of these types of compounds and can be correlated with the exhibited antimicrobial activity.Most of the studies assessed in this review focused on methanolic and ethanolic extracts of the Vitex species' leaf plant part.This may indicate that natural compounds present in this plant part are the main responsible ones for the antimicrobial activity exhibited and that these compounds must have high polarity rates since methanol and ethanol are polar solvents with high affinity for polar compounds.Regarding specific chemical compound isolation, studies conducted on Vitex species have identified diterpenoids and flavonoids.Since Vitex species have phytochemical profiles with high amounts of diterpenoids, these may be responsible for the exerted antimicrobial activity.However, further research should be conducted to better understand and characterize this activity.
The drug development of new antimicrobial agents is a major challenge for the pharmaceutical industry.There are limited mechanistic pathways that can be followed for antibacterial, antifungal, and antiviral activities, and some microorganisms can have intrinsic specific resistances that render them immune to some of these pathways.Moreover, the development of new drugs "from scratch" is a highly expensive and time-consuming process that often does not move from theory to practice due to limitations like synthesis yields, formulation compatibilities, bioavailabilities, and other technological aspects.Using natural products from plants with antimicrobial activities can circumvent some of these limitations since pharmaceutical engineering processes can use core molecular skeletons of these secondary metabolites to develop new therapeutic options with clinical significance.For example, amikacin, a semisynthetic aminoglycoside broad-spectrum antibiotic active against Pseudomonas aeruginosa and most Gram-negative aerobes, is derived from kanamycin A, which is a natural product isolated from Streptomyces kanamyceticus [137][138][139].
Numerous studies have explored the efficacy of different plant parts of Aloe vera, namely leaf extracts, against a range of bacterial and fungal species.Results showed high antibacterial activity against E. coli, with inhibition zones indicating significant activity against drug-resistant strains [140].On the other hand, Artemisia has shown antibacterial activity against drug-resistant bacterial strains.For example, Artemisia absinthium L. leaf extracts exhibit antibacterial activity against S. aureus [141].Given the results of our review, we believe that the limited nature of the direct evidence on the antimicrobial activities of Vitex species, when compared to other genera, is due to the fact that Vitex species have been less extensively studied for their antimicrobial properties, and more research is needed to understand their full potential.

Conclusions
This review summarizes the main traditional uses of plants belonging to the Vitex genus as anti-infective medicines in different traditional medicinal systems and the in vitro antimicrobial activity demonstrated by extracts made from these medicinal plants against a wide variety of bacterial, fungal, and protozoal strains.In vitro studies demonstrate that Vitex extracts very often exhibit antimicrobial activity against different bacterial, fungal, and protozoal species.Some of the natural compounds present in these extracts, most likely the main ones, can be responsible for this biological activity.The results analyzed contribute to legitimizing the traditional use of Vitex species in traditional medicinal systems.A better understanding of Vitex-genus medicinal plants and their natural compounds can constitute a valuable natural source for discovering antimicrobial drugs and helping fight and prevent infectious diseases.

Figure 1 .
Figure 1.Data screening based on PRISMA methodology.

Figure 1 .
Figure 1.Data screening based on PRISMA methodology.

Figure 2 .
Figure 2. Symptoms of disease treated with Vitex plants.

Figure 2 .
Figure 2. Symptoms of disease treated with Vitex plants.

Figure 5 .Figure 5 .
Figure 5. Solvents used for plant extraction for antibacterial activity essays.

Figure 5 .
Figure 5. Solvents used for plant extraction for antibacterial activity essays.

Figure 6 .Figure 6 .
Figure 6.The microorganisms were extensively studied for their interactions with Vitex species.

Table 2 .
In vitro antibacterial activity studies on Vitex species.

Table 2 .
Cont.Vitex species studied for their in vitro antibacterial activity.Plant parts used in antibacterial studies.

Table 3 .
In vitro antifungal activity studies on Vitex species.

Table 4 .
In vitro antiviral and antiprotozoal activity studies on Vitex species.

Table 5 .
Isolated chemical compounds from Vitex species and their antimicrobial activity.

Table 5 .
Isolated chemical compounds from Vitex species and their antimicrobial activity.
1 January 1980 and 23 May 2023, applying several keywords: Vitex, Vitex AND Traditional Use, Vitex AND Ethnomedicine, Vitex AND Biology, and Vitex AND Antimicrobial activity.