Bioactive Natural Products for Chemical Control of Microorganisms: Scientific Prospecting (2001–2021) and Systematic Review

The inappropriate use of synthetic antibiotics has become a global public health problem. Therefore, the study of new alternatives for the treatment of infectious diseases is relevant and natural bioactive products are on the rise. This study conducted a scientific prospection of bioactive natural products with promising applications in the chemical control of microorganisms. A systematic review of the most recent articles was performed according to the following three steps: (i) eligibility assessment, (ii) screening, and (iii) inclusion of articles and information extraction. There has been an increase in the number of scientific publications on bioactive natural products for microbial control in the CAPES and SciELO databases (2001–2021). Seventeen relevant articles were included, most of which focused on extracts. Ascorbic acid, chlorogenic acid, chrysin, and quercetin were the most cited compounds. Natural products were shown to be effective in inhibiting more than 30 microorganisms. A discussion was presented on the research trends.


Introduction
Injudicious use of conventional synthetic antibiotics in the treatment of infectious diseases has become a global public health problem. Pathogenic microorganisms evolve by developing adaptation and resistance mechanisms, such as production of inactivating enzymes, decreased membrane permeability, and efflux pumps for antibiotics. Multidrug resistance has been reported in several clinically relevant bacteria, including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus [1][2][3].
The World Health Organization (WHO) monitors global trends in evidence-based healthcare and has supported the implementation (2014-2023) of traditional and complementary medicine strategies. Traditional and complementary medicine is an important health resource for modern therapy and in it might lie important solutions to the development and evolution of multidrug-resistant strains [4,5]. For instance, Brazil has a great diversity of medicinal plants widely investigated for scientific and technological applications and intensively used as herbal medicines [6,7].
Several chemical compounds can be produced and extracted from a single species [8,9]. Medicinal plants can be used to produce standardized extracts, essential oils, and purified phytochemicals with biological activities [10,11]. Bioactive natural products, which can be used separately or in combination, induce changes in membrane permeability or damage proteins and nucleic acids in microbial cells [12]. Studies have demonstrated that several bioactive natural products show effective antagonistic activity against foodborne pathogens and spoilage microorganisms [13,14].
This study aimed to conduct a scientific prospection and systematic review of promising bioactive natural products for chemical control of microorganisms. Two databases were selected and strategies were applied for the search, collection, and treatment of prospection data . Protocols were developed for the selection of the most recent articles and data extraction, comprising the following three steps: (i) eligibility assessment, (ii) screening, and (iii) article inclusion and information extraction. A discussion was presented on the main sources of extraction, importance of natural bioactive products, most cited bioactive natural compounds, microorganisms and inhibition effectiveness, and perspectives on research trends.

Scientific Prospecting of Data
The results of the searches carried out in CAPES and SciELO databases are presented in Table S1 (Supplementary Materials). The keywords with the highest quantitative contribution were ("essential oil" OR extract) AND antib* for the CAPES database and ("essential oil" OR extract) AND antimicr* for the SciELO database, both in English. Search strategies containing English keywords retrieved a larger number of articles, given the international nature of this language. From a total of 266,936 scientific articles retrieved, 256,780 were published in peer-reviewed journals indexed in the CAPES database. The CAPES database had a higher number of published articles in both languages, as expected, because this database also includes publications indexed by SciELO. Sample representativeness was the lowest when the keyword "fitoquímico" (phytochemical) was used as a descriptor in term 1, with a reduction in the number of retrieved papers according to the following order of descriptors in term 2: antimicr* > antib* > bactericidal > biocidal > "chemical agent".

Annual Evolution of Studies (2001-2021)
The annual distribution (2001-2021) of articles retrieved from the CAPES database by using the keywords ("essential oil" OR extract) AND antib* is depicted in Figure 1. Initial and final cycles were defined as the first and last 5 completed years, respectively, of the study temporal range (21 years).
The number of publications in the CAPES database increased from 2001 to 2021. An increase in the number of studies in relation to the previous number occurred every year, except in 2016 (n = 18,329) and 2019 (n = 23,539). More than 20,000 articles were published in the last 5 completed years (final cycle). This number was at least four times higher than that in the initial cycle (2001)(2002)(2003)(2004)(2005). It was estimated that more articles will be published in subsequent years than in previous years, given that, up to the first quarter of 2021, 7234 studies had been published. Thus, >26,000 articles on the use of bioactive natural products for microbial control are expected to be published in 2021. WHO's incentive for countries to generate evidence-based policies and strategic plans for the use of medicinal plants might be related to the increase in research in recent years [4].
The annual distribution (2001-2021) of scientific articles indexed in the SciELO database, identified by the keywords ("essential oil" OR extract) AND antimicr*, is depicted in Figure 2.

Ranking of Thematic Areas and Journals
Ranking of the thematic areas and journals according to the number of scientific articles published in CAPES and SciELO databases is presented in Table 1. The thematic areas Chemistry (23.52%, n = 35,797), Engineering (16.71%, n = 25,428), and Bacteria (15.98%, n = 24,329) had the highest number of publications in the CAPES database. More specific areas, such as antibacterial agents, antibacterial activity, and antimicrobial agents, were also identified. The microorganisms E. coli and S. aureus ranked 5th and 10th, respectively, as thematic areas, suggesting that they are the major species investigated in inhibition tests with bioactive natural products. The journal Molecules had more than 5000 publications, representing about 70% of the total number of articles (n = 7846). The other journals with the highest number of publications on the subject were Journal of Applied Microbiology (6.93%, n = 517), Journal of Essential Oil-Bearing Plants (4.86%, n = 363), and Industrial Crops and Products (4.41%, n = 329).
Articles retrieved from SciELO were classified into the major thematic areas Agricultural Sciences (29.48%, n = 278), Health Sciences (26.30%, n = 248), and Biological Sciences (25.03%, n = 236). These thematic areas were comprehensive, with the global field of sciences accounting for over 800 publications. Articles can be classified into more than one area of publication or theme, generating duplicity of citations and variation in the number of themes. Of the scientific journals indexed in the SciELO database, Revista Brasileira de Pharmacognosia was the one with the highest number of publications on the subject (25.97%, n = 100), in agreement with the thematic area ranking 2nd (health sciences). The three journals with the largest numbers of scientific articles were discontinued from the SciELO database, namely Revista Brasileira de Pharmacognosia, Revista Brasileira de Plantas Medicinais, and Brazilian Journal of Microbiology. The scopes of the ranked journals are in accordance with the main thematic areas. All journals reported the impact factor, as identified in the 2019 Journal Citation Reports, with the exception of Revista Brasileira de Plantas Medicinais. Nevertheless, the impact factor was not directly proportional to the number of published articles.  A total of 6 countries had the highest number of publications, totaling 822 articles. Brazil accounted for 72% (n = 594) of the publications, which was expected, given that the SciELO database comprises a wide range of Brazilian scientific journals. Mexico, Colombia, Chile, Argentina, and Peru were the following major publishing countries, demonstrating the importance of research on bioactive natural products for microbial control in these countries. Only 4% (n = 35) of publications stemmed from other countries.

Relevant Recent Research
Initially, 822 records were identified using the advanced search table. In the first stage of selection, 66 articles were pre-selected, 22 of which were excluded. In the second stage (full-text reading), 27 articles were excluded. In the third stage, 17 relevant articles were selected. The information of interest was extracted, grouped, and presented in Table 2 and Figure S1 (Supplementary Materials).

Most Cited Bioactive Natural Compounds
Bioactive products were categorized into classes, subclasses, and specific compounds. A higher frequency of flavonoids and terpenoids was observed. Sesquiterpenes and caryophyllenes were the most frequently identified subclasses of terpenes. The 3D structure and characteristics of the most cited compounds are shown in Table 3. Ascorbic acid, chlorogenic acid, chrysin, and quercetin were associated with various bioactive properties, such as antioxidant, anti-inflammatory, antiviral, antiallergic, hepatoprotective, neuroprotective, antiasthmatic, antidiabetic, and antidepressant properties [34][35][36][37]. It is important to note that the identification of some constituents may be limited by the analytical method used. Some identification techniques depend on analytical standards and may not be able to screen all major compounds of extracts and essential oils. Therefore, quantified constituents may or may not be the most important compounds in terms of composition and bioactivity.

Microorganisms and Inhibition Effectiveness
Inhibition effectiveness was tested against more than 30 microorganisms. Cunha et al. [20] found that the foliar extract of Senna rugosa was not effective in inhibiting Aspergillus niger ATCC 10535, Candida albicans ATCC 90028, Klebsiella oxytoca ATCC 49131, Penicillium expansum ATCC 1117, or Salmonella Typhimurium ATCC 14028. The other relevant articles reported partial or total efficacy of bioactive natural products, demonstrating the high potential of biocompounds for the control of microorganisms. Efficacy in inhibition tests is directly associated with biocompound concentration, treatment time, and microbial resistance. Various studies have carried out tests against multi-drug resistant strains, such as S. aureus (Gram-positive) and E. coli (Gram-negative), explaining the high rank of these thematic areas with regard to the number of published studies (Table 1). C. albicans was the most studied fungus [15,17,[19][20][21]26,27].

Perspectives on Research Trends
Relevant articles demonstrated the promising action of bioactive natural products in microbial control. The following aspects should be considered and further explored: 1. Inclusion of extract or essential oil in food matrices may contribute to the bioactivity, bioavailability, and bioaccessibility of bioactive compounds of interest; compounds can be introduced into matrices via alternative techniques, such as marinating, sprinkling, soaking, and brushing. 2. In vitro and in vivo results are important to substantiate claims of the functional properties of bioactive ingredients in foods, contributing to healthiness and ensuring consumers' rights to information. 3. Extraction of bioactive compounds and development of bioactive natural products from food industry by-products has high scientific, technological, socioeconomic, and environmental appeal; these materials can also be used in active and intelligent packaging. 4. Reports of the direct application of purified phytochemicals and bioactive natural dyes/pigments for food enrichment are still scarce; viable systems may be developed to strengthen the metabolic activity of beneficial microorganisms, whether added, natural, or autochthonous (acid-lactic bacteria and/or yeasts, probiotic or not). 5. Compound protection technologies, such as microencapsulation, nanoencapsulation, and nanoemulsion, can optimize bioactivity, bioavailability, and bioaccessibility, thereby enhancing product stability and ease of transport, storage, and distribution.

Material and Methods
This study used a mixed method, consisting of scientific prospecting followed by a systematic review of the literature (Figure 4) based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [38], including procedures adapted from the work of Melo et al. [39] and Bezerra and Pinheiro [40].

Sources of Information
The following two electronic databases were searched: the CAPES database (Portal de Periódicos CAPES of the Brazilian Federal Agency for Support and Evaluation of Graduate Education), which provides democratic and easy access to several databases of high scientific relevance [41], and the Scientific Electronic Library Online (SciELO) database, which is the main cooperative electronic portal for open-access scientific journals in Latin America [42]. No contact was made with the authors to identify additional studies.

Search Strategies
Searches for articles on bioactive natural products for microbial control, written in Portuguese or English, were carried out in the advanced search tab of each database. Truncated terms, quotation marks, and Boolean operators were used to construct the search strategy, according to the particularities, supported characters, and instructions provided by the databases. Conference proceedings, technical books, monographs, dissertations, and theses were excluded by applying specific filters. Searches were performed between 27 April and 6 May 2021. Time of publication was restricted to the past 21 years. The combination of keywords and descriptors, presented in detail in Table S2 (Supplementary Materials), can be summarized as follows: bioactiv* AND (antib*; antimicr*; bactericidal; biocidal; "chemical agent"), phenol* AND (antib*; antimicr*; bactericidal; biocidal; "chemical agent"), phytochemical AND (antib*; antimicr*; bactericidal; biocidal; "chemical agent"); and "essential oil" OR extract AND (antib*; antimicr*; bactericidal; biocidal; "chemical agent").

Data Processing
The keyword and language with the highest quantitative contributions were used for data processing. The data were tabulated and analyzed in terms of the following quantitative variables: annual number of publications (2001-2021), ranking of thematic areas, journals with the highest number of articles published, and country of origin of journals (SciELO database only). The results are presented in tables or figures, constructed using Microsoft ® Excel 2013.

Systematic Review Protocol
Publications were identified and selected using the keyword and language with the highest quantitative contribution in the SciELO database. A protocol was used to select the most recent articles and extract the data, consisting of the following three steps: (i) eligibility assessment, (ii) screening, and (iii) article inclusion and extraction of information. The risk of bias was not assessed. Eligibility was defined as meeting the totality of the following criteria (yes = 100%): 1. Was the scientific paper published between 2020 and the current date (2021)? 2. Can the overall objective of the research be easily identified? 3. Is the source of bioactive natural products specified? 4. Was the bioactive natural product used in the form of an extract or essential oil? 5. Is the preparation procedure described clearly and in detail? 6. Has at least one compound responsible for biological activity (rather than a group or class) been identified using adequate analytical techniques? 7. Were inhibition tests performed against at least one specific microorganism? 8. Is the analytical method or technique described clearly and in detail? 9. Is the effectiveness of microbial inhibition reported? 10. Are the results and/or conclusions aligned with the overall objectives of the study?
In the first step, articles that did not meet the eligibility criteria were excluded. In the second step, articles were screened by two reviewers, who read the texts in full in a double-blind system and selected relevant articles. Disagreements were discussed until consensus was reached and the opinion of a third reviewer was requested for final decision making. In the third step, articles were included on the basis of shared decisions, and the following information was extracted: natural source of bioactive products (species and organ), type of bioactive natural product (extract or essential oil, solvent, and extraction method), main bioactive compounds and major components of the natural product, and inhibition test (microorganism species and efficacy assessment).
Efficacy was categorized as yes, partial, or no. Compounds were classified as effective (yes) when they achieved complete efficacy at a given concentration and/or during a given incubation time; as partial when they showed limited efficacy against at least one type of microorganism, regardless of the concentration and/or incubation time tested; and as ineffective (no) when they did not achieve significant results at any concentration or incubation time against any microorganism. Extracted data were organized in tables and/or figures and analyzed in terms of main sources of extraction, importance of natural bioactive products, most cited bioactive natural compounds, microorganisms and inhibition effectiveness, and perspectives on research trends.

Conclusions
The research trends indicated that more than 26,000 articles on the potential of bioactive natural products for chemical control of microorganisms will be published in the coming years in journals indexed by CAPES. E. coli and S. aureus were the main species investigated in inhibition tests with bioactive natural products. In the SciELO database, more than 800 articles categorized into the field of Sciences were identified, and the Revista Brasileira de Pharmacognosia was the major publishing journal, with 100 publications (25.97%). Scientific interest in the subject has increased in Brazil and in countries such as Mexico, Colombia, Chile, Argentina, and Peru.
The major extraction sources were of plant origin, mainly leaves, peels, and aerial parts. The importance of extracts and essential oils was evidenced. These materials were obtained mainly by maceration and hydrodistillation, respectively, with ethanol and water as solvents. Ascorbic acid, chlorogenic acid, chrysin, and quercetin were the most cited bioactive compounds. Partial and/or total inhibition of at least 30 different microorganisms by bioactive natural products was identified, being related to compound concentration, treatment time, and microbial resistance. Relevant articles demonstrated the promising action of bioactive natural products in the chemical control of microorganisms. Future trends were discussed.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/molecules27185917/s1, Table S1. Results of the keyword searches for articles in Portuguese (PT) and English (EN); Table S2. List of keywords used to search for scientific articles on bioactive natural products for 15 microbial control in Portuguese and English. Figure S1. Graphical abstract of scientific information on bioactive natural products for microbial control.

Conflicts of Interest:
The authors declare no conflict of interest.