Medicinal plants with mosquitoes repellent activity: a systematic review

Repellents are important allies in trying to prevent diseases transmitted by mosquito bites. Plants have been a source of various repellents and insecticides because they have a large reservoir of bioactive substances, in addition, their products have already been tested and are gaining space in research as a low-cost and easy-access alternative. A systematic review was carried out in the databases, Science Direct, Scielo, SciFinder, Springer, PubMed / Medline, Scopus and Web of Science, evaluated by three reviewers. Titles, abstracts and articles were analyzed in full in English, Spanish and Portuguese. To identify the articles, the following descriptors from the Medical Subject Headings (MeSH) were used: “insect repellents”, “essential oils”, “plants extract”, “repellent activity”, mosquitoes and plants. The bibliographic search resulted in 2274 records. Of these, 27 abstracts were considered potentially relevant, and at the end of the selection, 17 articles were included in their entirety. It was found that medicinal plants showed repellent activity against various species of mosquitoes, however due to the little standardization of the methodologies used and the products obtained it is necessary to develop standardized methods to guarantee the quality and reproducibility of clinical trials with medicinal plants.


Introduction
Mosquitoes are considered a serious public health problem due to serving as vectors for many important pathological conditions as well as inflicting significant discomfort through bites (Rueda, 2008;Ali et al., 2012). There are approximately 3500 mosquito species throughout the world, the majority of which are found in tropical and subtropical regions. However, only 10% have medical and veterinary relevance (Reiter, 2001). The most prevalent mosquitoes that act as vectors of human diseases are Aedes (Chikungunya, Zika, Dengue, and Yellow fever), Anopheles (malaria and filariasis), and Culex (Japanese encephalitis, West Nile virus, and filariasis) (Ghosh et al., 2012;Naseem et al., 2016). Recently, the Zika virus is creating damage in several parts of the world, including Brazil, Africa, Pacific Island, and Southeast Asia. According to Pan American Health Organization (PAHO), more than 40 countries or territories in the Americas recorded an epidemic of Zika virus in 2015-2016, with over 500,000 Zika suspected and confirmed cases (WHO, 2016).
Current and past strategies for mosquito control are based on synthetic insecticides, such as dichlorodiphenyltrichloroethane (DDT), temephos, and malathion. Although chemical agents have been successfully utilized in mosquitoes control during the last decades, the continuous use of these agents has resulted in the development of resistance, environmental impacts, and undesirable effects on non-target organisms, including humans (Deletre et al., 2019). All these factors have created a need for new biodegradable and renewable alternative insecticides. In this context, the use of plantderived natural products for vector control has various attractive characteristics, including biodegradability, availability at affordable prices, smaller toxicity, and broad-spectrum target-specific activities against different mosquito species (Ghosh et al., 2012;Varun et al., 2013;Wilke & Marrelli, 2015).
Essential oils, also known as essences, volatile oils, or etheric oils, are defined as complex mixtures of several volatile and lipophilic compounds, being constituted principally by terpenes (mono and sesquiterpenes) and phenylpropanoids Research, Society andDevelopment, v. 11, n. 1, e2611124142, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i1.24142 3 (Guenther, 1972;Sangwan et al., 2001). A great number of essential oils extracted from different aromatic plants has shown great potential for pest management, since these oils possess high repellency against several arthropod species (Geetha & Roy, 2014). As an example, we can highlight the citronella (Cymbopogon nardus) essential oil, one of the most common essential oils in mosquito repellent patents (Pohlit et al., 2011). In the United States, the U.S. Environmental Agency (U.S. EPA), responsible for repellent products regulation, has recognized and registered citronella oil as an insect repellent ingredient for human applications since 1948 (EPA, 1999). Given the above, we present here a systematic review of clinical articles on medicinal plants with mosquito repellent activity.

Methodology
Protocol of this study was registered in the International Prospective Register of Systematic Reviews (PROSPERO), an open access database of systematic reviews administered at the Centre of Reviews and Dissemination, University of York, UK PROSPERO under number CRD42017070923 (BOOTH et al., 2012).

Article search strategy
The search strategy was carried out based on studies available in scientific literature until August 2021. There was no initial time limit. The bibliographic literature search was conducted in different scientific databases, including Science Direct, Scielo, SciFinder, Springer, PubMed/Medline, Scopus, and Web of Science. In addition, a manual search was performed by analyzing the references of the included articles. The literature search was performed in English, Spanish and Portuguese. For identification of articles, the following descriptors of the Medical Subject Headings (MeSH) were used: "insect repellents", "essential oils", "plants extract", "repellent activity", "mosquitoes" and "plants". The descriptors were adapted to each database and combined through the Boolean operators (OR, AND, and NOT).
The titles and abstracts were evaluated according to predefined inclusion criteria to determine the relevance of this topic: clinical trials evaluating the use of medicinal plants as mosquito repellents; clinical trials with medicinal plant essential oils and clinical trials with plant products. The exclusion criteria were comments, editorials, articles not published in Portuguese, Spanish, and English, and articles not available in full.
Initially, the records were exported to Mendeley program 1.16.3 ©. Data extraction and initial assessment of the relevant records followed by the abstracts and full text was conducted by two independent reviewers (ARS and AMS). Then, selection divergences were resolved by a third reviewer (CASS) and consensus-building. After a consensus meeting, articles that were not within the scope of this review were excluded. Cohen's Kappa statistic was used to measure reliability among the evaluators (Mchugh, 2012).

Evaluation of repellent activity and repellent product quality
To evaluate interventions with medicinal plants, the following criteria were used: randomization, blinding of the participants (double-blind), description of the methods of randomization, and report of losses and exclusions after randomization. The used features to evaluate the quality of the products tested in the clinical trials were: characteristics of the medicinal plant; botanical identification; plant parts; type of product (fresh, dry, or extracts) and standardization of products (dose of active constituents / biomarkers) (Gagnier et al., 2006).

Quality of clinical trials of repellent activity with medicinal plants
This evaluation was performed according to the Main Items for Reporting Systematic Reviews and Meta-analyzes Research, Society andDevelopment, v. 11, n. 1, e2611124142, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i1.24142 4 (PRISMA). This statement provides essential information on the methodology and development of systematic reviews, as follows: terminology, research question formulation, study identification and data mining, study quality and results of publication (Moher et al., 2009).

Results
Initially, 2,274 records were found in the databases used. Of these, 27 abstracts were considered potentially relevant and, at the end of the selection, 17 articles were included in this systematic review according to the adopted inclusion and exclusion criteria (Figure 1). Most studies came from India 23% (n = 4), followed by Thailand 17% (n = 3), Colombia, Johannesburg, Ethiopia, Nigeria, Nepal, Malaysia, Bolivia, Benin, USA, Guatemala, and Peru.
The control groups present in the research were volunteers who did not apply any odorous or repellent substance on the arm.
The mean duration of the study was (μ) = 1.3 months with a standard deviation of (σ) = 0.9 Table 2.
As for the methodology of clinical trials, the main negative points were the lack of randomization, blinding of the participants, and no description of losses and exclusions Table 1. The main products used were essential oil with 59% (n = 10), followed by crude extract with 29% (n = 5) Table 3. The study included eighteen species of vectors, with an emphasis on the species Aedes aegypti, which was present in 28% of the analyzed studies Table 1.
The leaf was the main part of the plant used, 47% (n=8) in the preparations of repellents and the most used pharmaceutical form was a solution present in 47% (n=8) of the studies. Among the 17 studies, only two standardized their products according to the substance/markers present Table 3. Furthermore, only 6% (n = 1) of the studies reported not having achieved significant findings, while 94% (n = 16) of the studies speculated positive repercussions regarding the efficacy of the medicinal plant tested.
Concerning the technological quality of clinical trials involving medicinal plants with repellent activity, the main absences verified were: characterization of the medicinal plant (64.70%), standardization of products (94.11%), concentration of used extraction solvent (52, 94%), and qualitative and purity tests (100%) Table 3.

Discussion
According to the World Health Organization, vector-borne diseases account for more than 17% of all infectious diseases, causing more than 700,000 deaths annually. Diseases such as malaria, dengue, schistosomiasis, African human trypanosomiasis, leishmaniasis, Chagas disease, yellow fever, Japanese encephalitis, and onchocerciasis are among the main vector-borne diseases (WHO, 2020).
Due to the burden of these diseases, chronic suffering, disability, and morbidity throughout life, the WHO elaborated the "Global Vector Control Response (GVCR) 2017-2030", which provides strategic guidance for urgent strengthening of vector control to prevent disease and respond to outbreaks. Among the recommendations are the development and evaluation of new tools, technologies, and approaches for vector-borne diseases, including disease control and management technologies (WHO, 2017).
In this context, the field of study of medicinal plants as repellents is important, since repellents must be safe, agreeable, and ecologically sustainable. Moreover, the cost of the production of repellents is another significant factor that corroborates to the development of research on flora.

Evaluation of repellent activity and quality of products used
Although studies have shown the effectiveness of herbal repellent products, when applied to the skin, the protection provided usually dissipates rapidly (Trongtokit et al., 2005;Li et al., 2013;). In the case of essential oils, this fact is related to high volatility, a property that may be enhanced with the development of formulations that could be able to keep the principles active for an extended period. Considering the high volatility of EO, repellent performance is heavily dependent on the product's composition and capacity to prolong the duration of action. According to Oliveira et al, 2020, formulations based on creams, polymers, mixtures, or controlled-release microcapsules provide better results in repellency.
According to a study by Choocote et al. (2007) assessed in this review, a compound combined with essential oil that was demonstrated to increase repellency time was vanillin. Additionally, the repellency time of pure oil was shorter when compared with the mixture of 10% vanillin. Keziah et al., (2012) analyzed the formulation of crème with crude extract of leaves Ocimum gratissimum and Lantana camara L. which presented repellent activity against Aedes aegypti L., without exhibiting adverse reactions in the volunteers. Therefore, fixing additives and the production of repellents combined are possibilities that can increase the effectiveness and economic value of essential oils with repellent activity.
The process of transforming a plant into a medicine must adhere to established norms to ensure the plant's chemical and pharmacological integrity, ensuring consistent biological action and user safety. (Han et al.,2019). When analyzing the articles in this study, there are a significant number of limitations and absences, where most of the articles did not present the characteristics of medicinal plants in the tests and there was hardly any standardization of their products according to their chemical markers, there was also scarce descriptions of qualitative and quantitative quality control tests (only two articles presented). Such absences will interfere with the quality of the articles as well as question the safety and efficacy of the final product.
In the present study, only two articles, Hill et al. (2007) and Govere et al. (2000), described a standardization of the repellent product based on a biomarker. The substance used as a marker was p-mentan-3,8-diol (PMD), an essential oil classified as monoterpene and that can be extracted from aromatic plants such as eucalyptus (Corymbia citriodora) and has proven insect repellent properties similar to DEET (Carroll & Loye, 2006).
Despite the findings, there are some difficulties inherent to the development of the herbal product, due to the complexity of the plant's constituents,the raw material, and the variability in the quality of the products obtained from the same plant species. These characteristics are related to the factors and conditions of the place of cultivation, the procedure of collection, handling, and processing of the vegetable raw material. Thereby, plant products will show variations, further increasing the requirement for standardization to guarantee efficacy, safety, and quality (Liu et al., 2018).
The standardization and quality control of herbal medicines is carried out based on the content of biomarker, which by definition is a substance or a group of them, which is present mainly in a certain plant species and is the responsible or one of those responsible for the plant's pharmacological activity. Additionally, biomarkers must be stable and capable of analysis by analytical and bioanalytical processes, must guarantee the results' reliability, and must be validated according to current legislation. Hence, the biomarker's presence in an appropriate quantity demonstrates that the other components will be equally represented allowing the reproducibility of new studies with the same plant. Therefore, biomarkers are indispensable requirements in the production of medicines, in the planning and monitoring of technological production, and for studies on the stability of intermediate and final products. Thus, standardization is a prerequisite for constant therapeutic effects and patient safety (Han et al., 2019;Liu et al., 2018).
From this perspective of standardizing a product of plant origin, only two executable works are standardization and quality control tests of the obtained products, and most of these works may have carried out these tests. This statement can be observed in Table 4, which presents findings in the literature on experiments for the identification and analysis of plant biomarkers that were used in the studies. Also in this table, the main compounds of the plants used in the studies are from the class of essential oils and the most used method was Gas Chromatography coupled with Mass Spectrometry (CG-MS). This fact is justified by the aromatic and low molecular weight which are characteristics of the main biomarkers present in the medicinal species analyzed (Adams, 2007).

Evaluation of clinical trials of medicinal plants with repellent activity
Several methods were used to assess mosquito repellent activity by the studies that were included in this review, mostly amounts of EO or extracts were previously dissolved in a fixed volume of solvent and applied evenly to arms, legs, mosquito nets, or cages. To calculate the percentage of repellency, most studies evaluated the number of mosquito bites at certain times, the number of mosquitoes that landed and the time elapsed until the first bite on the arm or lower limbs.
Due to the variability in the methodologies found in the tests to assess repellency (different conditions, variables, and parameters), comparisons between the results of different clinical trials are not possible. Consequently, the results can only be compared with positive or negative control, in the same clinical trial, in order to classify the degree of repellency.
In this context, effective assessment is necessary following the adaptation of the CONSORT instrument (Table 5) for clinical trials with medicinal plants, since it allows the assessment of various aspects that interfere with the transparency of the study report with medicinal herbs. Many studies did not describe some methods (purity tests, biomarker standardization) and who performed them (e.g.name of the laboratory). Furthermore, they also failed to inform whether a product sample (eg, retention sample) was kept and, if so, where it was kept or deposited.
The standardized extracts were of high quality containing consistent levels of compounds, submitted to rigorous control. The analysis of the chemical profile is essential since using the appropriate techniques can provide a precise report of its constituents, besides providing qualitative and quantitative information (Folashade, Omoregie, & Ochogu, 2012). In view of the fact that the studies do not report the sample calculation and the size of the sample, these studies may be speculated to have small chances of being statistically significant (Sakpal, 2010).
The World Health Organization Guidelines for testing the efficacy of mosquito repellents for human skin (2009), recommends that the method of choice be performed in humans for producing results relevant to the conditions of use. In this review, the study by Hill et al., (2007), endorsed Eucalyptus maculata citriodon with p-menthane 3.8 diol (PMD), applied in mosquito nets making the evaluation of the product's repellency efficacy not possible.

Conclusion
Most studies included in this review were of medicinal plants rich in essential oils which demonstrated good repellent activity against several species of mosquitoes, however due to the high volatility of these compounds, the protection time is reduced when compared to positive control. Therefore, despite these products' potential, the development and standardization of products that increase the repellency time are required to improve mosquito repellent efficiency.
The absence of standardization of the methodologies was an obstacle to the evaluation of the articles included in this review. As a result, a standardized protocol capable of comprehensively evaluating the quality of clinical trials of medicinal plants with mosquito repellent activity is recommended. We hope that this review will contribute to future studies of medicinal plants with repellent activity, given the great necessity for new products to prevent diseases transmitted by mosquitoes.