BACKGROUND: Plantago major is a medicinal plant that has been used for centuries to treat various health conditions including wounds. PURPOSE: To investigate the effectiveness of the topical use of P major in healing skin wounds in animal models. METHODS: Using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, a systematic review was conducted. Seven (7) electronic databases (Virtual Health Library, Public/Publisher MEDLINE, Scopus, Web of Science, Embase, Cumulative Index of Nursing and Allied Health Literature, and CAB Direct) were searched for controlled studies published in English from January 2006 to March 2020. The Collaborative Approach to Meta Analysis and Review of Animal Data from Experimental Studies tools guided the evaluation of the studies and determined their quality. The Systematic Review Center for Laboratory Animal Experimentation was used to assess the risk of bias. RESULTS: Of the 176 publications identified, 4 met the inclusion criteria. Studies included 20 to 100 animals and varying concentrations of P major.  There were no reports of losses during research. Wound healing was assessed between 17 and 21 days following wounding. The best response in terms of wound contraction rate occurred with 10%, 20%, and 50% concentrations when compared with control groups. One (1) study did not have an animal ethics committee review. All studies had a high risk of bias and a lack of methodological rigor. CONCLUSION: The results of this review did not find evidence about the in vivo effectiveness of  P major for wound healing. More rigorous preclinical studies with adequate sample sizes are required to identify the best concentrations and formulations as well as increase understanding about the mechanisms of action of P major in wound healing.  

The search to elucidate the potential therapeutic effects of medicinal plants has been expanding worldwide. Data from the Global Report on Complementary and Traditional Medicine showed that 88% of World Health Organization member states recognize the use of complementary and traditional medicine as essential and are developing guidelines for the creation of policies, laws, regulations, and programs that involve the use of medicinal plants in health systems.¹ Due to their great potential to treat and prevent diseases, medicinal plants have been used traditionally in wound management.^2,3 In Brazil, medicinal plants are mainly used in primary health care because of a considerable lack of availability of  new technologies to treat wounds.⁴ Because of this, in 2009, the Brazilian Ministry of Health created the National List of Medicinal Plants of Interest to the Unified Health System (Relação Nacional de Plantas Medicinais de Interesse ao Sistema Único de Saúde or RENISUS), seeking alternatives for the treatment of common diseases that affect the population.² Plantago major belongs to the Plantaginaceae family and has 185 accepted species.⁴ It plays an essential role in complementary and traditional medicine in different cultures of the world, and it is used to treat diseases related to the skin, digestive system, circulatory system, urinary system, and skin lesions.⁵

The benefits of P major are related to the use of its leaves and seeds, which have been shown to have analgesic, anti-inflammatory, antioxidant, immunomodulatory, antifungal, anticancer, and wound healing effects in in vivo and in vitro studies.^5,6 These outcomes are believed to be related to the various bioactive compounds, such as flavonoids, alkaloids, terpenoids, phenolic compounds, iridoid glycosides, fatty acids, polysaccharides, and vitamins.^6,7 P major has been recommended for human use in various forms such as roasted seeds, decoction, syrup, liniment, gargle, rectal enema, vaginal suppository, and eye and nasal drops.⁵

Human skin is the main barrier against the environment, and acute and chronic problems can occur because of exposure to damaging factors. Tissue repair is physiological but can be compromised^8,9; therefore, there have been many efforts to investigate the possible therapeutic effect of medicinal plants in the treatment of wounds.^8,9

In their study utilizing a scratch assay, Zubair et al¹⁰ researched 5 populations of P major collected in different regions of Sweden and identified plantamajoside among the most expressive phenolic compounds, followed by 4 new compounds identified as PLMA 1 to PLMA 4. Compounds such as plantamajoside and iridoid glycosides have an anti-inflammatory and antioxidant action, which may contribute to the healing of skin wounds.^11,12 Results of other in vitro, in vivo, and ex vivo studies have supported the medicinal effects of various Plantago species via their ability to decrease the inflammatory cascade induced by nuclear factor-kappa B, nitric oxide, cyclooxygenase-2, and B4 leukotrienes as well as to improve wound healing.^10,13–15

Despite the use of  P major to treat diseases related to skin, respiratory organs, digestive organs, and reproduction as well as its use against infections, Plantago’s ability to heal wounds has only been evaluated in preclinical studies.¹⁰ Research on the topical use of P major in skin wounds has been limited in clinical practice, although it has been used in folk medicine. However, a recent clinical trial testing a P major solution versus a bicarbonate and chlorhexidine solution in the treatment of oral mucositis demonstrated that there were no differences in outcomes between the groups, and the authors suggested that the use of P major extract in the management of oral mucositis could be considered for clinical practice.¹⁶ In this context, the present systematic review aimed to evaluate the effectiveness of the topical use of P major in the healing of skin wounds in animal models.

The guidelines from the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guided this study.¹⁷ The protocol was registered in PROSPERO CRD42019121962 and previously published.¹⁸

Focus question. The main question to be answered in this systematic review was the following: What is the evidence in the literature of the effective use of P major for wound healing in animal models?

Search strategies. The databases accessed were the Virtual Health Library (BVS), Public/Publisher MEDLINE (PubMed), SciVerse Scopus (Scopus), Web of Science, Excerpta Medica Database (Embase), Cumulative Index of Nursing and Allied Health Literature (CINAHL), and CAB Direct. The study considered the period between January 2006 and March 2020. The year 2009 was chosen because it was in that year that the National List of Medicinal Plants of Interest to the Unified Health System was created. The survey was conducted in English, and the search terms used are shown in Table 1.

Inclusion and exclusion criteria. The inclusion criteria were 1) animal studies assessing the effect of P major in wound healing, 2) control group receiving treatment with the vehicle, 3) acute or chronic cutaneous wound models, and 4) studies must describe the initial and final wound size, number of animals per group, time of treatment, and concentration and formulation used in the treatment of the wound. The exclusion criteria were 1) use of other drugs in the same formulation with P major, 2) in vitro studies, and 3) literature reviews.

Outcomes. The primary outcome measure was the wound healing rate. The second outcome measure was histological scoring of P major in wounds.

Study selection. Figure 1 shows the selection process. Duplicate records were identified and removed before the screening. Two (2) independent reviewers evaluated study selection by reading the title and abstract according to the inclusion criteria. Disagreements were discussed with a third examiner until consensus was achieved.  After reading the studies in full, a consensus was reached on which relevant studies met the inclusion criteria. However, a third independent reviewer was involved if the disagreements could not be resolved.

Quality assessment. The quality improvement publications were assessed using the Collaborative Approach to Meta Analysis and Review of Animal Data from Experimental Studies (CAMARADES) tool,¹⁹ which includes 1) publication in a peer-reviewed journal, 2) statement of control of temperature, 3) randomization to treatment or control, 4) blinded induction of ischemia (ie, concealment of treatment group allocation at the time of induction of ischemia), 5) blinded assessment of outcome, 6) avoidance of anesthetics with marked intrinsic neuroprotective properties, 7) use of animals with hypertension or diabetes, 8) sample size calculation, 9) statement of compliance with regulatory requirements, and 10) statement regarding possible conflicts of interest. The included studies were evaluated by the Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) risk of bias tool,²⁰ which consists of the following 10 domains: 1) sequence generation, 2) baseline characteristics, 3) allocation concealment, 4) random housing, 5) researcher blinding, 6) random outcome assessment, 7) researcher blinding, 8) incomplete outcome data, 9) selective outcome reporting, and 10) other sources of bias. 

Data extraction and analysis. The primary information extracted from the selected studies were 1) author; 2) basic information about the animals such as species, sex, number, and weight; 3) wound initial size; and 4) time of treatment and dosage of P major for treatment, formulation, and control treatment.

Due to the extensive heterogeneity of methods among the studies identified, a narrative review was conducted instead of a meta-analysis.

The authors identified 176 studies using the search strategies. One-hundred (100) articles were duplicates in databases, 71 studies did not meet the inclusion criteria, and 1 study did not have a control group; this left a sample of 4 studies. 

Characteristics of included studies.  Table 2 summarizes the characteristics of the included studies. Animals used were mostly male healthy rats. The species used were Sprague Dawley,²¹ Swiss,²² and Wistar²³; 1 study did not report the species.²⁴ The initial wound area was 2 cm for most studies, and in 1 study a third-degree burn wound model covering 20% of the body area was used.²⁴

The studies presented 5 treatment groups. Of these, 3 studies used only the negative control in treatment^21,22,24; one (1) study also investigated the positive control in addition to the negative control in treatment.²³

The concentrations of P major were different among the studies; 20% and 50% in cream-based aqueous solution,²⁴ 5% and 10% in aqueous extract incorporated in an oily base,²⁵ 10% in hydroalcoholic extract and incorporated in oily base,²² and 20 µm and 40 µg in gel base.²³ The number of animals described ranged from 20 to 100, and there were no reports of losses during research. The follow-up time for tissue repair ranged from 17 to 21 days. One (1) study did not report an animal ethics committee review.²¹

Wound contraction rate. In the macroscopic assessment, the best response to the reduction of the area of the wound to treatment occurred between the ninth and 11th day in the P major treatment groups.^21–24 The concentration of 40 µg performed better than the concentration of 20 µg in terms of reduction of the wound area.²³ The study that investigated the concentration of 20% and 50%²⁴ found no statistically significant difference between the groups.

Microscopic evaluation was described in 2 studies.^22,24 In the first study,²² the analysis occurred on the ninth day after the wound was made. Edema was classified as moderate in the P major and control groups: in the group treated with Siparuna guianensis, it varied from low to moderate, and in the group treated with ointment base (lanolin and liquid paraffin), it was from severe to moderate and with an accumulation of extravascular fluid in the connective tissue. Inflammatory infiltrates were lower in the group treated with S guianensis: for the P major and control groups, it was moderate, and for conventional ointment, it was moderate to severe with a significant presence of neutrophils and lymphocytes. Mast cells were present in all treatments around the wound. Concerning reepithelization, the group treated with S guianensis showed a rate of 42.9%, followed by 33.3% in the P major group, 14.3% in the control group, and an absence of reepithelization in the conventional ointment group.²²

The second study²⁴ found no difference between the study groups in morphometric evaluation on the 7th and 14th days. However, on the 21st day after wounding in the groups treated with 20% and 50% P major, better organization of granulation tissue and reepithelization was found, with the epidermis showing structured layers without crust, few inflammatory cells, and an increased number of capillaries compared with the control treatment (untreated). The morphometric findings of the animals treated with 1% sulfadiazine were similar to the animals treated with P major.

Risk of bias and methodological quality assessments. No study fully met the evaluated methodological criteria. Regarding bias risk,²⁰ the information related to questions 1, 3, 5, and 8 from SYRCLE domains were not reported in the selected studies. For question 2, all studies reported the information. A total of 25% of the articles reported random housing (question 4). All studies (100%) were not clear about random outcome assessment and researcher blinding for detecting bias (questions 6 and 7, respectively). Three (3) studies (75%) presented an unclear risk for selective outcome reporting (question 9), and all studies had high potential sources of bias (question 10) (Figure 2). The quality assessment supported by the CAMARADES tool,¹⁹ had a score ranging from 3 to 6, with an average of 4.25 (Table 3).

This systematic review sought current evidence in preclinical studies on the use of P major and its effectiveness in tissue repair. The results showed that the lack of rigorous methodological study designs as well as varying concentrations and delivery methods of P major made it impossible to systematically analyze the data or draw conclusions about the effectiveness of P major in the healing of in vivo wounds.  

Bias risk assessment is a fundamental part of a systematic review that affects its analysis and conclusion. The authors evaluated studies using the SYRCLE tool,²⁰ and the main flaws observed were lack of allocation sequence, adjustments of confounding factors, concealment for allocation, and blind reading of the results. The studies did not investigate the anti-inflammatory and antioxidant effects that are described in the literature as being beneficial actions of P major in the healing process, further limiting the ability to elucidate its potential therapeutic effects and the indications of use.

In vitro studies have demonstrated that iridoid glycosides are active metabolites present in P major leaves with  excellent antioxidant²⁶ and anti-inflammatory²⁷ activity. In an in vivo study, Kartini et al²³ studied aucubin, a biologically active compound (major iridoid glycoside) known to be a specific inhibitor of nuclear factor-kappa B activation in mast cells, which explains its anti‑inflammatory properties.²⁷ The macroscopic evaluation in their study showed a significant reduction in the wound area of hyperglycemic animals compared with normoglycemic animals. Considering that the wounds of persons with diabetes have a prolonged inflammatory phase, which contributes to a delay in healing, this is particularly helpful regarding anti-inflammatory effects.²⁸ The use of aucubin was effective in healing the wounds of hyperglycemic animals, but the inflammatory markers were not investigaed, which could contribute to clarifying the mechanism of action of P major.²³

The mutagenic activity of the P major plant extract was analyzed in 1 study included in the current systematic review. Thomé et al²² observed no mutagenicity, which is positive for the safe use of medicinal plants via topical use.²⁶ This is because the presence of mutagenicity can impair cell proliferation, which happens at a high rate in the epithelium during tissue repair.²⁷

The small number of studies that met inclusion criteria, incomplete methodological quality, and design limitations (ie, varying dosages and formulations, a lack of standardized wound measurements, and absence of investigation of inflammatory cytokines in the scar tissue of wounds treated with P major) prevented the authors of the present study from comparing the results among studies and drawing conclusions. Preclinical studies need to be improved to optimize the use of experimental animals and provide safe and accurate information on the therapeutic effects of the topical use of P major in wound healing. The use of medicinal plants is an essential alternative in the treatment of wounds, especially in areas or among populations with limited resources. Therefore, studies must be designed using established methods and procedures so that their results can be translated safely for use in patients. 

Although a systematic approach was used for the literature search, it is recognized that there are limitations to this review. These limiations include the differences in methodological approaches in the studies and the use of only English language publications.  

A systematic review was conducted to explore the effect of topical P major on wound healing in preclinical studies. Four (4) publications met the inclusion criteria. Study heterogenicity prevented the use of meta-analysis techniques. All studies used P major in an oily or gel  base, and concentrations ranged from 5% to 50%; 1 study compared 20 µg and 40 µg. The best response in wound contraction rate occurred with 10%, 20%, and 50% concentrations when compared with the control groups. Due to the high risk of study bias, more rigorous preclinical studies with adequate sample sizes are required to identify the best concentrations and formulations as well as increase the understanding of the mechanisms of action of P major in wound healing.

Ms. Cardoso is a nurse,  Ms. Breder is a nurse, Dr. Apolinário is a nurse, Mr. Oliveia is a statistician, Dr. Saidel is an assistant professor, Dr. Dini is an assistant professor, Dr. Oliveira-Kumakura is an assistant professor, and Dr. Lima is an associate professor, University of Campinas, Campinas, Sao Paulo, Brazil. Address all correspondence to Maria Helena Lima, RN, PhD, University of Campinas, Campinas, Sao Paulo, Brazil; email: mhmelolima@gmail.com.