Cupidon of the Pahouins: Transition of Musanga cecropioides R.Br. and Alchornea cordifolia Müll.Arg. from Traditional Use to a Rational Anti-Infective Phytodrug. Preclinical and Clinical Efficacy in Treating Microbial and Fungal Infections

1. Introduction

Human beings have always relied on nature to meet their healthcare needs, using plants, animals, marine organisms, insects, and fungi. Over time, people have discovered numerous plant species with medicinal properties, accumulating vast ethnopharmacological knowledge that has improved their quality of life (Colalto 2018; Marshall 2011; Yuan et al. 2016). Phytotherapy remains the primary form of treatment for a significant portion of the population in many developing countries, including approximately 80% of people in Asia and Africa. This reliance is due to the unavailability or high cost of other forms of treatment. Traditional medicine provides accessible care and is trusted by many (WHO 2013). Information from traditional medicine plays a critical role in drug discovery. For instance, among 122 plant-derived compounds used as drugs, 80% are employed for the same or similar purposes as their source plants (Fabricant and Farnsworth 2001).

Traditional medicine among the Pahouins is highly developed, offering holistic healing for both body and mind. Günther Tessmann, a German explorer, botanist, linguist, and ethnologist, spent time in Central Africa between 1912 and 1916. He published in 1913 that the Pahouin people were exceptionally clean, more so than any in Europe, frequently washing and bathing daily. They mixed oil with a perfumed powder used by both men and women, although its production was a female prerogative. They applied this mixture to their skin as often as possible, citing its pleasing fragrance and its ability to provide skin elasticity and firmness (Tessmann 1913).

Among the plant species long used in Pahouin traditional medicine, Alchornea cordifolia and Musanga cecropioides are known for treating various ailments such as malaria, dermatitis, arthritis, ulcers, impetigo, scabies, chronic wounds, coughs, and typhoid (Balde et al. 2015; Mabeku, Roger, and Louis 2011; Lawal et al. 2022). The Cupidon of Pahouin (CP), containing these two plants, is part of the women's secret recipes. Research has demonstrated the antimicrobial properties of A. cordifolia and M. cecropioides (Mabeku, Roger, and Louis 2011; Agboke et al. 2020; Akoto et al. 2019). No clinical trials have been reported.

Currently, there is a rise in multidrug-resistant bacteria, such as extended-spectrum beta-lactamases (ESBL)-producing Escherichia coli and Klebsiella pneumoniae, Pseudomonas aeruginosa, vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus, (Alekshun and Levy 2007; Levin et al. 1999). These resistant organisms result from the prolonged use of multiple antibiotic treatments for various infections, underscoring the urgent need for new active substances to prevent resistance (Alekshun and Levy 2007; Blair et al. 2015). Medicinal plants, which contain bioactive compounds with multiple modes of action, are thus crucial for reducing the risk of resistance (Mickymaray 2019).

This study aims to investigate the antimicrobial activity of A. cordifolia and M. cecropioides aqueous extracts, both alone and in combination, against various microbial strains in vitro. Additionally, clinical trials were conducted to demonstrate the effectiveness of this plant mixture in real clinical prescriptions.

2. Materials and Methods

2.1. Chemicals Products

All reagents were purchased and used for research purposes only. The following chemicals were obtained from Sigma (Sigma–Aldrich, France): Berberine chloride (BRB), Sodium Lauryl Sulfate solution (SLS), Methotrexate, Etoposide, Doxorubicin, Doxycycline, Amikacin, Cotrimoxazole, Monensin, and Erythromycin.

2.2. Plant Material

Plants were collected from the south region of Cameroon, specifically the Valley of Ntem division, and were identified at the National Herbarium (see Table 1).

The selected plant organs were dried in the shade and in the open air and stored in a dry place until further use.

2.3. Aqueous Extraction

Leaves of A. cordifolia, terminal buds of M. cecropioides, and stem barks of M. arboreus and A. klaineanum were mixed with distilled water at a ratio of 1:20 (w/v) and heated on a hot plate at 70-80 °C for one hour. The mixtures were then filtered, and the filtrates were oven-dried at 70 °C until dry extract powders were obtained. These extracts were then stored in closed flasks, protected from light and humidity, until further use.

2.4. Antimicrobial Activity Testing

The antimicrobial activity of the aqueous plant extracts was evaluated using the agar dilution method (CLSI 2012). This method allows for determining the minimum inhibitory concentration (MIC) of each extract against 32 microorganisms in in vitro culture. Bacterial and fungal strains were provided by the Laboratory of Bacteriology, Faculty of Pharmacy, Lille University, France. The selected microorganisms are involved in various nosocomial or opportunistic infections. They were grown at 37 °C for 24 hours in tubes containing inclined Mueller-Hinton Agar (MHA) culture medium (Bacto™ Agar, Le Pont de Claix, France; Mueller-Hinton Broth Oxoid™, Basingstoke, United Kingdom). Ten milliliters of Ringer Cysteine (RC) liquid (Merck™, Darmstadt, Germany) were then added to the tubes and mixed to thoroughly suspend the cultured microorganisms. A single drop from each suspension was added to a dilution tube containing 10 mL of RC solution. The final suspension used for the test had an estimated turbidity of 0.5 McFarland. Each well of the inoculum replicator plate was then filled with the suspension from one of the dilution tubes.

2.4.1. Antimicrobial Activity of Individual Extracts

The activity of individual extracts was determined as previously described by Boutahiri et al., 2022. Aqueous extracts were first dissolved in an ethanol/water (3:7) mixture, then combined with Mueller-Hinton Agar (MHA) and poured into Petri dishes. The final concentrations tested were 0.075, 0.15, 0.30, 0.60, and 1.20 mg/mL. After cooling, the MHA-extract mixtures were inoculated with microorganisms using the previously prepared inoculum replicator and incubated at 37 °C for 24 hours. Minimum Inhibitory Concentration (MIC) values correspond to the lowest extract concentrations that completely inhibited the growth of specific germs. A negative control was tested using the ethanol/water solvent. Three antibiotics were used as positive controls: amoxicillin, vancomycin, and gentamicin. When MIC ≤ 4 mg/L, the studied strains were considered susceptible to the antibiotics used; when MIC > 16 mg/L, they were considered resistant to amoxicillin and vancomycin, and when MIC > 8 mg/L, they were resistant to gentamicin (CLSI 2012).

2.4.2. Pre-Formulation of Polyherbal Combination

Determination of different antimicrobial phytomedicinal candidates was realized using combination analysis (Eto 2019).

Where n represents the total number of plants used in the traditional formulation and kk the number of extracts per formulation. The traditional formulation contains four plant extracts: A. cordifolia (1), M. cecropioides (2), M. arboreus (3), and A. klaineanum (4). Equation 1 gives six different possible formulations containing two extracts to be tested: (1,2), (1,3), (1,4), (2,3), (2,4), (3,4). The most active extract was considered as the functional unit (score of activity against bacterial strains), and the functional ratio (Fr) used to determine the quantity of each extract in the polyherbal pre-formulation (PHF) was obtained by the ratio between the score of the most active extract and the score of the combined extract (Mamadou et al. 2011).

2.4.3. Checkerboard Assay for Antimicrobial Activity

The checkerboard assay was used to study the antimicrobial activity of mixed extracts. This method allows for examining all possible combinations within the range of the studied concentrations (0.075, 0.15, 0.30, 0.60, and 1.20 mg/mL). The agar dilution method was also used to determine the Minimum Inhibitory Concentration (MIC) of an extract mixture. The MIC corresponds to the lowest concentration of extracts in the mixture that inhibits the growth of a germ. This method involves calculating the Fractional Inhibitory Concentration Index (FICI), which is the sum of the Fractional Inhibitory Concentration (FIC) of each extract. The FICI indicates the effect on a specific microbial strain when combining two plant extracts (Fratini et al. 2017). The combination of extracts A and B is calculated as follows:

When FICI < 1, the effect of the combination is synergistic, when FICI = 1, the effect is commutative, when 1 < FICI ≤ 2, the effect is indifferent, and when 2 < FICI, the effect is antagonistic.

2.5. Effect of PHF on Inhibition of Protein Synthesis

The mechanism of action of several antibiotics involves the inhibition of protein synthesis. This is the case for antibiotics from the aminoglycoside family, macrolides, phenicoles, fusidic acids, and oxazolidinones. The same applies to certain anticancer agents. For this reason, the activity of the polyherbal formulation (PHF) (A. cordifolia and M. cecropioides in a ratio of 1:1.38) on protein synthesis was evaluated using the in vitro seed germination inhibition assay of Lepidium sativum, as previously described by Outman et al. 2023. The ratio 1:1.38 was determined according to the functional ratio.

Seeds were first placed on Whatman filter paper moistened with water and kept in the dark for 24 hours to induce seed pre-germination. Seeds that had begun to germinate were selected for the test. Fifteen seeds were then placed in Petri dishes containing Whatman filter paper in the presence of different concentrations (10^-4 to 10³ µg/mL in distilled water) of CP, anticancer drugs (Methotrexate (MTX), Etoposide (ETP), Berberine (BRB), and Doxorubicin (DOX)), and antibiotics (Doxycycline (DOC), Amikacin (AMK), Cotrimoxazole (TMP/SMX), Monensin (MON), and Erythromycin (ERY). The Petri dishes were kept in total darkness for 72 hours, after which rootlet lengths were measured. The negative control was achieved with distilled water.

This test allows for the evaluation of the antineoplastic activity of PHF and to determine one of its mechanisms of action as an antibacterial and antifungal agent.

2.6. Transdermal Passage Study

Franz diffusion chambers, which help maintain the physiological condition of a skin biopsy, are used to study transdermal passage. Briefly, rat skin biopsies were mounted in a special Franz diffusion chamber (Laboratories TBC, France). The isotonic Ringer's solution used throughout the experiments consists of 115 mM NaCl, 25 mM NaHCO₃, 1.2 mM MgCl₂, 1.2 mM CaCl₂, 2.4 mM K₂HPO₄, and 0.4 mM KH₂PO₄. Ringer's solution was used in the two bath reservoirs located on either side of the skin biopsy, defining the two compartments: the donor compartment and the receiver compartment, separated by the skin biopsy. The passage of CP through the skin was evaluated by measuring transdermal fluxes. Ringer's solution containing 3 mg of CP was then inserted into the donor compartment. A 1 mL sample from the receiver compartment was taken at 0, 1, 2, 4, 20, and 24 hours and replaced each time with 1 mL of Ringer's solution (Iliopoulos et al. 2020). The solution obtained from the receiver compartment (1 mL) was used for UV measurements between 200 and 500 nm.

2.7. Toxicity Assessment

CP is an antimicrobial for external use. It can be used as a mouthwash, eye drops, for skin infections, and feminine hygiene. Several toxicity studies have been carried out on A. cordifolia and M. cecropioides. All these studies show that these two plants are not toxic (Adeneye et al. 2006; Djimeli et al. 2017; Mahama et al. 2022). In this study, the toxicity assessment of the formulated product (CP) was carried out using the tests required for products for external use, such as the primary skin irritation test, the eye irritation test, and the challenge test. All the toxicological studies were conducted at Laboratoires TBC-TransCell-Lab, University of Paris Diderot - Paris 7, Faculty of Medicine Xavier Bichat, Paris, France.

2.7.1. Primary Skin Irritation Test (PSIT)

The PSIT is commonly used in cosmetic and pharmaceutical studies to assess the potential of a substance to cause skin irritation. In this study, the PSIT was used to evaluate the potential of CP to cause irritation to human skin and mucosa. Briefly, patches containing a solution of CP (10 mg/mL), a positive control (2% Sodium Lauryl Sulfate solution or SLS), or a negative control (Saline solution, 0.9% NaCl) were applied to the forearm of 12 healthy human volunteers for 4 hours. The standardized scale for evaluating irritation (e.g., Draize scoring system) was used. The Primary Irritation Index (PII) was calculated by averaging the scores of all test subjects and all observation points. The PII of the test substance was then compared with that of the control substances to determine the irritation potential (see Table 2).

2.7.2. Corneal Fibroblast Cytotoxicity Test:

Corneal fibroblast cells are used to evaluate the potential cytotoxic and irritant effects of substances on the cornea (OECD 2019). In this test, the human corneal fibroblast cell line (HCF) was used. CP was tested at a concentration of 5 mg/mL. For viability and cytotoxicity assessments, MTT reagent and LDH assay kits were utilized. Benzalkonium chloride was used as a positive control, and saline solution (0.9% NaCl) was used as a negative control. CP and controls were incubated for 24 hours. Test interpretation is given in Table 3 according to OECD Guidelines.

2.7.3. Challenge Test

The agar medium counting technique was used to evaluate the resistance of CP to bacterial and fungal contamination and to assess the efficacy of preservatives in plant extracts in the formulation (USP-NF 2024; Vu N, Nguyen K, and Kupiec TC 2014). Briefly, to perform this test, standard bacterial (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli) and fungal (Candida albicans, Aspergillus niger) strains were used (see Table 4). Suitable media for growing the microorganisms were prepared (e.g., Agar for bacteria, Sabouraud Dextrose Broth and Agar for fungi).

Inoculum Preparation: Suspensions of the microorganisms were prepared at a specified concentration (e.g., 10⁵ to 10⁶ CFU/mL).

Incubation: The plates were incubated under appropriate conditions (e.g., 30-35°C for bacteria, 20-25°C for fungi).

Sampling and Enumeration: Sampling and microbial enumeration were conducted at specified time intervals (e.g., 0 hours, 24 hours, 7 days, 14 days, 28 days), according to OECD Guidelines.

2.8. Galenic Formulation of Cupidon of Pahouins

The comparative method of determining the commercial dosage form (CDF) is empirical. It is based on a simple comparison with the existing medicine prescribed in the clinic, with the same effects as possible and the same mechanisms of action. This method is always recommended when pharmacological parameters are available such as the MIC. The method is also known as the equivalent ratio (Eto 2019). In this study, gentamycin (GN) is used as the clinically prescribed reference drug.

Where CDF_cp represents the commercial dosage form of CP (combination) and CDF_gn the commercial dosage form of gentamycin (180 mg/2mL). MIC_cp and MIC_gn represent the mean of the lowest concentrations of CP and gentamycin, respectively, that completely inhibited the growth of a specific microbe.

2.9. Clinical Study

2.9.1. Study Design

This is a multicenter observational cohort study conducted by Etobiotech. 451 patients were included between June 2015 and April 2022 (Cameroon). All patients gave their informed consent to participate in the observational study in the real situation of prescription of traditional herbal product external used and received CP (Table 5). CP was provided by Laboratoires TBC (Paris, France).

2.9.2. Patients and Procedures

Patients were eligible after a medical examination to confirm their pathology. In the clinical study, the effect of CP administration on different pathologies was evaluated. All patients received vials containing CP according to their pathology and the duration of treatment (Figure 1). The method of use was explained for each patient according to the disease (Table 5).

2.9.3. Clinical Study Endpoints

A clinical examination was realized at the end of the treatment to assess the efficacy of the treatment with CP.

2.10. Statistical Analysis

Data are presented as means ± standard deviations. Statistical analysis was performed using GraphPad Prism version 8.0 for Windows (GraphPad Software, San Diego, California, USA). A multiple t-test was used to compare MIC values, with statistical significance defined as p < 0.05. Rootlet lengths as a function of concentration are presented as mean values ± SE (standard error) for separate experiments using n=15 seeds. Graphs of concentration-response curves were generated using non-linear regression and fitted to the Hill equation through an iterative least-squares method to provide estimates of the maximum effective concentration IC₅₀ (the negative logarithm of the agonist concentration producing 50% of maximum inhibition). For the comparison of the different effects against the control, one-way analysis of variance (ANOVA) was use followed by multiple comparison t-tests. Statistical significance was defined as p < 0.05.

3. Results

3.1. Antimicrobial Activity of Aqueous Extracts

Aqueous extracts of A. cordifolia, M. cecropioides, M. arboreus and A. klaineanum were first tested individually to assess their antimicrobial activity. This screening is essential to select the most active extracts. The results (Table 6) show that the aqueous extract of A. cordifolia is the most active (activity against 29 strains), followed by M. cecropioides (activity against 21 strains), then the extract of A. klaineanum (activity against 14 strains), and finally M. arboreus is the least active extract (activity against 7 strains).

Based on the results in Table 6, the functional ratio (Fr) of the aqueous plant extracts was determined. It shows that the best pre-formulation of PHF is a combination of A. cordifolia and M. cecropioides (CP).

Following the results found, the most active aqueous extracts (A. cordifolia and M. cecropioides) were chosen to be tested in combination against 32 microorganisms. The different results of their antimicrobial activity are shown in Table 7. Remarkably, A. cordifolia was active against most of the tested strains (29 among 32), including Gram-negative ones often multidrug-resistant (Table 8), while M. cecropioides was active against 21 strains. Both individual extracts were active against all the studied staphylococci and streptococci strains, as well as against Corynebacterium striatum, Citrobacter freundii, Proteus mirabilis and Pseudomonas aeruginosa. The activity of both extracts against the same strain was not significantly different. Moreover, M. cecropioides was active against one of the two Candida albicans strains tested (ATCC 10231), and A. cordifolia extract was active against Enterococcus faecalis, Enterobacter aerogenes, Klebsiella pneumoniae, Salmonella sp. and three Escherichia coli strains (ATCC 25922, T20A1 and 8138). The lowest MIC obtained after using A. cordifolia extract was 0.25 ± 0.07 mg/mL against S. epidermidis T46A1, and 0.30 ± 0.00 mg/mL was the lowest MIC obtained by M. cecropioides against different staphylococci strains.

After combining extracts of A. cordifolia and M. cecropioides, some improvement in the antimicrobial activity was observed. The concentration of each plant extract in the mixture varies depending on the tested germ. Synergistic effects were observed on half of the strains, and antagonistic effects were absent. Moreover, this combination was active against 30 of the 32 strains tested.

3.2. Effect of New CP on Inhibition of Protein Synthesis

3.2.1. Comparison with Antineoplastic Medicine

The antineoplastic effect of CP was compared with that of medically prescribed anticancer drugs that inhibit protein synthesis. The IC₅₀ of the new CP was 2.92 ±1.26 mg/mL, while that of methotrexate (MTX) was 42.42± 8.70 ng/mL, etoposide (ETP) 7.11 ± 0.58 µg/mL, berberine (BRB) 38.33 ± 3.21 µg/mL and doxorubicin (DOXO) 85.75 ± 7.74 µg/mL (Figure 2).

3.2.2. Comparison with Antibiotic Medicines

The effect of CP on the inhibition of LS seed germination was compared with that of different medically prescribed antibiotics. In this study, only antibiotics inhibiting proteins synthesis were used. The IC₅₀ of the new CP was 2.92 ± 1.26 mg/mL, while that of doxycycline (DOC) was 2.41 ± 1.78 µg/mL, amikacin (AMK) 128.80 ± 4.25 µg/mL, cotrimoxazole (TMP/SMX) 2.75 ± 1.03 µg/mL, monensin (MON) 53.28 ± 4.25 µg/mL, and erythromycin (ERY) 78.79 ± 3.75 µg/mL (Figure 3).

3.3. Transdermal Passage of CP

The objective of the transdermal passage study was to test the penetration of CP through the rat skin barrier. Crossing the rat skin barrier suggests that CP can cross human skin, and the vaginal and oral mucosa. The result shows that CP does not cross the rat skin barrier after 24 hours of exposure to CP (Figure 4).

3.4. Toxicological Studies

3.4.1. Primary Skin Irritation Test

The primary irritation skin test is used to assess the irritant potential of products for external use or intimate care. Contact of the CP solution to be tested (20 mg/mL) with the epidermis of healthy volunteers for 4 hours did not cause skin irritation. As the skin irritant power of 0.05 is extremely low, it can be concluded that CP has no irritating effect on the skin.

3.4.2. Eye Irritation Test

The potential irritant effect of a product for external use or an intimate care product for women was determined in vitro on a cell culture of corneal fibroblasts. Under the experimental conditions described above, the results obtained show that the ocular irritation index is 6, i.e. extremely low, leading to the conclusion that CP is very slightly irritating to the ocular mucosa.

3.4.3. Challenge Test

In the challenge test, after 14 days of contact with the CP solution, no revivable microorganisms were detected, demonstrating the absence of subsequent proliferation. This shows the excellent protection of CP against the five microbial species used. Table 9 shows the results of the challenge test, representing the number of microorganisms per millilitre.

3.5. Clinical Study

The clinical benefits of CP were evaluated on different bacterial and fungal infections such as dermatosis, pruritus, skin rashes, ringworm, fungus, achromic eczema, smallpox, varicella, conjunctivitis, acne, vaginitis, vaginosis, Vulvitis, intimate comfort and firming of the vaginal mucosa, oral infections, genital herpes, genital candidiasis, diaper rash in babies, otitis, athlete's foot, epiglottitis, cervical cancer with bleeding, rectal infection, beard button and postpartum toilet.

Different concentrations were used according to the ailments. No adverse effects were reported. The efficacy of CP was demonstrated against all the ailments tested (Table 11 and Table 12).

4. Discussion

In the present study, the antimicrobial activity of aqueous extracts of A. cordifolia and M. cecropioides was evaluated and showed strong antimicrobial potential, either alone or in combination (CP). To our knowledge, this is the first study demonstrating the enhanced antimicrobial activity resulting from the combination of A. cordifolia and M. cecropioides. Several antimicrobial studies have been carried out on A. cordifolia and M. cecropioides under different experimental conditions or using different plant parts (Ajayi et al. 2020; Fomogne-Fodjo et al. 2014). Some studies were conducted using organic extracts rather than aqueous extracts. It should be noted that organic solvents, apart from ethanol, are prohibited in the development of herbal medicines and plant-based food supplements. Moreover, the molecules extracted and isolated with organic solvents are not always the same as those obtained with aqueous extractions. They are often toxic or have no pharmacological effects. Most medicinal plants used traditionally are prepared as aqueous decoctions (Chan 2003). It, therefore, seems unwise to justify traditional uses with organic extracts that are not used by traditional healers.

Regarding the antimicrobial effects of A. cordifolia leaves, Agboke and his collaborators investigated the antibacterial activity of aqueous and ethanolic extracts obtained by maceration of Nigerian A. cordifolia leaves. MIC values were obtained using the broth dilution method and ranged from 1.95 to 15.62 mg/mL (Agboke et al. 2020). These values are much higher than those obtained in our study. In another study, the aqueous extract of A. cordifolia leaves from Cameroon was obtained by maceration and tested on two strains of E. coli using the broth microdilution method. The MIC values obtained were 1.50 and 0.75 mg/mL (Djimeli et al. 2017), while those obtained in our study were 1.20 mg/mL. Moreover, aqueous and ethanolic extracts of A. cordifolia leaves from Ghana were tested on S. aureus, E. coli, P. aeruginosa, and C. albicans. Although both extracts were active against all the tested germs, the MIC values (ranging from 2.50 to 10.00 mg/mL) were higher than those obtained in our study, except for C. albicans, which was resistant to the tested extract (Agyare et al. 2014). This difference in results can be explained by the method used to obtain the leaf extracts. Some researchers pick fresh leaves and dry them before use. Others prefer to use young leaves for their studies. In the Pahouin tradition, only dead leaves collected from the ground are used to prepare CP. We found that the UV spectra of extracts from dead leaves differed from those of young or fresh leaves. The same observations on the different methods of obtaining extracts also apply to M. cecropioides (personal communication).

A study was conducted to evaluate the antibacterial activity of M. cecropioides stem bark extracts. MIC values were determined using the broth microdilution method. The hydroalcoholic extract was active against E. coli with an MIC value of 0.31 mg/mL, while the methanol extract was active against K. pneumoniae with an MIC of 1.25 mg/mL (Mabeku, Roger, and Louis 2011). In another study carried out by Fomogne-Fodjo et al. 2014, the antibacterial activity of the methanol/dichloromethane extract of M. cecropioides leaves from Cameroon was evaluated. The respective MIC values obtained for S. aureus and K. pneumoniae were 4.00 and 1.00 mg/mL (Fomogne-Fodjo et al. 2014). To our knowledge, this is the first study conducted on the terminal buds of M. cecropioides. The results show that the extract was not active against E. coli and K. pneumoniae, but it was highly active against S. aureus with MIC values ranging from 0.30 to 0.60 mg/mL.

The novelty of this work lies in the combination of aqueous extracts of A. cordifolia and M. cecropioides to create an antimicrobial phytodrug from an ancestral traditional recipe. The new formulation proposed (CP) is more effective as an antimicrobial than the individual plants used alone or even than the traditional recipe. This work confirms the previous research carried out by our laboratory team, which shows that the rational combination of plant extracts using ACP, along with the functional approach, enables the development of the most effective products with minimal side effects (Boutahiri et al. 2022; 2021; Mamadou et al. 2011).

The chemical composition of A. cordifolia and M. cecropioides has already been studied (Awwad et al. 2021; Sinan et al. 2021). The results revealed the presence of apigenin and derivative compounds. Apigenin is a flavonoid with demonstrated antimicrobial activity. It has been found to promote antibacterial activity by regulating the production of superoxide anions and nitric oxide (Kim, Woo, and Lee 2020). The antifungal activity of this compound is due to its ability to induce fungal apoptosis through the disruption of calcium homeostasis (Lee, Woo, and Lee 2019). In addition, apigenin possesses anticancer potential by reducing Akt (protein kinase B) phosphorylation, which promotes cell growth inhibition (Harrison et al. 2014). Other chemical compounds identified in extracts of A. cordifolia and M. cecropioides include gallic acid, ellagic acid, caffeic acid, vanillic acid, shikimic acid, rutin, quercetin, myricetin, kaempferol, luteolin, and naringenin (Awwad et al. 2021; Sinan et al. 2021). Several of these compounds have been studied for their biological activities, revealing significant antimicrobial and anticancer potential (Azeem et al. 2023; Bangar et al. 2023; Choubey et al. 2015; Matejczyk et al. 2018).

Furthermore, it has been reported in several studies that combining chemical compounds from plants can generate more potent activity (Lewandowska et al. 2014; Vaou et al. 2022). Additionally, there are interactions between compounds within crude plant extracts, enhancing their effectiveness (Rasoanaivo et al. 2011). These findings could confirm the synergistic effects observed in this study after combining aqueous extracts of A. cordifolia and M. cecropioides. This would validate the results of the clinical study, which demonstrated the efficacy of CP in treating several infectious diseases.

5. Conclusions

In conclusion, this study confirms the efficacy of CP in treating or preventing bacterial and fungal infections. It demonstrates that ACP can be used to enhance the efficacy of traditional treatments.