LC-MS Phytochemical Profiles of Phenolic Compounds and Antimicrobial, Antioxidant, and Antiplasmodial Activities of Ethanol Extracts of Pycnostachys erici-rosenii R.E.Fr and Leucas martinicensis (Jack.) R.Br (Lamiaceae) ()
1. Introduction
In African countries, plants are the main medicinal resources for public health care. Among these plants, is the Lamiceae (Labiatae) family, from the Latin (Labia: lip) which includes about 258 genera and 7000 species spread especially from the Mediterranean Basin to Central Asia. The Lamiaceae are herbaceous, annual or perennial and aromatic plants. This family is an important source of essential oils for aromatherapy, perfumery and the cosmetics industry [1] [2] .
In the traditional African pharmacopoeia, plants of the Lamiaceae family are used as diuretics, anti-syphilitics, anti-diarrhoea, healing, antiseptic and in the treatment of many ailments such as intestinal problems or meteorism (bloating of the belly due to gas) [1] . P. erici-rosenii R.E.Fr and L. martinicensis are among the plants of the Lamiaceae family, which are the subject of the present study.
P. erici-rosenii and L. martinicensis are used in traditional medicine of Democratic Republic of Congo (DRC) to treat diseases of bacterial origin and those due to oxidatives stress, but also many others such as hypogastritis, cough, malaria, colibacillosis, asthma, tuberculosis, panaria, boils, food poisoning, snake bites, nervous disorders, liver disorders, diarrhoea, etc. P. erici-rosenii and L. martinicensis are frequently used in combination with other plants like Crassocephalum montuosum (Asteraceae), Cinchona ledgeriana (Rubiaceae), Bridelia bridelifolia (Phyllanthaceae), Centella asiatica (Apiaceae), Psidium guajava (Myrtaceae), Leonotis naepetifolia (Lamiaceae), Ageratum conyzoides (Asteraceae), Phyllanthus nuriri (Phyllanthaceae), Solanum nigrum (Solanaceae), etc.
Actually, the phenomenon of bacterial resistance concerns all families of antibiotics. Bacterial species develop different mechanisms according to their initial sensitivity and their capacity to express the various resistances. This bacterial resistance is increasing at an alarming rate and is responsible for more than 700,000 deaths each year worldwide [3] and if nothing is done, by 2050, it will cause 10 million deaths annually [4] .
Regarding malaria, the number of malaria cases worldwide was estimated at 247 million in 2021 in 84 malaria-endemic countries, up from 245 million in 2020. Most of this increase occurs in the WHO African Region Countries. In 2015, the baseline year for the Global Technical Strategy for Malaria 2016-2030, there were an estimated 230 million malaria cases. The incidence of malaria cases fell from 82 in 2000 to 57 in 2019, before rising to 59 in 2020. The incidence of cases did not change between 2020 and 2021. The increase in 2020 was associated with the disruption of services during the Coronavirus Disease 2019 (COVID-19) pandemic. About 96% of global malaria deaths occurred in 29 countries, and four countries accounted for just over half of all global malaria deaths in 2021: Nigeria (31%), the Democratic Republic of the Congo (13%), Niger (4%) and the United Republic of Tanzania (4%) [5] .
Thus, in view of the different uses of P. erici-rosenii and L. martinicensis in traditional medicine, the ethanol extracts from aerial parts of these two species were scripted for antimicrobial, antioxidant and antiplasmodial agents.
The investigation of the chemical composition of the aerial parts of P. erici-rosenii and L. martinicensis for the antimicrobial, antioxidant, and antiplasmodial properties of the ethanol extracts is useful in light of recent epidemiological studies. Those studies have suggested that certain plant extracts containing polyphenols may be helpful in preventing the development or slowing infectious diseases. Therefore, we investigated these two plants, which have not been investigated before for these purposes.
2. Materials and Methods
2.1. Plant Material
The aerial parts of P. erici-rosenii (synonym: Coleus erici-rosenii (R.E. Fr.) A.J. Paton) and L. martinicensis (Synonym: Leonotis martinicensis (Jacq.) J.C. Manning & Goldblatt) were collected in November 2020 in the territory of Kabare, South Kivu Province, in the East of the Democratic Republic of Congo. After the identification of plants in the Herbarium of the “Centre de Recherche en Sciences Naturelles de Lwiro (CRSN/Lwiro)”, the aerial parts were collected, dried at room temperature and then crushed to obtain the plant powders. The powders were kept in sealed bags at room temperature for further analysis (Figure 1).
2.2. Preparation of Extracts
The ethanol extracts of P. erici-rosenii and L. martinicensis were prepared by
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Figure 1. Photos of aerial parts of P. erici-rosenii (a) and L. martinicensis (b) (Credit: AfricanPlant Data Base).
maceration using ethanol 96% v/v for 48 hours. After filtration, the different filtrates were concentrated by evaporation using rotavapor BUCHI Switzerland Heating Bath B-100 under reduced pressure (Vacuum Pump V-100). The extracts were carefully preserved for further phytochemical analysis and for antimicrobial, antioxidant and antiplasmodial tests.
2.3. Total Phenolic Content
The Total Phenolic Contents of ethanol extracts of P. erici-rosenii and L. Martinicensis were measured using the Folin-Ciocalteu method with a visible wavelength of 765 nm. A calibration curve with various concentrations of Gallic acid (1 mg/100mL; 0.5 mg/100mL; 0.25 mg/100mL; 0.125 mg/mL, and 0.0625 mg/mL) was established and had an equation y = 2.364x + 0.0649 with R2 = 0.9965. Wells of 3 mL was used, containing 2.35 mL distilled water, 0.05 mL of the extract, 0.15 mL Folin-Ciocalteu reagent, and 0.45 mL Na2CO3 (7.5%). The samples were left for 2 hours in darkness before measuring the absorbance on the UV-Vis spectrometer at 765 nm to determine total polyphenols expressed in mg Gallic acid equivalents (GAE)/100g dry weight (DW) through the calibration curve. The analysis was completed three times and done using the SPECTROstar® Nano microplate spectrophotometer (from BMG Labtech, Ortenberg, Baden-Württemberg, Germany) [6] [7] .
2.4. Total Flavonoid Content
The total flavonoid content was measured using the Plant Flavonoids Colorimetric Assay Kit, based on the reaction between flavonoids and aluminum ions, resulting in a red complex. The concentration of flavonoids in the sample was determined by recording absorbance at 510 nm. We prepared a standard curve following the instructions in the kit, diluting a 1 mg/mL standard solution with double-distilled water for concentrations of 150, 120, 100, 80, 60 and 20 µg/mL. The calibration curve was established and had an equation y = 0.0056x − 0.0126 with R2 = 0.9936. After that, samples have been prepared and their absorbances read against double-distilled water as a blank. The result obtained was expressed as mg Quercetin equivalent (QE)/100g DW and a spectrophotometer SPECTROstar® Nano (BMG Labtech, Ortenberg, Baden-Württemberg, Germany) was used [8] .
2.5. Total Antioxidant Capacity
2.5.1. FRAP (Ferric Reducing Antioxidant Power) Assay
The FRAP method relies on the change in the color of a complex with Fe+3 ion of the 2,4,6-tri(2-pyridyl)-1,3,5-triazine (TPTZ) radical by the reduction of the ferric ion to the ferrous ion (Fe+2) in this complex. To 2.5 mL of 10 mM TPTZ solution in 40 mM HCl, 2.5 mL 20 mM ferric chloride solution and 25 mL acetate buffer (pH = 3.6) were added. This mixture is the FRAP reagent. To 4.0 mL extract up to 0.8 mL water and 6.0 mL FRAP reagent were added. The blank solution was prepared in the same manner using water instead of a sample. Trolox was used as a reference. The color change was correlated with the antioxidant capacity by measuring absorbance at 450 nm. Using a calibration curve (R2 = 0.992), the result was converted to µM Trolox equivalents/100mL extract [9] [10] [11] .
2.5.2. DPPH Radical Scavenging Activity
DPPH (2,2-diphenyl-picrylhydrazil) radical scavenging assay is a spectrophotometric quantitative method based upon the reaction between the antioxidant compounds of the extract and DPPH● reagent in an alcoholic solution. 2 mL (at different concentrations) of the extract was added to 2 mL of a 0.1 g∙L−1 DPPH● methanol solution. After 30 minutes in a thermostatic bath at 40˚C, the variation of the absorbance was measured at 517 nm (R2 = 0.997). The percent of DPPH● scavenging ability was calculated as follows: DPPH scavenging ability % = (A control − A sample/A control) × 100, where A control is the absorbance of DPPH● radical + methanol (containing all reagents except the sample) and A sample is the absorbance of DPPH radical + sample extract. The DPPH radical scavenging activity of the extract was expressed as IC50 (μg/mL) [9] [10] [11] .
2.6. Antimicrobial Properties Assay
The antimicrobial activities of ethanol extracts of P. erici-rosenii and L. martinicensis were evaluated on seven bacterial strains: Staphylococcus aureus 29213, Listeria monocytogenes ATCC 13932, Enterococcus faecalis ATCC25922, Escherichia coli ATCC51299, Bacillus cereus ACTT 11778, Pseudomonas aeruginosa QC76110 and Candida albicans ATCC 10231. The bacteria were replicated in 45 mL sterile nutrient broth (Oxoid Ltd., Basingstoke, Hampshire, UK) at 37˚C for 24 h, and C. albicans was grown at 30˚C. Purity of the inoculum was confirmed by plating on appropriate media and viewing under a microscope (Optika microscope, B252, M.A.D; Apparecchiature Scientifiche, Milan, Italy). Plates were incubated for 24 h at 30˚C for C. albicans, and 37˚C for other strains. Stock cultures were kept at refrigeration temperatures to obtain freshly cultured suspensions, which were later used to assess antimicrobial activity via the disk-diffusion method. Several colonies from standard cultures cultivated on Mueller-Hinton agar (Oxoid Ltd., Basingstoke, Hampshire, UK) were transferred in saline solution (8.5 g/L) and then adjusted to match a McFarland 0.5 standard, before preparing a bacterial suspension of 1.5 × 106 CFU/mL. 15 mL Mueller-Hinton agar was poured into Petri dishes and 100 µL culture suspension was distributed over the plates using a Drigalski spatula [12] [13] .
To create an inoculum, a 0.5 McFarland microbial suspension was diluted 1:150 to achieve 106 CFU/mL. This solution underwent a further dilution of 1:2, resulting in a final inoculum of 5 × 105 CFU/mL. Six test tubes received 0.1 mL of varying concentrations of the tested compound, ranging from 8 to 0.25 mg/mL; each was also given 0.4 mL of Mueller-Hinton broth with 2% - 5% lysed horse blood (for Streptococcus spp.), and 0.5 mL of the microorganism suspension at ~0.5 × 105 CFU/mL. After incubating at 37˚C for 24 hours, the minimum inhibitory concentration (MIC) was recorded (MIC is the lowest concentration without visible growth). In a control tube containing ethanol instead of a tested compound; 0.5 mL of microbial suspension and 0.4 mL Mueller-Hinton broth were added [13] [14] .
To determine the minimum bactericidal concentration (MBC), 1 µL from tubes with no growth was taken and applied to Columbia agar with 5% blood. The minimum fungicidal concentration (MFC) was assessed by inoculating a Sabouraud with chloramphenicol medium both incubated at 37˚C for 24 hours afterwards [12] [13] [14] .
2.7. Antiplasmodial Properties Assay
2.7.1. Culture Protocol for the Plasmodium falciparum Strain
The culture technique used was that of Trager and Jensen [15] . The chloroquine-sensitive Plasmodium falciparum strain Pf3D7 was grown in fresh human red blood cells, group O Rhesus positive at 4% haematocrit in complete RPMI medium [500 mL RPMI 1640 (Gibco, UK) supplemented with 25 mM HEPES (Gibco, UK), 0.50% Albumax I (Gibco, USA), 1X hypoxanthine (Gibco, USA) and 20 µg/mL gentamicin (Gibco, China)] and incubated at 37˚C in a humidified incubator consisting of 92% N2, 5% CO2 and 3% O2. The medium was replaced daily with a complete RPMI medium to facilitate the growth of parasites in culture. Subsequently, fine blood smears were taken and stained with Giemsa and then observed under a microscope at 100× objective with immersion oil in order to follow all the steps of the cell cycle and to evaluate the parasitemia.
2.7.2. Synchronisation of Cultures
Before each test for antiplasmodial activity, parasitic cultures containing the majority of ring stages (>80%) were synchronised to the same evolutionary stage (ring stage) by treatment with 5% (w/v) sorbitol for 10 min according to the protocol of [16] . The use of synchronised cultures at the same evolutionary stage compared to mixed stage cultures allows the evaluation of the plant extract’s effect on all three evolutionary phases (rings, trophozoite, schizont) of the 48 hours life cycle of P. falciparum [17] [18] .
2.7.3. Preparation of Plant Extracts and References (Artemisinin and Chloroquine)
Stock solutions were prepared in 10% DMSO at concentrations of 10 mg/mL and 1 mM for each plant extract, and artemisinin, and chloroquine references respectively. For this purpose, 10 mg of extract from each plant was dissolved in 100 µL of dimethylsulphoxide (DMSO) followed by vortexing for at least 15 minutes to get a homogeneous solution. After dissolution, the volume was made up to 1 mL to obtain a 10 mg/mL solution. The solution obtained was filtered and the filtrate was used for the anti-plasmodial activity tests [17] [18] .
Intermediate concentrations of each sample were achieved by adding 10 µL of stock solution of each plant extract (10 mg/mL) and 20 µL of stock solution of each positive control (100 µM) into 190 µL and 180 µL of incomplete RPMI1640 medium respectively in a 96-well microplate followed by a geometric dilution of order 5. Intermediate concentrations ranged from 8 to 5000 μg/mL for the extracts of each plant and from 0.016 to 10 µM for the positive controls [17] [18] .
2.7.4. In Vitro Test for P. falciparum Growth Inhibition Based on SYBR Green Fluorescence
The anti-plasmodial activity assay of the extracts from each plant and the positive controls were performed in 96-well flat-bottom microplates based on the fluorescence of SYBR green I [17] . The ability of SYBR green I to produce strong fluorescence only in the presence of DNA is the basis for its use in assessing cell proliferation. The lack of cell nuclei within human red blood cells in which the parasite proliferates allows specific monitoring of plasmodial growth using SYBR green I. Experimentally, 90 µL of the parasite suspension synchronised to the ring stage at 2% parasitaemia and 1% haematocrit was incubated with 10 µL of different concentrations of pre-diluted plant extracts, artemisinin and chloroquine. The plates were then incubated at 37˚C in a humidified incubator consisting of 92% N2, 5% CO2 and 3% O2 for 72 h. The final concentrations in the test plates varied from 0.8 to 500 μg/mL (DMSO < 1%) for the extracts of each plant and from 0.0016 to 1 µM (DMSO 0.1%) for the positive controls in the final volume of 100 µL. The experiments were performed in duplicate [17] [18] .
After 72 hours of incubation, 100 μL of SYBR Green I buffer 6 μL of 10,000 × SYBR Green I (Invitrogen) + 600 µL of red cell lysis buffer [Tris (25 mM; pH 7.5)] + 360 µL of EDTA (7.5 mM) + 19.2 µL of parasite lysis solution {saponin (0.012%; wt/vol)} and 28.8 µL of Triton X-100 (0.08%; vol/vol)]} were gently added to each well, followed by incubation at 37˚C for 1 hour in the dark. After 1 hour of incubation, fluorescence was measured using the Infinite M200 (Tecan) Microplate reader at an excitation and emission wavelength of 485 and 538 nm, respectively. The fluorescence values obtained were used to calculate the percent inhibition using Microsoft Excel. Subsequently, the 50% inhibitory concentrations (IC50) were determined from the concentration-response curves obtained by plotting the logarithm of the concentration versus the percentage inhibition using GraphPad Prism software [17] [18] .
2.8. HPLC-DAD-MS-ESI+ Analysis
2.8.1. Chemical Reagents and Materials
Acetonitrile, HPLC-gradient, was provided by Merck (Germany) and water was purified with a Direct-Q UV system by Millipore (USA). The pure standard of gallic, chlorogenic acid (purity 99% HPLC) and rutin (purity 99% HPLC) was purchased from Sigma (USA) [19] [20] .
2.8.2. Chromatographic Condition
To evaluate polyphenolic compounds, the HP-1200 liquid chromatography was used, fitted with a quaternary pump, autosampler, DAD detector, and MS-6110 single quadrupole API-electrospray detector (Agilent Technologies, Santa Clara, CA, USA). Detection was done in the positive ionization mode. Different fragments or voltages were applied in the range of 50 - 100 V. Separation was achieved with an Eclipse XDB-C18 (5 µm; 4.5 × 150 mm i.d.) column (Agilent) and a mobile phase consisting of 0.1% acetic acid in water (A) and 0.1% acetic acid in acetonitrile (B). A multistep linear gradient was employed for elution from 5% to 90% B over 20 minutes, followed by a 4-minute hold at 90% B and a 6-minute return to 5% B keeping the flow rate at 0.5 mL/min and temperature at 25˚C ± 0.5˚C. Phenolic compounds in the extracts of P. erici-rosenii and L. martinicensis were identified by comparing retention times, UV visible spectra and mass spectra to the database. Data acquisition was conducted using the scan mode on a mass spectrometer set at 3500˚C, 7 L/min nitrogen flow, nebulizer pressure 35 psi, capillary voltage 3000 V, fragment or 100 V and m/z 120 - 1200 chromatograms recorded at λ = 280 and 340 nm were processed through Agilent ChemStation software [19] [20] .
3. Results
3.1. Phytochemicals Content of P. erici-rosenii and L. martinicensis
Table 1 below presents the results of the total phenolic compounds content, and the total flavonoid content of the aerial parts of P. erici-rosenii and L. martinicensis.
3.2. Antioxidant Activities Assays of P. erici-rosenii and L. martinicensis
The in vitro antioxidant capacities of ethanol extracts of aerial parts of P. erici-rosenii and L. martinicensis were evaluated by two different methods: 2,2-diphenyl-picrylhydrazil (DPPH•) scavenging assay and ferric-reducing antioxidant power (FRAP) (Table 2).
3.3. Antimicrobial Activities of P. erici-rosenii and L. martinicensis
Table 3 presents the antimicrobial activities of the ethanol extracts of aerial parts of P. erici-rosenii and L. martinicensis. Two methods were used to assess these activities: the disk diffusion method, indicated by the diameter of the inhibition zone, and the dilution method, indicated by the MIC and the MBC/MFC.
3.4. Antiplasmodial Properties Assay
Table 4 presents the results obtained for antiplasmodial properties assay of the
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Table 1. Phytochemicals Content of the aerial parts of P. erici-rosenii and L. martinicensis.
SD: Standard Deviation, QE : Quercetin Equivalents, GAE: Gallic Acid Equivalents.
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Table 2. Antioxidant activities assays of P. erici-rosenii and L. martinicensis.
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Table 3. The diameter of inhibition growth zone, the minimum inhibitory concentration, and minimum bactericidal concentration of P. erici-rosenii and L. martinicensis.
MIC: Minimum Inhibitory Concentration; MBC: Minimum Bactericidal Concentration; MFC: Minimum Fungicidal Concentration.
ethanol extracts of aerial parts of P. erici-rosenii and L. martinicensis on Pf 3D7 (CQ-sensitive) (Tested concentration range: 0.78125 - 100 µg/mL; Assay type: SYBR Green I-based growth inhibition assay; Activity: IC50 ≤ 20 µg/mL).
3.5. HPLC-DAD-ESI+ Analysis
Figures 2-5 and Table 5 present the results obtained for the HPLC-DAD-ESI+ analysis of phenolic compounds found in the ethanol extracts of aerial parts of P. erici-rosenii and L. martinicensis.
4. Discussion
The Total Phenolic Contents of the aerial parts of P. erici-rosenii and L. martinicensis were 209.0 ± 16.1 mg GAE/100g DW for P. erici-rosenii and 175.3 ± 1.6 mg GAE/100g DW (Table 1). For the Total Flavonoids Contents of the aerial parts of P. erici-rosenii and L. martinicensis, the values were 33.4 ± 1.8 mg QE/100g DW and 39.6 ± 0.6 mg QE/100g DW respectively (Table 1). When comparing these obtained values to other values from some other Lamiaceae species (169 mg GAE/g DW of aqueous extract from Salvia sclarea, 170 mg GAE/g
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Table 4. Antiplasmodial test results of ethanol extracts of P. erici-rosenii and L. martinicensis.
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Figure 2. HPLC-DAD chromatogram of ethanol extract of P. erici-rosenii at 280 nm (1: 2-hydroxybenzoic acid; 2: gallic acid; 3: 4-hydroxybenzoic acid; 4: kaempferol-rutinoside; 5: vanilic acid; 9: quercetin-glucoside; 10: coumaroylquinic acid)
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Figure 3. HPLC-DAD chromatogram of ethanol extract of P. erici-rosenii at 340 nm (1: 2-Hydroxybenzoic acid; 2: gallic acid; 4: kaempferol-rutinoside; 5: vanilic acid; 9: quercetin-glucoside; 11: quercetin)
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Figure 4. HPLC-DAD chromatogram of ethanol extract of L. martinicensis at 280 nm (1: 2-hydroxybenzoic acid; 3: 4-hydroxybenzoic acid; 5: vanilic acid; 7: ferulic acid; 8: feruloylquinic acid; 10: coumaroylquinic acid).
DW aqueous extract from Salvia raeseri ssp; 138 mg GAE/g DW of aqueous extract from Mentha pulegium), it can be noticed that ethanol extracts of these two species from the Eastern Democratic Republic of Congo were rich in phenolics and flavonoids content. This would explain the high antioxidant capacity
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Figure 5. HPLC-DAD chromatogram of ethanol extract of L. martinicensis at 340 nm (3: 4-hydroxybenzoic acid; 6: p-coumaric acid; 7: ferulic acid; 8: feruloylquinic acid; 10: coumaroylquinic acid; 11: quercetin).
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Table 5. Quantification and identification of phenolic compounds (µg/mL dry vegetal product) in the ethanol extracts of aerial parts of P. erici-rosenii and L. martinicensis by HPLC-DAD-MS-ESI+ analysis.
found in the ethanol extracts of these two species (Table 2). According to Boizot and Charpentier [21] , polyphenols are secondary metabolites characterised by the presence of an aromatic ring with free hydroxyl groups or hydroxyl groups linked to a carbohydrate. Phenolic compounds are a class of antioxidant agents that act as free radical scavengers and are responsible for antioxidant activity in medicinal plants. They are present in all parts of higher plants (roots, stems, leaves, flowers, pollen, fruits, seeds and wood) and are involved in non-enzymatic defense systems against free radicals. Flavonoids have biological and pharmaceutical properties such as anti-allergic [22] , anti-inflammatory [23] , antihypertensive [24] , antifungal [25] , antiviral [26] , antiulcer [22] , anti-free radical [27] , and they are also recognised as having antimalarial activity [28] .
For investigating the antioxidant capacity of the extracts, two methods were used, DPPH and FRAP (Ferric Reducing Antioxidant Power) methods, since more than one method of antioxidant capacity assessment needs to be performed to take into account the various modes of action of antioxidants [29] . Both DPPH and FRAP antioxidant assays showed that all the tested extracts possess antioxidant activities. For the DPPH assessment, the results showed the IC50 of 56.17 μg/mL and 78.43 μg/mL for the ethanol extract of the aerial parts of P. erici-rosenii and L. martinicensis respectively, while those values of IC50 were of 112.03 μM TE/ 100mL and 90.46 μM TE/100mL for FRAP assessment. Similar IC50 values have been reported in other studies for Lamiaceae species. Previous studies on extracts of different plant species show that the Lamiaceae family has the strongest antioxidant activities [30] [31] [32] . This would explain the encouraging results for the ethanol extracts of aerial parts of P. erici-rosenii and L. martinicensis in this study (Table 2).
In this study, the greatest inhibition zone diameters (12.5 mm, 12.2 mm, and 11.9 mm) were obtained for the ethanol extracts of P. erici-rosenii on the L. monocytogenes ATCC 13932, S. aureus 29213, and E. faecalis ATCC25922 strains respectively. Inhibition zone diameters for other strains were 10.0 mm-10.7 mm for the ethanol extract of P. erici-rosenii. Concerning the L. martinicensis ethanol extract, the greatest inhibition zone diameters (10.6 mm) were obtained on the L. monocytogenes ATCC 13932 strains and 10.0 mm for other strains.
According to the extract activity classification scale established by Kuete et al. [33] based on MICs (Significant: MIC ˂ 100 μg/mL; Moderate: 100 μg/mL ˂ MIC ≤ 625 μg/mL and Negligible: MIC ˃ 625 μg/mL); it can be noted that: the ethanol extract of P. erici-rosenii showed moderate activity on all trains with 0.125 mg/mL on L. monocytogenes ATCC 13932, E. faecalis ATCC25922, and C. albicans ATCC 10231 as the best activity. The same observations have been made for ethanol extract of L. martinicensis with 0.250 mg/mL on S. aureus 29213 and E. faecalis ATCC25922 as the best activity. Overall, the results showed that the ethanol extract of the aerial parts of P. erici-rosenii and L. martinicensis weren’t bactericides on E. coli ATCC51299, B. cereus ACTT 11778, C. albicans ATCC 10231 because no MBC/MFC was observed on these strains (Table 3).
These results are in agreement with the work carried out by Sarac and Ugur [34] which showed that several plants belonging to the Lamiaceae family were found to be potentially antimicrobial. Among those plants, Phlomis fruticosa, Salvia verbenaca, Salvia argentea, Teucrium chamaedrys ssp. lydium, Teucrium divaricatum ssp. villosum, Teucrium polium, Stachys cretica ssp. smyrnaea, Stachys annua ssp. annua, Stachys albiflora, Sideritis leptoclada, Melissa officinalis ssp. officinalis, Micromeria juliana and Prunella vulgaris can be mentioned.
In the present study, two species (P. erici-rosenii and L. martinicensis) from the Lamiaceae family were evaluated for their antimalarial potencies. The ethanol extracts of these two species have been analyzed for the first time against P. falciparum. The ethanol extract of P. erici-rosenii was found to be more potent (IC50 3D7: 4.07 μg/mL) than the ethanol extract of L. martinicensis (IC50 3D7: 12.29 μg/mL). In the study of Murugan et al. [35] , the leaf extracts of Senna occidentalis (Cassiae) and Ocimum basilicum (Lamiaceae) were evaluated for antimalarial activity against P. falciparum strains 3D7 and INDO. Senna occidentalis IC50 was 48.80 μg/ml (CQ-s) and 54.28 μg/mL (CQ-r), while O. basilicum IC50 was 68.14 μg/mL (CQ-s) and 67.27 μg/mL (CQ-r); IC50 of chloroquine was 85 μg/mL (CQ-s) and 91 μg/mL (CQ-r). However, it should be pointed out that the activity of these plant extracts can be affected by the region of origin and the growing conditions. For instance, the ethanol leaf extract of S. occidentalis from Congo showed a higher in vitro antimalarial activity against P. falciparum CQ-sensitive strains with an IC50 < 3 μg/mL [36] . The ethanol extracts of P. erici-rosenii and L. martinicensis could therefore be used against malaria, since they are effective at lower doses (Table 4) and could be advantageous sources of metabolites to build newer and safer antiplasmodial compounds.
The antimicrobial, antioxidant, and antiplasmodial results of the ethanol extracts of the aerial parts of P. erici-rosenii and L. martinicensis (Lamiaceae) have been reported for the first time in this study.
The HPLC-DAD-MS-ESI+ analysis has clearly shown significant differences concerning the quantity of chemical compounds in the ethanol extracts of P. erici-rosenii and L. martinicensis. Eleven polyphenols have been identified and quantified in all analyzed extracts (Table 5). These eleven polyphenols were free or glycosylated chemical compounds belonging to various therapeutically important structural classes: hydroxybenzoic acid (2-hydroxybenzoic acid, gallic acid, 4-hydroxybenzoic acid and vanilic acid), flavonol (kaempferol-rutinoside, quercetin-glucoside and quercetin), and hydroxycinnamic acid s (p-coumaric acid, ferulic acid, feruloylquinic acid and foumaroylquinic acid). Ferulic acid, feruloylquinic acid and p-coumaric acid were only present in ethanol extract of L. martinicensis while gallic acid, kaempferol-rutinoside, quercetin-glucoside, and quercetin were only present in ethanol extract of P. erici-rosenii (Table 5).
Furthermore, significantly high amounts of certain identified compounds, namely 2-hydroxybenzoic acid (4.976 μg/mL), gallic acid (1.140 μg/mL), 4-hydroxybenzoic acid (1.080 μg/mL), kaempferol-rutinoside (3.519 μg/mL), vanilic acid (2.210 μg/mL), quercetin-glucoside (5.153 μg/mL), coumaroylquinic acid (9.995 μg/mL), and quercetin (1.811 μg/mL) were observed in the case of the ethanol extract of P. erici-rosenii. For the ethanol extract of L. martinicensis, 2-hydroxybenzoic acid (3.608 μg/mL), p-coumaric acid (3.309 μg/mL), vanilic acid (0.931 μg/mL), ferulic acid (5.345 μg/mL), feruloylquinic acid (6.630 μg/mL), and coumaroylquinic acid (6.142 μg/mL) were observed.
These results are in agreement with the work carried out by Maria et al. [37] , in Lamiaceae family (Ocimum basilicum L., Origanum vulgare L., and Thymus vulgaris L.). They found gallic acid, caftaric acid, chlorogenic acid, 4-OH phenylacetic acid, 4-OH benzoic acid, caffeic acid, isoquercetin, vanillic acid, syringic acid, chicoric acid, p-coumaric acid, ferulic acid, rutin, rosmarinic acid, o-coumaric acid, cinnamic acid and quercetin.
Although the literature does not give detailed information regarding the chemical profile of phenolic compounds from the ethanol extracts of P. erici-rosenii and L. martinicensis. Thus, this work is original because it provides for the first time the profile of polyphenolic compounds in the ethanol extract of these two species and their biological activities such as antimalarial activity.
Therefore, the antimicrobial, antioxidant, and antiplasmodial results may suggest that the ethanol extracts of the aerial parts of P. erici-rosenii and L. martinicensis (Lamiaceae) contain compounds with antimicrobial, antioxidant, and antiplasmodial properties, which can be searched in new drug discovery for treatment of infectious diseases.
5. Conclusion
This study looked at the phytochemical and biological activities of ethanolic extracts of the aerial parts of P. erici-rosenii and L. martinicensis (Lamiaceae) from Eastern of Democratic Republic of Congo. The extracts had a high concentration of polyphenols and flavonoids, and showed significant antimicrobial, antioxidant and antiplasmodial activities.
However, further studies are needed. Among these studies, some are to identify the pure compounds responsible for these biological activities, toxicity tests and obtaining more information regarding the practical effectiveness of the extracts in animal models.
Acknowledgements
The authors thank the Laboratories of the Yaounde-Bielefeld Bilateral Graduate School Natural Products with Antiparasitic and Antibacterial Activity (YaBiNaPA) in partnership with “Institut Pasteur de Yaoundé/Cameroon” for the antiplasmodial test (Project: 57316173/2021-2025) and The University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca, Romania, for antimicrobial, and antioxidant activity assessment and HPLC-DAD-MS-ESI+ Analysis (Project: “Bourses “Eugen Ionescu” 2021-2022”).
Author Contributions
Conceptualisation, plants and extracts preparation: U. S. H., N. L. and P. T. M. Biological tests and acquisition of HPLC-DAD-MS-ESI+ data: U. S. H., D. C. V., D. H., I. M. and S. A. Writing original draft preparation: U. S. H., N. L. and P. T. M. Writing and editing: U. S. H., N. L., P. T. M., N. I. O., N. J. and S. A. All authors read and agreed to the published version of the manuscript.