Phytochemical screening, UPLC analysis, evaluation of synergistic antioxidant and antibacterial efficacy of three medicinal plants used in Kinshasa, D.R. Congo

doi:10.21203/rs.3.rs-4724430/v1

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Phytochemical screening, UPLC analysis, evaluation of synergistic antioxidant and antibacterial efficacy of three medicinal plants used in Kinshasa, D.R. Congo

https://doi.org/10.21203/rs.3.rs-4724430/v1

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Plant extracts are used worldwide for treating microbial diseases due to their biologically active compounds. This study investigated the phytochemical constituents and the synergistic antimicrobial and antioxidant potential of three medicinal plants namely Ocimum gratissimum, Tetradenia riparia, and Dysphania ambrosioides. Antibacterial studies against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were performed using the broth dilution method. Antioxidant activity was assessed using the DPPH method. UPLC analysis identified several metabolites in the plant extracts, including phenolics and flavonoids. The phytochemical analysis revealed the presence of alkaloids, saponins, flavonoids, iridoids, and anthraquinones in all extracts. The extract of T. riparia had the highest phenolic content (299.146 ± 0.143 mg GAE/g extract), while O. gratissimum had the highest flavonoid content (138.256 ± 0.277 mg QE/g extract). Decocted extracts of O. gratissimum exhibited the highest antioxidant activity. The combination of O. gratissimum + T. riparia demonstrated synergistic antioxidant activity (CI = 0.57). Antibacterial activity was highest with percolated extracts of O. gratissimum and T. riparia against S. aureus (MIC = 500 µg/mL), with their combination showing additive antibacterial activity (FICI = 1). This study concludes that these plant extracts are promising sources of natural antimicrobial and antioxidant agents for pharmaceutical and food industries.

Biological sciences/Biological techniques

Biological sciences/Chemical biology

Biological sciences/Drug discovery

Health sciences/Diseases

Physical sciences/Chemistry

Bio-active compounds

antioxidant activity

antibacterial activity

Ocimum gratissimum

Tetradenia riparia

Dysphania ambrosioides

Despite substantial advancements in modern medicine, microbial diseases persist as significant global threats¹. Among these diseases, the acute respiratory infections are one of the leading causes of medical consultations and main cause of mortality for children. The prevalence and severity of acute respiratory infections are constantly on the rise and constitute a public health problem. They are the sixth leading cause of all-age mortality, and the leading cause of death for children under five². Regarding this matter, the World Health Organization emphasizes on herbal medicines as a primary healthcare modality in numerous low-income countries. Globally, approximately 80% of the world population relies on medicinal plants for disease treatment, with a much more pronounced prevalence in African nations³.

The Democratic Republic of the Congo, recognized for its vast equatorial forest and mega biodiversity as well as profound traditional knowledges of medicinal plants significantly incorporates these resources into its healthcare system. A large proportion of the Congolese population uses traditional medicine, which places medicinal plants at the heart of these practices and emphasizes their role in the treatment of various infectious and non-communicable diseases. Indeed, plants are vital resources for drug discovery due to their extensive use in traditional medicine⁴. Throughout history, natural compounds from plants have served as medicine and numerous studies attest their biological activities against various infectious agents. The Food and Agriculture Organization report indicates that at least 25% of pharmaceutical drugs in modern pharmacopoeia are plant-derived. Most of them are synthetic analogues derived from plant prototype compounds⁵. Fundamental methods for assessing bioactive properties and identifying compounds from plant extracts include phytochemical screening and antibacterial activity testing^6,7,8.

In our previous ethnobotanical data conducted on herbal use and preparation of the medicinal plants, several plants were highly reported as frequently used for treating oral microbial diseases⁹. It was also described that three of them, Dysphania ambrosioides, Ocimum gratissimum and Tetradenia riparia were often used in combination. Previous research has explored the phytochemical properties and antibacterial activity of oils from these three plants^{10,11,12,13,14,15,16,17}. However, there is no investigation available concerning the bioactivity of their combinations. Therefore, this current study aims to evaluate the phytochemical composition, antioxidant and antibacterial activities of these three plants, taken alone and in combination.

i. Collection and preparation of plant material

In a recent ethnobotanical data collection, we identified Dysphania ambrosioides, Ocimum gratissimum and Tetradenia riparia as key medicinal plants used in multi-species recipes for treating oral microbial infections within the district of Mont-Ngafula, Kinshasa, DRC⁹. Based on this foundational research, fresh leaves of Ocimum gratissimum and Tetradenia riparia as well as stem and leaves of Dysphania ambrosioides were collected between January and February 2023 in the Ngansele district of the Mont-Ngafula in the city of Kinshasa for further analysis. The geographical coordinates are as follows; for Dysphania ambrosioides 4°26'20''S 15°17'14''E; for Tetradenia riparia 4°26'20''S 15°17'13'E and for Ocimum gratissimum 4°26'20''S 15°17'13'E. The plant collection adhered to all guidelines and the permission was obtained from the Faculty of Sciences and Technologies of the University of Kinshasa. The voucher specimen N° 001/23; N° 002/23 and N° 003/23 were also deposited at the Herbarium of Kinshasa (IUK), which is part of the National Institute for Agronomic Research (INERA-RDC).

The collected plant material was air-dried at room temperature and each of them was then milled to powder using an electric grinder (Blinder Butterfly B-592). The powder was accurately weighed using an electronic weighing balance, then packed into polyethylene bags to prevent air entry and contamination. Finally, the bags were stored in a securely closed container, appropriately labeled, to facilitate further extraction processes. For combinations of the different specimens, a ratio of 1:1 (m/m) was used for the mixture containing O. gratissimum and T. riparia (OT); O. gratissimum and D. ambrosioides (OD) as well as T. riparia and D. ambrosioides (TD). The mixture containing all the three plant materials (OTD) was prepared using the ratio 1:1:1 (m/m/m).

Table 1

Ethnobotanical data on medicinal plants selected for phytochemical analysis and antibacterial activity.
Scientific name	Family name	Local names	Parts used	Locally used in the study area to treat	Frequencies of citations
Ocimum gratissimum L.	Lamiaceae	Dinsusu nsusu, Mazulu	Leaves, roots	Pneumonia, cough, diarrhea, skin pathology, fever, headaches, conjunctivitis, dysentery, rheumatism, menorrhagia.	103
Tetradenia riparia (Hochst) Codd	Lamiaceae	Mutuzo, Mutubya	Leaves, roots	Headaches, cough, stomachache, diarrhea, fever, dengue, respiratory problems.	53
Dysphania ambrosioides L.	Amaranthaceae	Lukunga-nioka, Lukunga bakishi, Kepamekusu, Diniioka nioka, Nkasi	Leaves	Worms (hookworms, roundworms, threadworms, ringworms), lice, stomachache, fever, cough, asthma, headaches, dysmenorrhea, eczema, itches.	67

ii. Preparation of extracts

The powder obtained from each dried plant material and their combination was extracted by decoction and maceration (percolation). For the decoction, 10 g of powder was mixed with 100 mL of water. After heating the mixture at 100°C for 5 minutes, it was cooled down to room temperature and then filtered through Whatman paper. For the maceration, 10 g of powder were allowed to stand in a mixture of methanol and ethyl acetate (50:50, v/v) for 48 hours. The percolate was paper filtered until exhaustion. The decocted solutions and percolates obtained were then evaporated and the dried extracts thus obtained were preserved in sterile vials and stored in a refrigerator (4°C) for subsequent analysis. The extraction yield (R) was calculated as the percentage of the plant dry matter (DM) according to the formula: R(%) = Mex/Mmv* 100 where Mex is the dry residue following the percolation or decoction extraction and Mmv is the mass of the starting dry plant matter¹³.

iii. Microorganisms

The antibacterial activity of plant extracts was evaluated against selected standard bacterial strains of the American Type Culture Collection (ATCC). The bacterial strains selected were representative of both classes of Gram-positive and Gram-negative bacteria. The tested gram-positive bacterial strain was Staphylococcus aureus (ATCC 25923), and the tested gram-negative bacterial strain were Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). The microorganisms were maintained in cryotubes on Triptosoya + 20% glycerol broth at -80 ̊C at the Microbial Unit of the Faculty of Pharmaceutical Sciences at the University of Kinshasa. Finally, the selected bacterial strains were cultivated using Mueller-Hinton broth¹⁸.

iv. Phytochemical screening

Phytochemical screening was conducted on the extracts using the methods outlined by Kilonzo and Munisi (2021), Shaikh and Patil (2020), María et al. (2018) and Wagner et al. (2018)^19,20,21,22 to determine the presence of secondary metabolites including phenolics, flavonoids, coumarins, anthraquinones, terpenoid, saponins and alkaloids. The selection of these metabolites was based on their superior potency in terms of antibacterial activity as well as the availability of reagents in our laboratory. The results were denoted as (+) for the presence and (-) for the absence of phytochemicals. The content of total polyphenols was determined using the Folin-Ciocalteu method^22,23. Gallic acid is the standard used to establish the calibration curve. Flavonoids contained in our extracts were quantified and determined according to the aluminum trichloride colorimetric method as described in Bahorun et al. (2016)²⁴. Aluminum trichloride (AlCl₃) forms a yellow complex with flavonoids that absorbs light at 415 nm²⁵.

v. Ultra-performance liquid chromatography-quadrupole time of flight-mass spectrometry (UPLC-QTOF-MS) analysis

The dry extracts (1 mg) of each plant and of their combinations were dissolved in 1 mL of methanol: water (1:1) and then filtered through a 0.22 µm nylon syringe filter (13 mm diameter). A Waters acquity UPLC System equipped with a binary solvent delivery system and an autosampler was used to run the samples. The chromatographic separation was achieved on a Waters BEH C₁₈ (2.1 mm x 100 mm, 1.7 µm) column with a gradient elution of solvents A (water + 0.1% formic acid) and B (methanol + 0.1% formic acid), applied as follow: 0 min 3% B, 0.10 min 3% B, 14 min 100% B, 16 min 100% B, 16.5 min 3% B, 20 min 3% B. The flow rate was set at 0.3 ml/min. The injection volume was 5 µl. The separated compounds were analyzed by a Waters Synapt G2 high definition QTOF mass spectrometer, which was run in electrospray ionization negative mode. The following MS source parameters were set for negative mode: source temperature 120 ºC, sampling cone 20 V, extraction cone 4.0 V, desolvation temperature 300 ºC, cone gas flow 10.0 L/h, desolvation gas flow 600 L/h, capillary 2.6 kV. It was constantly infused at a rate of 3 µl/min through a separate orthogonal ESI probe to compensate for experimental drift in mass accuracy. The trap collision energy was 28 V. Compounds were tentatively identified by generating their respective molecular formula from MassLynx V 4 and comparing their fragmentation patterns with that of matching compounds from various search databases including Dictionary of Natural Products and MetFrag.

vi. Antioxidant activity

The free radical scavenging ability of our extracts and their combination was tested by the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay as described by Bahorun et al. (1996)²⁴.

- Determination of antioxidant combination index (CI)

To investigate the possible synergistic antioxidant activity between the different plants used, an isobologram analysis based on the median effect principle (IC₅₀) was performed. The classical isobologram-combination index equation (CI) was used for analyzing the data²⁶. CI = CI₁ + CI₂ ; where CI₁ = (D)₁/ (D_X)₁ and CI₂ = (D)₂/ (D_X)₂ ; (D)₁ and (D)₂ are the doses (IC₅₀ values) of two plants in combination; (D_X)₁ and (D_X)₂ are the doses (IC₅₀ values) of two plants taken individually. Results were interpreted as synergistic interaction (CI < 1), additive interaction (CI = 1), or antagonistic interaction (CI > 1).

vii. Antibacterial assay

The antibacterial activity test to determine the minimum inhibitory concentration (MIC) of organic and aqueous extracts was carried out using the broth dilution method on 96-well polyester microplates. The MIC of plant extracts was determined according to the protocol established by Golus et al. (2016)²⁷.

To prepare stock solutions of plant extracts, 20 mg of each extract was dissolved in 250 µL 5% DMSO, then 9.75 mL Mueller Hinton broth was added, giving a concentration of 2 mg/mL for each solution. Triplicate wells were prepared in 96-well microplates for each bacterial strain, with 900 µL of Muller Hinton broth (MHB) added to each well. Next, 100 µL of extract was added to the first row and serial dilutions were made using multichannel micropipettes from column 1 to column 9. Thus, the dilution series ranged from 2000 µg/mL to 7.8125 µg/mL. Column 10 was left empty, while 200 µL of MHB was placed in column 11 as a growth control, and 200 µL of 2 mg/mL extract was placed in the last column as a sterility control for the culture medium.

Reference strains were stored in cryotubes in a freezer at -20°C. To obtain the active culture, the stored strains were sub-cultured overnight at 37°C on MacConkey for Escherichia coli ATCC 25922, on Mannitol Salt Agar (MSA) for Staphylococcus aureus ATCC 25922 and on ketrimide agar for Pseudomonas aeruginosa ATCC 9027. A few bacterial colonies were transferred from subculture tubes to tubes containing sterile Mueller Hinton agar using a platinum loop. Incubation for 24 hours yielded colonies of 10⁸ CFU/mL, with an optical density (OD) corresponding to 0.5 MacFarland. These three or four looped colonies were diluted in 2 mL of physiological water (1:100 dilution) to give suspensions containing around 10⁶ CFU/mL, tested at a concentration of around 1.0×10⁶ CFU/mL, and prepared by diluting the normalized inoculum in MHB. Within 15 minutes of normalization, 2 µL of the test bacterial suspensions (1.0×10⁶ UFC/mL) were added to each well of the microdilution plate, with the exception of the sterility control (column 12). The plate was then incubated at 37°C for 18 to 24 hours. After incubation, 2 µL solution of 7-hydroxy-10-oxidophenoxazin-10-ium-3-one sodium (resazurin) 1% was added to each well as an indicator of microbial growth and incubated at 37°C for 4 hours. MIC values were determined by visually observing the presence or absence of pink color to the naked eye after the 4-hour incubation period. The MIC was recorded as the lowest concentration of each extract that showed no visible pink color, indicating the absence of microbial growth. MIC values were determined by performing measurements in triplicate. For each strain, one sterility control well and one growth control well were included in the study. To assess bacterial sensitivity, parallel experiments were carried out using ciprofloxacin as a positive control, starting at a concentration of 64 µg/mL in sterile water (range 64 µg/mL to 0.0009765625 µg/mL).

- Synergistic effect: Fractional Inhibitory Concentration (FIC)

Synergistic effects of plant extract combinations were evaluated using a checkerboard synergy assay to obtain fractional inhibitory concentration (FIC) values as published by Cicco et al. (2009)²⁸. One extract was serially diluted along the abscissa, while another extract was serially diluted along the ordinate. The total volume of the combination was 100 µL per well. Then 100 µL of each bacterial suspension (1x10⁶ CFU/mL) was added to each well of a 96-well microplate. The microplate was sealed and incubated at 37°C for 18h. Then, a second incubation was conducted for 4 h at 37°C after adding 2 µL of resazurin to each well. Experiments were conducted in triplicates. The FIC index (FICI) was calculated using the following equation: FICI = FIC_A + FIC_B where FIC_A is the MIC of extract A in combination/MIC of extract A alone and FIC_B is MIC of extract B in combination/MIC of extract B alone. Results were interpreted as synergistic interaction (FICI ≤ 0.5), partial synergy (0.5 < FICI ≤ 0.75), additive interaction (0.75 < FICI ≤ 1.0), indifferent (1.0 < FICI ≤ 4.0), or antagonistic interaction (FICI > 4.0).

viii. Statistical analysis

All tests were carried out in triplicates. Data were presented as mean ± standard deviation. The statistical significance of differences in means between experimental parameters was established by analysis of variance (ANOVA) using the t-test and P values < 0.05 were considered statistically significant. GraphPad Prism 9.5.1.733 was used for all statistical analysis.

Qualitative phytochemical screening

Phytochemical screening is used to evaluate the constituents of plant extracts, and their predomination. This preliminary step for the search for bioactive constituents is very helpful in the production of therapeutic drugs²⁹. In this study, qualitative phytochemical analysis of methanolic extracts of Ocimum gratissimum, Tetradenia riparia and Dysphania ambrosioides was carried out as shown in Table 2. Alkaloids, saponins, flavonoids, iridoids and anthraquinones were detected in all the three plant extracts. Coumarins were only present in O. gratissimum while terpenes were present both in O. gratissimum and in T. riparia. Our results corroborate those of Ahmed et al. (2019) and Olamilosoye et al. (2018)^30,31. The therapeutic potential of these three plants is probably due to the presence of these phytochemicals. Flavonoids are known to have antioxidant activities, inhibiting the initiation, promotion and progression of tumors³². Alkaloids obtained from beta-carboline group are known to exhibit strong antimicrobial, anti-HIV and antiparasitic activities³³. Terpenoids show multiple pharmacological activities such as anti-inflammatory, anticancer, antioxidant and antibacterial activities ^32,34,35.

Table 2

Phytochemical screening of methanolic extracts of *Ocimum gratissimum*, *Tetradenia riparia*, and *Dysphania ambrosioides* [+ indicates presence, − indicates absence].
Phytochemical compounds	O. gratissimum	T. riparia	D. ambrosioides
Alkaloids	+	+	+
Saponins	+	+	+
Flavonoids	+	+	+
Iridoids	+	+	+
Anthraquinones	+	+	+
Coumarins	+	-	-
Terpenes	+	+	-

Total phenolic and flavonoid content

Phenolic and flavonoid contents are very important criteria for evaluating the plant extracts quantitatively and their biological strength because they play a crucial role in different physiological processes^32,33. The total phenolic and flavonoid contents of Dysphania ambrosioides, Ocimum gratissimum, and Tetradenia riparia methanol extracts are shown in Table 3. The result showed that T. riparia had significantly the highest total phenolic content (299.146 ± 0.143 mg GAE/g extract) followed by D. ambrosioides (155.602 ± 1.712 mg GAE/g extract) and O. gratissimum (153.597 ± 5.277 mg GAE/g extract). On the other hand, O. gratissimum had the highest flavonoids content (138.256 ± 0.277 mg QE/g extract) followed by T. riparia (112.843 ± 0.255 mg QE/ g extract) and D. ambrosioides (63.925 ± 0.494 mg QE/ g extract).

Our results are a bit higher than those found by Panda et al. (2022) and Olamilosoye et al. (2018)^10,31. The discrepancy can be due to several factors such as the harvesting season of the plants, the pH of the soil, the choice and stage of drying conditions, the geographical location, the chemotype or subspecies and the choice of plant part or genotype or extraction method. Phenolics are known to possess antioxidant antimutagenic, and anti-carcinogenic activities as well as the ability to modify gene expression. Flavonoids are active constituents performing various biological activities such as resisting microbial, ulcer, arthritis, angiogenic, and cancerous diseases along with inhibiting the formation of mitochondrial adhesion³⁰.

Table 3

Total phenolics and flavonoid content of the methanolic extracts of *Ocimum gratissimum*, *Tetradenia riparia*, and *Dysphania ambrosioides*
Extracts	Phenol content (mg GAE/g extract)	Flavonoid content (mg quercitin/g extract)
T. riparia	299.146 ± 0.143	112.843 ± 0.255
O. gratissimum	153.597 ± 5.277	138.256 ± 0.277
D. ambrosioides	155.602 ± 1.712	63.925 ± 0.494

UPLC analysis

Ultra-performance liquid chromatography-quadrupole time of flight-mass spectrometry (UPLC-QTOF-MS) analysis is one of the most frequently applied techniques to detect different metabolites present in plants. This technique was used to tentatively identify the metabolites present in the decocted and percolated extracts of O. gratissimum, T. riparia, D. ambrosioides and those of their combinations including O. gratissimum + T. riparia (OT), O. gratissimum + D. ambrosioides (OD), T. riparia + D. ambrosioides (TD) as well as O. gratissimum + T. riparia + D. ambrosioides (OTD). The chromatograms of the percolated extracts are shown in Fig. 1 while those of the tested decocted extracts are in supplementary data file (Figure S1). The tentatively identified compounds in the percolated extracts are depicted in Table 4 while those of the decocted extracts are shown in Table S1.

The major compounds identified in the percolated extract from O. gratissimum were rosmarinic acid, cirsimaritin and xanthomicrol (Figures S2, S3). Rosmarinic acid was also identified in the percolated extract from T. riparia (Figure S2) while several flavonoids such as apigenin 7-glycosides, kaempferitrin (a 3,7-dirhamnoside of kaempferol) and kaempferol 3,7-diglycosides were identified in D. ambrosioides (Figures S4, S7). Furthermore, rosmaniric acid and xanthomicrol were detected in the extract combination of O. gratissimum + T. riparia (Figures S2 and S3). Rosmarinic acid, kaempferitrin, vitexin and xanthomicrol were identified in the percolated extract combination containing O. gratissimum and D. ambrosioides (Figures S2, S3, S4 and S5). For the percolated extract combination including T. riparia and D. ambrosioides, luteolin 7-O-glucoside, rosmarinic acid, apigenin 7-glycosides and kaempferitin were putatively identified (Figures S2, S4, S7 and S8). Finally, in the percolated extract combination containing all three plants, epigallocatechin 3-gallate, 3-galloylcatechin epicatechin 5-gallate, rosmarinic acid, xanthomicrol and apigenin 7-glycosides were identified (Figures S2, S3, S7, S9 and S10). Rosmarinic acid had previously been identified as one of major compounds in O. gratissimum methanolic, hydromethanolic and ethanolic extracts³⁶. Likewise, cirsimaritin and xanthomicrol had previously been detected in O. gratissimum³⁷. Further, several quercetins and kaempferol derivatives had been identified in D. ambrosioides³⁸.

Table 4

Tentative identification of the major compounds in the percolated plant extracts.
Medicinal plants	RT min	Acquired [M-H]⁻ m/z	Theoretical [M-H]⁻ m/z	Molecular Formula	Tentative Identification
O. gratissimum	10.06	359.0836	359.0767	C₁₈H₁₆O₈	Rosmarinic acid
	13.27	313.0800	313.0712	C₁₇H₁₄O₆	Cirsimaritin
	13.68	343.0911	343.0818	C₁₈H₁₆O₇	Xanthomicrol
T. riparia	10.05	359.0836	359.0767	C₁₈H₁₆O₈	Rosmarinic acid
D. ambrosioides	10.77	563.1530	563.1401	C₂₆H₂₈O₁₄	Apigenin 7-glycosides
	10.86	577.1682	577.1557	C₂₇H₃₀O₁₄	Kaempferitrin
	11.67	605.1561	605.1506	C₂₈H₃₀O₁₅	Kaempferol 3,7-diglycosides
O. gratissimum + T. riparia	10.10	359.0836	359.0767	C₁₈H₁₆O₈	Rosmarinic acid
O. gratissimum + T. riparia	13.71	343.0911	343.0818	C₁₈H₁₆O₇	Xanthomicrol
O. gratissimum + D. ambrosioides	9.97	359.0836	359.0767	C₁₈H₁₆O₈	Rosmarinic acid
	10.76	577.1578	577.1557	C₂₇H₃₀O₁₄	Kaempferitrin
	12.25	431.1033	431.0978	C₂₁H₂₀O₁₀	Vitexin
	13.61	343.0880	343.0818	C₁₈H₁₆O₇	Xanthomicrol
T. riparia + D. ambrosioides	9.94	447.0977	447.0927	C₂₁H₂₀O₁₁	Luteolin 7-O-glucoside
	10.07	359.0836	359.0767	C₁₈H₁₆O₈	Rosmarinic acid
	10.78	563.1015	563.1401	C₂₆H₂₈O₁₄	Apigenin 7-glycosides
	10.86	577.1578	577.1557	C₂₇H₃₀O₁₄	Kaempferitrin
	15.26	661.4565	661.4468	C₄₂H₆₂0₆	Ziziphorin B
T. riparia + D. ambrosioides + O. gratissimum	7.07	457.0755	457.0771	C₂₂H₁₈O₁₁	Epigallocatechin 3-gallate
	8.17	441.0869	441.0880	C₂₂H₁₈O₁₀	3-Galloylcatechin Epicatechin 5-gallate
	9.86	359.0823	359.0767	C₁₈H₁₆O₈	Rosmarinic acid
	10.57	563.1446	563.1401	C₂₆H₂₈O₁₄	Apigenin 7-glycosides
	13.46	343.0870	343.0818	C₁₈H₁₆O₇	Xanthomicrol

Antioxidant activity

The scavenging of free radicals through the DPPH is an approved technique commonly used to determine the antioxidant activity of extracts taken from plants. The method requires less time for analysis and therefore, is used widely for measuring the antioxidant activity of plant extracts. Due to their high hydrogen-donating ability, DPPH is considered as an efficient antioxidant. Removal of free radicals is vital to inhibit their toxic roles in various diseases, including cancer and bacterial diseases ^29,39,40,41.

The results of the antioxidant activities of the percolated and decocted extracts of D. ambrosioides, O. gratissimum and T. riparia as well as of their combinations using the DPPH method are shown in Tables 5 and 6, respectively. The extracts obtained through decoction demonstrated higher antioxidant activity than the extracts obtained through percolation. When considering the extracts obtained through decoction, the extract from O. gratissimum exhibited the highest antioxidant activity (11,744 ± 0,584 µg/mL) followed by that from T. riparia (12,916 ± 0,972 µg/mL). The extract from D. ambrosioides displayed the lowest antioxidant activity (203,492 ± 9,285 µg/mL). However, when looking at the extracts obtained through percolation, the extract from T. riparia exhibited the highest antioxidant activity (20,157 ± 3,125 µg/mL) followed by that from O. gratissimum (22,747 ± 1,139 µg/mL). Again, the extract from D. ambrosioides displayed the lowest antioxidant activity (275,053 ± 13,20 µg/mL). The differentiating factor might be the absence of terpenes. The terpenes had been found in T. riparia and O. gratissimum (Table 1) and they have been reported to have efficient antioxidant activity ^34,35.

Table 5

Antioxidant combination effects of decocted extracts of *O. gratissimum*, *T. riparia* and *D. ambrosioides* using the DPPH method.
Decocted extracts	IC50 (µg/mL)	CI₁	CI₂	CI₃	CI	Interpretation
O. gratissimum	11,744 ± 0,584	-	-	-	-	-
D. ambrosioides	203,492 ± 9,285	-	-	-	-	-
T. riparia	12,916 ± 0,972	-	-	-	-	-
O. gratissimum + D. ambrosioides	13,050 ± 0,239	1,11	0,06	-	1,17	Additive
O. gratissimum + T. riparia	3,529 ± 0,362	0,30	-	0,27	0,57	Synergistic
D. ambrosioides + T. riparia	45,450 ± 3,013	-	0,22	3,51	3,73	Antagonistic
O. gratissimum + T. riparia + D. ambrosioides	11,953 ± 0,183	1,02	0,92	0,06	1,99	Antagonistic
Quercetin	3,21 ± 1,0

On the basis of antioxidant combination index (CI), the decocted extract from the combination of O. gratissimum + T. riparia showed synergy (CI = 0.57) and depicted the highest antioxidant among all combinations whereas that of O. gratissimum + D. ambrosioides showed additive effects (CI = 1.17). All other combinations showed antagonistic effects (Tables 5 and 6). This indicates that the proton donating ability of the decocted extract from the combination of O. gratissimum + T. riparia was higher than that of the extracts taken individually. This synergism can be explained by several mechanisms such as the formation of stable intermolecular complexes between the antioxidants which then exhibits higher antioxidant activity than that of the parent compounds. Unpredictable interactions between the examined compounds could also explain the synergism effect⁴². The additive effect produced from the decocted extract of O. gratissimum + D. ambrosioides combination might indicate the absence of interactions between the antioxidants during the oxidation. This effect occurs when single antioxidant acts independently of each other, one antioxidant does not disturb the action of another antioxidant occurring in the complex mixture⁴³. Several mechanisms can explain the antagonism observed with all the other combinations. It might be that antioxidants polymerization, formation of complex and adduct between antioxidants are causing the decrease in the overall antioxidant properties⁴². Several investigations need to be undertaken in order to understand the exact mechanism of action of the synergism, additive and antagonistic effects revealed in this study.

Table 6

Antioxidant combination effects of percolated extracts of *O. gratissimum*, *T. riparia* and *D. ambrosioides* using the DPPH method.
Percolated extracts	IC50 (µg/mL)	CI₁	CI₂	CI₃	CI	Interpretation
O. gratissimum	22,747 ± 1,139	-	-	-	-	-
D. ambrosioides	275,053 ± 13,20	-	-	-	-	-
T. riparia	20,157 ± 3,125	-	-	-	-	-
O. gratissimum + D. ambrosioides	48,134 ± 0,858	2,12	0,17	-	2,29	Antagonistic
O. gratissimum + T. riparia	49,068 ± 31,011	2,16	-	2,43	4,59	Antagonistic
D. ambrosioides + T. riparia	197,560 ± 2,079	-	9,80	0,72	10,52	Antagonistic
O. gratissimum + T. riparia + D. ambrosioides	43,287 ± 0,316	1,90	2,15	0,16	4,21	Antagonistic
Quercétine	3,21 ± 1,0

The CI index (CI) = CI₁ + CI₂, or (CI₂ + CI₃) or (CI₁ + CI₃) or (CI₁ + CI₂ + CI₃) where CI₁ = (D)₁/ (D_X)₁ and CI₂ = (D)₂/ (D_X)₂, CI₃= (D)₃/ (D_X)₃ ; (D)₁, (D)₂ and (D)₃ are the doses (IC₅₀ values) of the plant extracts in commination; (D_X)₁, (D_X)₂ and (D_X)₃ are the doses (IC₅₀ values) of the plant extracts taken individually. Results were interpreted as synergistic interaction (CI < 1), additive interaction (CI = 1), or antagonistic interaction (CI > 1).

Antibacterial activity

Due to the emergence of multi-drug resistant microorganisms worldwide, researchers are currently looking for new constituents from natural resources to combat microbial diseases⁴⁴. The plants are, without any doubt, a valuable resource of bioactive compounds of important medicinal value^39,40. In this study, the antimicrobial potential of percolated and decocted extracts from O. gratissimum, T. riparia and D. ambrosioides was analyzed against gram-positive bacteria (S. aureus) and gram-negative bacteria (E. coli and P. aeruginosa). The minimum inhibitory concentration (MIC) is defined as the least concentration of an antimicrobial agent that can stop the visible increase in the microorganisms’ growth after an overnight incubation. The MIC is usually used to evaluate the antimicrobial activity of new compounds or extracts by measuring the effect of decreasing antimicrobial concentration. Compounds with lower MIC values are more effective and vice-versa^33,40.

According to our findings, the percolated extracts showed significant inhibitory effects (Table 7) as opposed to the decocted extracts (Table S2). The decocted extracts were less potent against all tested microorganisms although several potential flavonoids and polyphenols were tentatively identified through UPLC (Table S1). The percolated extracts of T. riparia and O. gratissimum had the lowest MIC (500 µg/mL) followed by those of D. ambrosioides (1000 µg/mL) against Staphylococcus aureus. However, these extracts were less potent (2000 µg/mL) against gram-negative bacteria (E. coli and P. aerogenes). Cells of Gram-negative bacteria possess an extra outer membrane, which provides them with a hydrophilic surface that performs as a permeability barrier for several substances including biological compounds⁴⁵. This might explain the lower potency of our extracts against the gram-negative bacteria. The antibacterial activity of our plant extracts might be due to the presence of high content phenols and flavonoids identified. Phenols such as the identified rosmarinic acid are known to disturb the function of cytoplasmic membrane and metabolism of energy, thus affecting the synthesis of nucleic acids.

They have anticancer, antidiabetic, antiaging, antidepressant and antimicrobial activities^41,42. Flavonoids such as cirsimaritin, xanthomicrol, kaemferitin, kaempferol 3,7-diglycosides, etc. have shown inhibitory activity against the bacterial DNA polymerase, RNA polymerase, reverse transcriptase, telomerase as well as antitumoral activities^43,44,46.

Table 7

Minimum inhibitory concentration (MIC) values of selected medicinal plants and control antibiotic against test bacterial strains.
Percolated extracts	MIC (µg/mL)
	Bacterial strains
	Staphylococcus aureus	Escherichia coli	Pseudomonas aerogenes
T. riparia	500	2000	2000
O. gratissimum	500	2000	2000
D. ambrosioides	1000	2000	2000
Ciproflaxine	0.125	0.03	0.25

The combined use of plant extracts can usually cause different interactions. This is because each extract possesses several types of compounds. The enhanced antibacterial activity of plant extract combinations had been reported previously^47,48,49. Some interactions are also known to decrease the efficacy of plant extract combination either by neutralizing each other, forming inactive complexes or by acting competitively for the same molecular target^50,51,52. It is always necessary to confirm the influence of the combination of plant extracts as reported by local communities.

In our study, checkboard synergy assays was performed to evaluate the synergistic effects of our three selected medicinal plants commonly used in combinations in the capital city of the Democratic Republic of the Congo. Fractional inhibitory concentrations (FICs) and their interpretations are presented in Table 8. There was no best synergistic interactions. However, percolated extracts from O. gratissimum (OG) and T. riparia (TR) showed additive effects (FICI = 1) against S. aureus; their MIC decreased by 2-fold respectively. This might be due to the flavonoids such as xanthomicrol and rosmaniric acid, identified in the UPLC chromatogram of the plant extract combination (O. gratissimum + T. riparia) (Fig. 1, S2 and S3). All others combinations showed indifferent effects (1.0 < FICI ≤ 4.0), suggesting that the combined effect is equal to the effects of the most active extract. No antagonism effect was found in any of the plant extract combinations. Our results clearly show that the potent activity of one extract might not necessarily lead to a high synergy with another extract but can potentially improve the overall antibacterial activity. Most of the time, antibacterial mechanisms of plant extract combinations are not fully understood. The inhibitory antibacterial effect of the OG + TR combination need to be further investigated in order to elucidate the mode of action and the pathways involved.

Table 8

Fractional inhibitory concentration (FIC) values of percolated extracts from selected medicinal plants in combination against test bacterial strains.
Bacterial strains	Medicinal plants			FIC values			FIC index	Interpretation
Bacterial strains	A	B	C	FIC_A	FIC_B	FIC_C	FIC index	Interpretation
Staphylococcus aureus	OG	TR		0.5	0.5		1	Additive
	OG		DA	2		1	3	Indifferent
		TR	DA		2	1	3	Indifferent
	OG	TR	DA	1	1	0.5	2.5	Indifferent
Escherichia coli	OG	TR		1	1		2	Indifferent
	OG		DA	1		1	2	Indifferent
		TR	DA		1	1	2	Indifferent
	OG	TR	DA	1	1	1	3	Indifferent
Pseudomonas aeruginosa	OG	TR		1	1		2	Indifferent
	OG		DA	1		1	2	Indifferent
		TR	DA		1	1	2	Indifferent
	OG	TR	DA	1	1	1	3	Indifferent

OG: Ocimum gratissimum, TR: Tetradenia riparia, DA: Dysphania ambrosioides. The FIC index (FICI) = FIC_A + FIC_B, or (FIC_B + FIC_C) or (FIC_A + FIC_C) or (FIC_A+FIC_B+FIC_C) where FIC_A was MIC of extract A in combination/MIC of extract A alone, FIC_B was MIC of extract B in combination/MIC of extract B alone and FIC_C was MIC of extract C in combination/MIC of extract C alone. FICI are interpreted as synergistic (FICI ≤ 0.5), partial synergy (0.5 < FICI ≤ 0.75), additive (0.75 < FICI ≤ 1.0), indifferent (1.0 < FICI ≤ 4.0), or antagonistic (FICI > 4.0).

The search for natural compounds that might possess antimicrobial and antioxidant potential is increasing everyday due to the prevalence of infectious diseases and the raise of antimicrobial resistance. The extracts obtained from Dysphania ambrosioides, Ocimum gratissimum and Tetradenia riparia as well as their combination have shown evidence of the presence of phenolic and flavonoids content exhibiting strong antioxidant activity and antimicrobial activity against Staphylococcus aureus. The decocted extract from the combination of O. gratissimum + T. riparia showed synergetic antioxidant activity while that of O. gratissimum + D. ambrosioides showed additive antioxidant effects. The percolated extracts from O. gratissimum and T. riparia showed additive antibacterial effects; their MIC decreased by 2-fold respectively.

The results from this study showed that the extracts from O. gratissimum and T. riparia have important antioxidant and antibacterial activity that was improved and strengthened when applied together. To the best of our knowledge, this is the first study revealing the nature of interactions from these medicinal plants commonly used in the Democratic Republic of the Congo. Further studies could be done to elucidate the exact mechanism of action and to test their possible application as antibacterial and antioxidant compounds when applied using different methods in both in vivo and in vitro models.

Data curation

Lyz Makwela Ngolo and Tania Bishola Tshitenge

Investigation

Lyz Makwela Ngolo, Francis Mubigalo Faraja, Sephora Mianda Mutombo and Tania Bishola Tshitenge

Visualization

Lyz Makwela Ngolo, Francis Mubigalo Faraja, Sephora Mianda Mutombo and Tania Bishola Tshitenge

Methodology

Lyz Makwela Ngolo, Sephora Mianda Mutombo and Tania Bishola Tshitenge

Project administration

Tania Bishola Tshitenge

Supervision

Tania Bishola Tshitenge, Odette Kabena Ngandu and Paulin Mutwale Kapepula

Writing – original draft

Tania Bishola Tshitenge

Writing – review & editing

Lyz Makwela Ngolo, Sephora Mianda Mutombo and Tania Bishola Tshitenge

Competing interests

The authors declare no competing interests.

Additional information

There are supplementary information available.

Correspondence and requests for materials should be addressed to Tania Bishola Tshitenge.

Figure 1.

ESI negative mode BPI chromatograms of percolated extracts of the tested plant species (A) and their combinations (B). The tentatively identified compounds are shown in Table 4.

Funding

acquisition: Lyz Makwela Ngolo and Tania Bishola Tshitenge

Author Contribution

LMN and TBT were responsible for conceptualisation, data curation and funding acquisition for the project. LMN, FMF, SMM and TBT were involved in investigation, visualisation and methodology. TBT, OKN and PMK supervised the work. TBT coordinated the project, wrote the first draft of the paper. TBT, LMN and SMM were involved in subsequent editing and review of the paper.

Acknowledgement

The authors are thankful to the Holger-Pöhlmann Foundation for granting Lyz Makwela Ngolo the “Small Research Grant 2023”, which provided funds for this study. This publication was also possible thanks to the Seed Grant for New African Principal Investigators (SG-NAPI) awarded to Tania Bishola Tshitenge, supported by the German Ministry of Education and Research (BMBF) through UNESCO-TWAS. We are indebted to the University of Kinshasa Clinics for providing the microorganisms.

Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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No competing interests reported.

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Phytochemical screening, UPLC analysis, evaluation of synergistic antioxidant and antibacterial efficacy of three medicinal plants used in Kinshasa, D.R. Congo

Status:

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Abstract

Figures

Introduction

Material and methods

i. Collection and preparation of plant material

ii. Preparation of extracts

iii. Microorganisms

iv. Phytochemical screening

v. Ultra-performance liquid chromatography-quadrupole time of flight-mass spectrometry (UPLC-QTOF-MS) analysis

vi. Antioxidant activity

- Determination of antioxidant combination index (CI)

vii. Antibacterial assay

- Synergistic effect: Fractional Inhibitory Concentration (FIC)

viii. Statistical analysis

Results and Discussion

Qualitative phytochemical screening

Total phenolic and flavonoid content

UPLC analysis

Antioxidant activity

Antibacterial activity

Conclusion

Declarations

Data curation

Investigation

Additional information

Figure 1.

Funding

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

Status:

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