# Triterpene and Steroids from Ludwigia abyssinica A. Rich (Onagraceae) Displayed Antimicrobial Activities and Synergistic Effects with Conventional Antibiotics

Arlette Meli Sonkoue, Irene Chinda Kengne, Stephen Tamekou Lacmata, Claudia Darille Jouogo Ngnokam, Mahamat Djamalladine Djamalladine, Laurence Voutquenne-Nazabadioko, David Ngnokam, Jean-De-Dieu Tamokou

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Arlette Meli Sonkoue, Irene Chinda Kengne, Stephen Tamekou Lacmata, Claudia Darille Jouogo Ngnokam, Mahamat Djamalladine Djamalladine, et al. Triterpene and Steroids from Ludwigia abyssinica A. Rich (Onagraceae) Displayed Antimicrobial Activities and Synergistic Effects with Conventional Antibiotics. Evidence-Based Complementary and Alternative Medicine, 2023, 2023, pp.1-8.
10.1155/2023/2975909. hal-04066307

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Research Article

# Triterpene and Steroids from *Ludwigia abyssinica* A. Rich
(Onagraceae) Displayed Antimicrobial Activities and Synergistic
Effects with Conventional Antibiotics

Arlette Meli Sonkou,<fnref n="1" /> Irene Chinda Kengne,<fnref n="2" /> Stephen Tamekou Lacmata ID,<fnref n="2" />

Claudia Darille Jouogo Ngnokam,<fnref n="1" /> Mahamat Djamalladine Djamalladine,<fnref n="1" />

Laurence Voutquenne-Nazabadioko,<fnref n="3" /> David Ngnokam,<fnref n="1" /> and Jean-de-Dieu Tamokou ID<fnref n="2" />

Correspondence should be addressed to Jean-de-Dieu Tamokou; jtamokou@yahoo.fr

Received 14 September 2021; Revised 14 December 2021; Accepted 23 March 2023; Published 10 April 2023

Academic Editor: Harish Chandra

Copyright © 2023 Arlette Meli Sonkou et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Difficulties encountered in treating drug-resistant pathogens have created a need for new therapies. Synergistic combinations of
antibiotics are considered as ideal strategies in combating clinical and multidrug-resistant (MDR) infections. In this study, the
antimicrobial activities of triterpenes and steroids from *Ludwigia abyssinica* A. Rich (Onagraceae) and their combined effects with
antibiotics were assessed. The associations between plant constituents and antibiotics were evaluated by determining their
fractional inhibitory concentrations (FICs). Sitost-5-en-3β-ol formate (1), 5α,6β-dihydroxysitosterol (2), and maslinic acid (3)
were isolated from the *L. abyssinica* ethyl acetate (EtOAc) extract. The EtOAc extract, compounds 1, 2, and 3 (MIC = 16–128 µg/
mL) would be the best antibacterial and antifungal agents. The antimicrobial activities of amoxicillin were relatively weak against
MDR *Escherichia coli* and *Shigella flexneri* and significant against *Staphylococcus aureus* ATCC 25923. However, when used in
association with plant constituents, it displayed an interesting synergistic effect. Among plant components-antibiotic combi-
nations, the EtOAc extract and compound 1 (steroid) showed a synergistic effect with amoxicillin/fluconazole against all the tested
microorganisms whereas the association of compound 3 (triterpenoid) and amoxicillin/fluconazole displayed an additive effect
against *Shigella flexneri* and *Escherichia coli* and a synergistic effect on *Staphylococcus aureus*, *Cryptococcus neoformans*, *Candida
tropicalis*, and *Candida albicans* ATCC 10231. Overall, the results of the present study demonstrated antibacterial and antifungal
activities of extracts and compounds isolated from *L. abyssinica*. The findings of the current study also showed that the potency of
antibiotics was improved when screened in combination with *L. abyssinica* components, supporting the drug combination
strategy to combat antimicrobial resistance.

**1. Introduction**

Regardless of the significant progress noted in medical
sciences, infections are still the main cause of illnesses and
deaths worldwide [1]. Due to the often inappropriate use of
antibiotic drugs by the population, microbes have developed

resistance strategies which have culminated to treatment
failure and further increased the cost of treatment. The mode
of action of antibiotic resistance, stemming from long du-
ration of use, is the generation of multiple novel genes, with
resistant attributes to one drug [2]. This mechanism of
action in a bacterial cell has culminated in the evolution of

<footnote n="1">¹Research Unit of Applied and Environmental Chemistry, Department of Chemistry, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon</footnote>

<footnote n="2">²Research Unit of Microbiology and Antimicrobial Substances, Department of Biochemistry, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon</footnote>

<footnote n="3">³Groupe Isolement et Structure, Institut de Chimie Moléculaire de Reims (ICMR), CNRS UMR 7312, Bat. 18 B.P. 1039, 51687 Reims Cedex 2, Reims, France</footnote>
bacteria with multiple resistances to drugs. Some diseases caused by MDR bacterial strains are without cure and are deadly as a result of the increased resistance rate in most of the therapeutic antibiotics [3].

Some methods have been developed to counter bacterial drug resistance. Generating new antibiotic substances is one common approach. Nonetheless, making new antibacterial substances can be challenging and new resistance tactics will arise following the large clinical use. A new way to counter the resistance is implementing combination therapy, where at least two antibacterial drugs are used to strengthen antibacterial actions against the resistant infectious microbes. Chemical substances obtained from plants are great sources for therapeutic combinations. The natural triterpenes and steroids are documented for their antibiotic attributes and activity. Proof of this is considered thoughtfully through their destructive effects on the microbial structure as well as functional interruption on cell membranes and cell walls [4]. An effective strategy to overwhelm antimicrobial drug resistance, chemical damage to cell, and prolong treatments with the antibiotics at hand is combinatorial therapy or synergistic interaction. Drug action effects in killing microbes such as inhibition, addition, or synergy have been observed following the combinations of triterpenes and antibiotics. For instance, a synergistic effect between oleanolic acid and ethambutol, rifampicin, or isoniazid targeting *Mycobacterium tuberculosis* have been reported in vitro [5]; while synergism has also been observed in many triterpenoids (betulinic acid, imberbic acid, rotundic acid, ulsolic acid, oleanic acid, betaboswellic acid, and cycloastragenol)-antibiotic combinations targeting multiresistant *Staphylococcus aureus* variants [6, 7]. Due to increasing antimicrobial resistance, the search for alternative compounds with potential microbicidal effects has become top-priority. In the traditional practice of medicine, plants are commonly used as therapeutic sources to treat infections.

*Ludwigia* is a large genus within the Onagraceae family, which is widespread in both temperate regions such as Europe, tropical and subtropical regions like Africa, Australia, and the West Indies [8]. Many species belonging to this genus constitute plants used for medicines in the traditional sector of practice [9, 10]. Early reports on the *Ludwigia* genus highlighted the presence of bioactive secondary metabolites such as flavonoids [11, 12], cerebroside, and triterpenoids [12, 13]. Many of them possess various pharmacological properties such as antidiarrheal [3], antitumoral and antibacterial [13, 14], antimicrobial, and antioxidant [15, 16] properties. Furthermore, recent studies have demonstrated that alcohol extracts of *L. abyssinica* leaves as well as those of *L. octovalvis* and *L. decurrens* do possess antioxidant, antimycotic, and antibacterial attributes [8, 17]. These studies revealed the antimicrobial potential of *L. abyssinica* but without any information on its chemical components as well as antimicrobial effects. As we seek for compounds with the microbe-interruptive biological activity from Cameroon medicinal plants, we carried out chemical and biological investigations of *L. abyssinica*. This present study focused on isolating and deciphering the structure of

two steroids: sitost-5-en-3β-ol formiate (1), 5α and 6β-dihydroxystosterol (2) and one triterpene: maslinic acid (3) along with the evaluation of their antibacterial and antifungal properties. This work also aimed at evaluating and finding any interactive relations between maslinic acid and sitost-5-en-3β-ol formiate isolated from *L. abyssinica* in the presence of either amoxicillin or fluconazole on MDR and reference strains as model organisms.

## 2. Materials and Methods

### 2.1. Spectroscopic and Chromatographic Analyses.
Infrared (IR) spectra of isolated compounds were recorded with a Shimadzu FT-IR-8400S spectrophotometer (Shimadzu, France). Nuclear magnetic resonance (NMR) for ¹H and ¹³C were recorded at 500 MHz and 125 MHz, respectively, with a Bruker Avance DRX-500 spectrometer (Bruker, Wissembourg, France) constituting a BBFO + 5 mm probe. NMR spectra measurement of ¹H at 600 MHz and ¹³C at 150 MHz were performed with a tetramethylsilane (TMS) internal standard-built Bruker Avance III-600 spectrometer (Bruker, Wissembourg, France) using CD₃OD as cryoplatform. Column chromatographic analysis was achieved through Merck silica gel with a pore size of 60 Å &70–230 mesh (VWR, France). Sephadex LH-20 chromatographic medium (VWR, France) was used for gel permeation chromatography. Thin layer chromatography was applied on precoated silica gel GF254. The plates were revealed by spraying with 10% of H₂SO₄ and heated at 100°C in an oven. Alternatively, using detection with ultraviolet visualization with the lamp at 254 and 365 nm was also fine.

### 2.2. Plant Material.
*Ludwigia abyssinica* whole plant was obtained in Dschang, a university city in the West Region of Cameroon during the month of September 2016. Plant validity was confirmed with reference N° 40753/HCN in the National Herbarium in Yaounde.

### 2.3. Plant Component Obtention and Isolation.
Five kilograms of the ambient-dried plant were crushed into powder and plant compounds were extracted with methanol (MeOH) for 72 hours. The extract was evaporated at low pressure and a 215 g dark crude extract was obtained. 210 g of this crude extract was dissolved in 400 ml of distilled water. Ethyl acetate (EtOAc) and *n*-butanol (*n*-BuOH) were used sequentially for further extraction of the dissolved extract. This yielded dried and concentrated ethyl acetate (80 g) and *n*-butanol (15 g) extracts. 75 g of EtOAc extract was purified over a silica gel column and hexane elution prepared with increasing EtOAc concentrations at 10 magnitude difference from 10 to 80%. Thin-layer chromatography was used to combine them in 9 subfractions: A, B, C, D, E, F, G, H, and I. Fraction B (3.58 g) was subjected over a silica gel column eluted and the hexane-EtOAc (90 : 10) purification yielded 14 g of compound 1. The purification of fraction F (4 g) over a silica gel column eluted with a hexane-EtOAc mixture (60 : 40) gave 10 mg of compound 2. The
elution of fraction E (8 g) over a silica gel column chromatography with a hexane-EtOAc mixture (70:30) yielded 30 mg of compound 3.

## 2.4. Antimicrobial Activity Test for Plant Components

### 2.4.1. Test Organisms.
Selected organisms for testing purified substances were three bacteria (Staphylococcus aureus ATCC 25923, Escherichia coli S2, and Shigella flexneri SDINT) and three yeasts (Candida albicans ATCC 10231, Candida tropicalis PK233, and Cryptococcus neoformans H99). All these microbes came from our laboratory bank of preserved microbes. These microbes were conserved at 4°C in nutrient agar (Conda, Madrid, Spain) for the bacteria and in Sabouraud dextrose agar (SDA, Conda, Spain) for yeasts before testing for antimicrobial properties.

### 2.4.2. Investigation of Inhibitory and Microbicidal Concentrations.
Antimicrobial activities of plant extracts and isolated compounds were assayed by analyzing their inhibitory, bactericidal, and fungicidal effects via minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), and minimum fungicidal concentrations (MFCs). The broth microdilution technique [18] with few modifications was used to establish MICs of the test samples. Obtained extracts/compounds were prepared in dimethylsulfoxide (DMSO, Fisher chemicals) to yield a solution stock. The latter was serially diluted two times in Sabouraud dextrose broth (SDB)/Mueller-Hinton broth (MHB) to attain concentration ranges of 512 to 0.50 µg/mL and 4096 to 32 µg/mL for extracts and isolated compounds, respectively. 10 µL of bacteria ($1 \times 10^6$ CFU/mL) and $1 \times 10^5$ spores/mL of yeast cells were prepared with each concentration in 90 µL MHB or SDB culture medium to a final volume of 100 µL. This yielded a final concentration span for pure compounds of 256 to 0.25 µg/mL and 2048 to 16 µg/mL for acquired extracts. Incubation ensued on a shaker are as follows: 24 h, 37°C for bacteria, 48 h, 30°C for Candida spp,

and 72 h, 30°C for Cryptococcus neoformans. To determine MIC, 50 µL of violet-colored p-iodonitrotetrazolium solution (0.2 mg/mL) was pipetted into wells and incubated for 30 minutes at 35°C. Viable microbes produced a pink coloration from the initial yellow one. The smallest sample concentration values (i.e. MIC) that altered this color change was considered to have offered maximum inhibition of bacteria or yeast growth. To establish minimum microbicidal concentration (MMC), 10 µL of solution from the aliquots of growth-negative wells was added on MHA and SDA and incubation for each organism done respectively as indicated earlier. The least concentration for which no growth was observed after subculturing was considered as MBCs or MFCs [19]. The antibiotic positive control for the bacteria was amoxicillin and fluconazole for yeasts, and these two products were from Sigma-Aldrich in the city of Steinheim in Germany. The negative control used was 20 µL of DMSO in the respective broth. Experiments were repeated thrice for each.

### 2.4.3. Interactions between Antibiotics and the EtOAc Extract, Compounds 1 or 3.
The antimicrobial properties of an association of antibiotics (amoxicillin and fluconazole) with test samples (the EtOAc extract, compounds 1 and 3), which showed the largest biological activity, were investigated using the checkerboard method as illustrated earlier [20]. The microbe seeding broth medium was made ready as directed earlier above. A plate of 96 wells was seeded with test microbes and stepwise dilutions were done with a pair of antimicrobial substances: antibiotic and the test sample. Each of the wells contained a mixture of the test sample with antibiotic at a defined concentration. Incubation of 96-well plates was done at 37°C for 24 h. MICs were obtained as indicated earlier above. All analyses were repeated three times. Interactive effects between antimicrobial substances were uncovered using the fractional inhibitory concentration (FIC) indices calculated as follows:

$$ \text{FIC} = \frac{\text{MIC of antibiotic tested in combination}}{\text{MIC of antibiotic tested alone}} + \frac{\text{MIC of extract or compound tested in combination}}{\text{MIC of extract or compound tested alone}}. \quad (1) $$

For FIC interpretation, a synergistic effect was indicated by a FIC value less than or equal to 0.5 (FIC ≤ 0.5). An addictive effect was identified when the FIC was less than or equal to 1 and more than 0.5 (0.5 < FIC ≤ 1). An indifferent effect was known when the FIC was less than or equal to 2 and more than 1 (1 < FIC ≤ 2). An antagonistic effect was declared when the FIC was above 2 (FIC > 2.0).

## 3. Results

### 3.1. Chemical Analysis.
In agreement with antimicrobial activities of the MeOH and EtOAc extracts, the EtOAc extract was further fractionated and purified. Three compounds were isolated and their structures (Figure 1) have

been determined on the basis of spectroscopic data ($^1$H and $^{13}$C NMR, $^1$H-$^1$H COSY, HSQC, HMBC, ROESY, and NOESY) and by comparison to those published in the literature. Hence, the isolates were identified as sitost-5-en-3$\beta$-ol formiate (1) [21]; 5$\alpha$,6$\beta$-dihydroxysitosterol (2), [22] and maslinic acid (3) [23] (Figure 1).

Sitost-5-en-3$\beta$-ol formiate (1): Colorless needles; $C_{30}H_{50}O_2$; $^{13}$C NMR (100 MHz, CDCl$_3$) $\delta_C$ 36.9 (C-1), 27.7 (C-2), 73.9 (C-3), 38.0 (C-4), 139.2 (C-5), 122.9 (C-6), 31.9 (C-7), 31.8 (C-8), 50.0 (C-9), 36.5 (C-10), 21.1 (C-11), 39.9 (C-12), 42.3 (C-13), 56.6 (C-14), 26.0 (C-15), 28.2 (C-16), 56.0 (C-17), 11.8 (C-18), 19.2 (C-19), 36.1 (C-20), 18.6 (C-21), 31.0 (C-22), 26.0 (C-23), 46.0 (C-24), 29.1 (C-25), 11.8 (C-26), 18.7 (C-27), 23.0 (C-28), 12.0 (C-29), 160.2 (OCHO,
<figure><img src="image_2.png" /><figcaption>FIGURE 1: Structures of the isolated compounds from the whole plant of *Ludwigia abyssinica*.</figcaption></figure>

C-3). Its HMBC spectrum allowed us to confirm the
sitostane skeleton through the correlations observed between
methyl groups and neighbouring carbons. This spectrum
also confirmed the location of the formiate group at C-3
through the correlation observed between the proton H-3 at
$\delta_H$ 4.76 ppm and the carbonyl group at $\delta_C$ 160.6 ppm
(OCHO) (Figure 2).

5α,6β-dihydroxysitosterol (2): Colorless needles; C₂₉H₅₀O₃; ¹³C NMR (100 MHz CDCl₃): 34.2 (C-1), 30.2 (C-2), 75.4 (C-3), 40.4 (C-4), 75.8 (C-5), 67.2 (C-6), 32.2 (C-7), 30.8 (C-8), 45.4 (C-9), 38.0 (C-10), 21.0 (C-11), 36.0 (C-12), 42.7 (C-13), 56.1 (C-14), 24.6 (C-15), 28.4 (C-16), 55.8 (C-17), 11.8 (C-18), 16.5 (C-19), 30.1 (C-20), 19.3 (C-21), 33.8 (C-22), 24.0 (C-23), 45.7 (C-24), 29.1 (C-25), 18.9 (C-26), 19.6 (C-27), 22.9 (C-28), 12.0 (C-29). The correlations observed on its HMBC spectrum between the H-3 (3.42 ppm), H-19 (1.10 ppm), and the carbon C-5 (75.8 ppm) on the one hand and between H-6 (3.97 ppm) and carbons C-4 (40.4 ppm) and C-6 (67.2 ppm) on the other allowed us to locate the hydroxyl groups at C-3, C-5, and C-6.

Maslinic acid (3): White crystals; C₃₀H₄₈O₄; ¹³C NMR (100 MHz CD₃Cl₃): 47.0 (C-1), 68.0 (C-2), 82.8 (C-3), 38.6 (C-4), 55.3 (C-5), 18.0 (C-6), 32.0 (C-7), 39.0 (C-8), 47.5 (C-9), 38.0 (C-10), 22.6 (C-11), 122.2 (C-12), 143.9 (C-13), 41.5 (C-14), 27.2 (C-15), 22.5 (C-16), 41.5 (C-17), 45.9 (C-18), 46.6 (C-19), 29.8 (C-20), 33.0 (C-21), 32.4 (C-22), 28.0 (C-23), 15.6 (C-24), 16.3 (C-25), 16.2 (C-26), 25.1 (C-27), 180.4 (C-28), 32.2 (C-29), 22.2 (C-30). The main correlations observed on its HMBC spectrum were in agreement with this structure and are shown in Figure 2.

**3.2. Antimicrobial Activities.** The antimicrobial activities of MeOH and EtOAc extracts and compounds isolated from the whole of *L. abyssinica* have been assessed against bacteria (*Shigella flexneri*, *Escherichia coli*, and *Staphylococcus aureus*)

and yeasts (*Cryptococcus neoformans*, *Candida tropicalis*, and *Candida albicans*) (Table 1) selected based on their relevance as human pathogens. The extracts and isolates showed different MICs depending on the test microorganism (Table 1). The MICs of the MeOH and EtOAc extracts against bacteria and yeasts varied from 64 to 512 µg/mL, demonstrating that the *L. abyssinica* can be a potent antimicrobial medicinal plant. Moreover, the antimicrobial activity of the EtOAc extract was greater than that of the MeOH extract. Compound 1 was the most active compound followed in decreasing order by compounds 2 and 3. The antifungal and antibacterial properties of all the tested substances were lesser than those of fluconazole and amoxicillin used as reference drugs. The lowest MIC (16 µg/mL) and MMC (32 µg/mL) values were recorded with compound 1 against *S. aureus*. The largest MIC (512 µg/mL) and MMC (1024 µg/mL) values were noted against *C. albicans* with the MeOH extract. In general, the bacterial species were more sensitive to the extracts/isolates in comparison to the fungal species.

**3.3. Association between Antibiotics and the EtOAc Extract, Compounds 1 or 3.** In order to determine the interaction effect between antibiotics and plant components (the plant extract and isolated compounds), FICIs were evaluated. The results (Table 2) showed an additive response in the case of compound 3 in combination with amoxicillin against multiresistant *E. coli* and *S. flexneri*. However, synergism was also recorded when this compound was used in conjunction with amoxicillin or fluconazole against *S. aureus*, *Candida albicans*, *C. tropicalis*, and *Cryptococcus neoformans*. Interestingly, the EtOAc extract and compound 1 displayed synergistic effects with antibiotics against all the tested pathogenic bacteria and yeasts. Among plant components-antibiotic combinations, the most effective were the EtOAc extract and amoxicillin (FICI = 0.093), compound 1 and
<figure><img src="image_3.png" /><figcaption>FIGURE 2: HMBC correlations of compounds 1–3.</figcaption></figure>

<table><caption>TABLE 1: Antimicrobial activity (MIC and MMC in µg/mL) of *Ludwigia abyssinica* components and reference antibiotics.</caption><thead><tr><th>Extracts/compounds</th><th>Inhibition parameters</th><th>E. coli</th><th>S. flexneri</th><th>S. aureus</th><th>C. tropicalis</th><th>C. albicans</th><th>C. neoformans</th></tr></thead><tbody><tr><td rowspan="3">MeOH extract</td><td>MIC</td><td>128</td><td>128</td><td>64</td><td>256</td><td>512</td><td>128</td></tr><tr><td>MMC</td><td>128</td><td>128</td><td>64</td><td>256</td><td>1024</td><td>256</td></tr><tr><td>MMC/MIC</td><td>1</td><td>1</td><td>1</td><td>1</td><td>2</td><td>2</td></tr><tr><td rowspan="3">EtOAc extract</td><td>MIC</td><td>64</td><td>64</td><td>64</td><td>128</td><td>128</td><td>128</td></tr><tr><td>MMC</td><td>128</td><td>64</td><td>64</td><td>256</td><td>256</td><td>128</td></tr><tr><td>MMC/MIC</td><td>2</td><td>1</td><td>1</td><td>2</td><td>2</td><td>1</td></tr><tr><td rowspan="3">1</td><td>MIC</td><td>32</td><td>32</td><td>16</td><td>64</td><td>64</td><td>32</td></tr><tr><td>MMC</td><td>64</td><td>64</td><td>32</td><td>&gt;256</td><td>&gt;256</td><td>64</td></tr><tr><td>MMC/MIC</td><td>2</td><td>2</td><td>2</td><td>—</td><td>—</td><td>2</td></tr><tr><td rowspan="3">2</td><td>MIC</td><td>32</td><td>64</td><td>128</td><td>64</td><td>64</td><td>32</td></tr><tr><td>MMC</td><td>64</td><td>128</td><td>&gt;256</td><td>&gt;256</td><td>&gt;256</td><td>&gt;256</td></tr><tr><td>MMC/MIC</td><td>2</td><td>2</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td rowspan="3">3</td><td>MIC</td><td>64</td><td>64</td><td>32</td><td>64</td><td>128</td><td>64</td></tr><tr><td>MMC</td><td>128</td><td>128</td><td>64</td><td>&gt;256</td><td>&gt;256</td><td>&gt;256</td></tr><tr><td>MMC/MIC</td><td>2</td><td>2</td><td>2</td><td>—</td><td>—</td><td>—</td></tr><tr><td rowspan="3">Ref<sup>a</sup></td><td>MIC</td><td>16</td><td>16</td><td>1</td><td>0.5</td><td>1</td><td>2</td></tr><tr><td>MMC</td><td>16</td><td>16</td><td>1</td><td>0.5</td><td>1</td><td>2</td></tr><tr><td>MMC/MIC</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr></tbody></table>

—: not determined; MIC: minimum inhibitory concentration; MMC minimum microbicidal concentration; *: fluconazole and amoxycillin for yeasts and bacteria, respectively.

amoxicillin (FICI = 0.156) against clinical strain *S. aureus* and the EtOAc extract and fluconazole, compound **1** and fluconazole against clinical strain *C. neoformans* (FICI = 0.093). Overall, the combinations between plant components and antibiotics showed 100% reduction in MICs of antibiotics when antibiotics were alone used.

## 4. Discussion

The present study shows antibacterial and antifungal activities of the MeOH extract from the whole plant *L. abyssinica*. These results corroborate those of the previous

studies which reported the antifungal and antibacterial activities of the extracts from the *L. abyssinica* leaves [8, 17] and from different plant parts of *Ludwigia erecta* [24]. These findings also demonstrate that the antimicrobial activities of the EtOAc extract are greater than those of the MeOH extract. This difference in activities can be explained by the fact that the two solvents have different polarities and, therefore, their extracted metabolites would be different [25]. The antimicrobial properties of the isolates were generally higher than those of their extracts. These results are in agreement to those of Nzogong et al. [26] who showed an increase of the antimicrobial activity with the fractionation
<table><caption>TABLE 2: Interactions of *L. abyssinica* components and antibiotics against bacteria and yeasts.</caption><thead><tr><th rowspan="2">Microorganisms</th><th colspan="5">EtOAc extract</th><th colspan="4">Compound 1</th><th colspan="4">Compound 3</th></tr><tr><th>FICA</th><th>FICEx</th><th>FICI</th><th>Interpretation</th><th>FICA</th><th>FIC2</th><th>FICI</th><th>Interpretation</th><th>FICA</th><th>FIC5</th><th>FICI</th><th>Interpretation</th></tr></thead><tbody><tr><td><i>E. coli</i></td><td>0.125</td><td>0.125</td><td>0.375</td><td>Synergistic</td><td>0.25</td><td>0.125</td><td>0.375</td><td>Synergistic</td><td>0.50</td><td>0.25</td><td>0.75</td><td>Additive</td></tr><tr><td><i>S. flexneri</i></td><td>0.125</td><td>0.062</td><td>0.187</td><td>Synergistic</td><td>0.25</td><td>0.125</td><td>0.375</td><td>Synergistic</td><td>0.50</td><td>0.125</td><td>0.625</td><td>Additive</td></tr><tr><td><i>S. aureus</i></td><td>0.062</td><td>0.031</td><td>0.093</td><td>Synergistic</td><td>0.125</td><td>0.031</td><td>0.156</td><td>Synergistic</td><td>0.25</td><td>0.125</td><td>0.375</td><td>Synergistic</td></tr><tr><td><i>C. tropicalis</i></td><td>0.25</td><td>0.125</td><td>0.375</td><td>Synergistic</td><td>0.25</td><td>0.125</td><td>0.375</td><td>Synergistic</td><td>0.25</td><td>0.25</td><td>0.50</td><td>Synergistic</td></tr><tr><td><i>C. albicans</i></td><td>0.125</td><td>0.125</td><td>0.25</td><td>Synergistic</td><td>0.125</td><td>0.062</td><td>0.187</td><td>Synergistic</td><td>0.25</td><td>0.125</td><td>0.375</td><td>Synergistic</td></tr><tr><td><i>C. neoformans</i></td><td>0.062</td><td>0.031</td><td>0.093</td><td>Synergistic</td><td>0.062</td><td>0.031</td><td>0.093</td><td>Synergistic</td><td>0.25</td><td>0.125</td><td>0.375</td><td>Synergistic</td></tr></tbody></table>

FICA: MIC of antibiotic tested in combination/MIC of antibiotic tested alone; FICEx: MIC of extract tested in combination/MIC of extract tested alone; FIC2: MIC of compound 1 tested in combination with antibiotic/MIC of compound 1 tested alone; FIC4: MIC of compound 3 tested in combination with antibiotic/MIC of compound 3 tested alone; FIC: MIC of antibiotic tested in combination/MIC of antibiotic tested alone + MIC of extract/compound tested in combination/MIC of extract/compound tested alone; antibiotics: ciprofloxacin for bacteria and fluconazole for yeasts.

of extracts from the medicinal plants. Our results also demonstrate that the MICs of the plant components are four times lesser than the MBCs on the corresponding microorganisms, highlighting the microbicidal effects of most samples tested [25]. Plant extracts are consistently classified as antimicrobials on the basis of susceptibility tests that produce MIC values [27]. Hence, the MeOH extract of *L. abyssinica* was highly (MIC < 100 µg/mL)/significantly (100 ≤ MIC ≤ 512 µg/mL) active against the test bacterial species and significantly active against all the yeasts. The EtOAc extract was highly active against the test bacterial species and significantly active against yeasts. Interestingly, this study is the first to report on the isolation and structural elucidation of two steroids: sitost-5-en-3β-ol formiate (1) and 5α,6β-dihydroxysitosterol (2) and one triterpene: maslinic acid (3) with their antibacterial and antifungal properties. With the exception of compound 2 which demonstrated a moderate activity against *C. neoformans*, a low antifungal activity was noted with all compounds against *C. albicans*, *C. neoformans*, and *C. tropicalis* at the concentration range between 100 and 1000 µg/mL. Based on the antimicrobial cutoff points defined in the literature [27], the antimicrobial activity of compounds 1 and 2 could be considered as moderate against bacteria and yeasts. Compound 3 exhibited a moderate activity against *E. coli*, *S. aureus*, *S. flexneri*, and *C. neoformans* and a low activity against *C. tropicalis* and *C. albicans*. The presence of hydroxyl (-OH) and formyl (-OCHO) groups in positions 6 and 1 of compounds 1 and 2, respectively, may explain the differences in the activity observed between these two compounds. The strains of *E. coli* S2 (1) and *Shigella flexneri* [28, 29] employed in this study are MDR clinical strains and they are resistant to commonly used antibiotics such as cotrimoxazole, tetracycline, streptomycin, ampicillin, furazolidone, and nalidixic acid. Most of the plant components displayed moderated antibacterial activities against these MDR strains, indicating that their administration may represent an alternative treatment against MDR *S. flexneri* and *E. coli*.

*Shigella* species, which are the causative organism of shigellosis, were formally sensitive to cotrimoxazole, chloramphenicol, ampicillin, and nalidixic acid but have presently become resistant to cephalosporins, azithromycin, and fluoroquinolones [30]. Most of the

reported cases of shigellosis have been due to resistant strains of *Shigella* species. Emergent *E. coli* presenting different MDR phenotypes to three or more different antimicrobial agents have been reported and are responsible for serious health problems [31–33]. Hence, it is crucial to analyze multidrug-resistant*Shigella*/*Escherichia* and find new treatment modalities. In this work, we assessed the possibility of using *L. abyssinica* extract, maslinic acid (pentacyclic triterpenoid), and sitost-5-en-3β-ol formiate (steroid) as natural adjuvants for antibiotics against bacteria and fungi. FICI results depict an additive/synergistic relationship between maslinic acid and antibiotics. A synergistic effect was also observed between the plant extract/sitost-5-en-3β-ol formiate and antibiotics. The sum of the results shows that the activity of both plant extract/isolated compounds and antibiotics increased when they are combined with each other. These results are in conformity with the earlier findings where myrcene, R-limonene, β-elemene, sabinene, and S-limonene were found to be synergistic with first-line tuberculostatic antibiotics against isolated *Mycobacterium tuberculosis* [34]. Studies have reported the synergistic activity of numerous combinations of triterpenoids (rotundic acid, imberbic acid, ulsolic acid, betulinic acid, oleanolic acid, cycloastragenol, beta-boswellic acid, and ursolic acid) and antibiotics against multiresistant *Staphylococcus aureus* strains [6, 7]. To the best of our knowledge, this is a pioneer report on the plant extract/maslinic acid/sitost-5-en-3β-ol formiate and antibiotic associations against clinical strains.

The tested triterpene and steroids are relatively weaker than antibiotics. However, when assessed in association with antibiotics, they show relevant a synergistic effect and thus can help in prolonging the viability of these antibiotics against bacterial and fungal infections. In addition, reduction in the MIC value of amoxicillin and fluconazole with the plant extract, compounds 1 and 3 indicates their potential use against MDR *S. flexneri* and *E. coli*. The activity of antibiotics was further enhanced against bacteria and yeasts when these antibiotics were used in combination with the plant extract, compounds 1 or 3. Test microorganisms were sensitive to amoxicillin and fluconazole when these antibiotics were mixed with plant components. Similarly, the amoxicillin activity was further increased against multi-resistant *E. coli* and *S. flexneri* strains when combined with
the plant components. Through MIC determinations, it was observed that both the plant extract/isolated compounds and antibiotics supplemented each other's effects. Amoxicillin is a beta-lactam antibiotic, which acts by binding to penicillin-binding proteins that inhibit the transpeptidation process, leading to the activation of autolytic enzymes in the bacterial cell wall [35], while fluconazole acts by inhibiting the conversion of lanosterol to ergosterol through the binding to fungal cytochrome P-450 leading to the disruption of fungal membranes [36]. The triterpenoids can theoretically act in a way that makes fungi/bacteria more sensitive to these antibiotics by destroying biofilms [37] or they can use some other pathways to exert the antimicrobial effect.

## 5. Conclusion

Overall, the results of this investigation demonstrate the antibacterial and antifungal activities of the extracts and compounds isolated from *L. abyssinica*. The EtOAc extract, compounds 1 and 3, act in synergy with amoxicillin/fluconazole and thus can help in prolonging the viability of these antibiotics against bacterial and fungal infections, particularly those caused by multidrug resistant *E. coli* and *S. flexneri*.

## Abbreviations

*¹³C-NMR:* Carbon thirteen nuclear magnetic resonance

*¹H-NMR:* Proton nuclear magnetic resonance

*2D NMR:* Two-dimension nuclear magnetic resonance

*ATCC:* American Type Culture Collection

*CC:* Column chromatography

*COSY:* Correlation spectroscopy

*DMSO:* Dimethylsulfoxide

*EtOAc:* Ethyl acetate

*HMBC:* Heteronuclear multiple bond connectivities

*HNC:* Herbier National du Cameroun

*HR-EI-MS:* High resolution electron impact mass spectrometry

*HR-:* High-resolution time of flight electrospray

*TOF-IMS:* Ionization mass spectrometry

*HSQC:* The heteronuclear single quantum coherence

*IR:* Infrared

*MBC:* Minimum bactericidal concentration

*MDR:* Multidrug resistant

*MeOH:* Methanol

*MHA:* Mueller-Hinton agar

*MHB:* Mueller-Hinton broth

*MIC:* Minimum inhibitory concentration

*MMC:* Minimum microbicidal Concentration

*NA:* Nutrient agar

*n-BuOH:* n-Butanol

*NMR:* Nuclear magnetic resonance

*Rf:* Retention factor

*TLC:* Thin-layer chromatography

*TMS:* Tetramethylsilane

*TOF-:* Time of flight electrospray ionization mass

*ESIMS:* Spectrometry

*UV:* Ultraviolet.

## Data Availability

The datasets used and analyzed to support the findings of this study are available from the corresponding authors upon request.

## Conflicts of Interest

The authors declare that there are no conflicts of interest with regard to the publication of this article.

## Authors' Contributions

AMS, ICK, STL, CDJN, and MDD contributed to data collection and analysis. JDT designated the study, carried out the biological assays, and helped in manuscript writing and editing. LVN, DN, and JDT supervised and revised the manuscript critically for important intellectual content. All authors read and agreed on the final version of the manuscript.

## Acknowledgments

The authors are grateful to the University of Dschang for financing some consumables used in this work; they also thank the Laboratory of Microbiology and Antimicrobial Substances, the Department of Biochemistry, the Faculty of Science, the University of Dschang; the “Service Commun d'Analyses” and “Groupe Isolement et Structure”; and the “Institut de Chimie Moléculaire de Reims” for the spectroscopic and spectrometric analysis on the ESIMS and NMR equipment of the PIAnet Platform. The EU-programme FEDER to the PIAnet CPER project was gratefully acknowledged.

## References

[1] R. C. Moellering, J. R. Graybill, J. E. McGowan, and L. Corey, “Antimicrobial resistance prevention initiative—an update: proceedings of an expert panel on resistance,” *The American Journal of Medicine*, vol. 120, no. 7, pp. 4–25, 2007.

[2] A. Haq, M. Siddiqi, S. Z. Batool et al., “Comprehensive investigation on the synergistic antibacterial activities of Jatropha curcas pressed cake and seed oil in combination with antibiotics,” *AMB Express*, vol. 9, no. 1, p. 67, 2019.

[3] H. Nikaido, “Multidrug resistance in bacteria,” *Annual Review of Biochemistry*, vol. 78, no. 1, pp. 119–146, 2009.

[4] J. D. D. Tamokou, M. F. Tala, H. K. Wabo, J. R. Kuiate, and P. Tane, “Antimicrobial activities of methanol extract and compounds from stem bark of Vismia rubescens,” *Journal of Ethnopharmacology*, vol. 124, no. 3, pp. 571–575, 2009.

[5] F. Ge, F. Zeng, S. Liu et al., “In vitro synergistic interactions of oleanolic acid in combination with isoniazid, rifampicin or ethambutol against *Mycobacterium tuberculosis*,” *Journal of Medical Microbiology*, vol. 59, no. 5, pp. 567–572, 2010.

[6] P. Y. Chung, P. Navaratnam, and L. Y. Chung, “Synergistic antimicrobial activity between pentacyclic triterpenoids and antibiotics against *Staphylococcus aureus* strains,” *Annals of Clinical Microbiology and Antimicrobials*, vol. 10, no. 1, p. 25, 2011.

[7] M. Hamza, M. Nadir, N. Mehmood, and A. Farooq, “In vitro effectiveness of triterpenoids and their synergistic effect with
antibiotics against *Staphylococcus aureus* strains,” *Indian Journal of Pharmacology*, vol. 48, no. 6, pp. 710–714, 2016.

[8] O. Oyedeji, M. Oziegbe, and F. O. Taiwo, "Antibacterial, antifungal and phytochemical analysis of crude extracts from the leaves of Ludwigia abyssinica A. Rich and Ludwigia decurrens Walter," *Journal of Medicinal Plants Research*, vol. 5, pp. 1192–1199, 2010.

[9] M. Baerts and J. Lehmann, "Healers and medicinal plants of the region ridges zaire-nile in Burundi. Royal museum for central Africa, tervuren, Belgium," *Annals of Ecology Sciences*, vol. 18, p. 218, 1989.

[10] T. Murugesan, L. Ghosh, K. Mukherjee, J. Das, M. Pal, and B. P. Saha, "Evaluation of antidiarrhoeal profile of Jussiaea suffruticosa Linn. extract in rats," *Phytotherapy Research*, vol. 14, no. 5, pp. 381–383, 2000.

[11] J. E. Averett, E. M. Zardini, and P. C. Hoch, "Flavonoid systematics of ten sections of Ludwigia (Onagraceae)," *Biochemical Systematics and Ecology*, vol. 18, no. 7-8, pp. 529–532, 1990.

[12] F. D. Mabou, P. L. F. Tebou, D. Ngnokam, D. Harakat, and L. Voutquenne-Nazabadioko, "Leptocarposide: a new triterpenoid glycoside from Ludwigia leptocarpa (Onagraceae)," *Magnetic Resonance in Chemistry*, vol. 52, no. 1-2, pp. 32–36, 2014.

[13] F. D. Mabou, D. Ngnokam, D. Harakat, and L. Voutquenne-nazabadioko, "New oleanane-type saponins: leptocarposide B-D, from Ludwigia leptocarpa (Onagraceae)," *Phytochemistry Letters*, vol. 14, pp. 159–164, 2015.

[14] B. Das, J. Kundu, and S. C. Bachar, "Evaluation of antitumor and antibacterial activity of Ludwigia hyssopifolia Linn," *Bangladesh Journal of Botany*, vol. 31, pp. 15–18, 2002.

[15] S. P. C. Fodouop, D. Gastsing, and G. N. Teke, "Chemical constituent, antibacterial and antioxidant activity crude extract and oil fraction of Ludwigia abyssinica," *International Journal of Phytomedical*, vol. 6, pp. 170–176, 2014.

[16] F. D. Mabou, J. D. D. Tamokou, D. Ngnokam, L. Voutquenne-Nazabadioko, J. R. Kuiate, and P. K. Bag, "Complex secondary metabolites from <i>&gt;Ludwigia leptocarpa&lt; /i>&gt; with potent antibacterial and antioxidant activities," *Drug Discoveries & Therapeutics*, vol. 10, no. 3, pp. 141–149, 2016.

[17] H. K. Yakob, S. F. Sulaiman, and A. M. Uyub, "Antioxidant and antibacterial activity of Ludwigia octovalvis on *Escherichia coli* O157:H7 and some pathogenic bacteria," *World Applied Sciences Journal*, vol. 16, pp. 22–29, 2012.

[18] T. B. L. Nyaa, A. L. Tapondjou, and L. Barboni, "NMR assignment and antimicrobial/antioxidant activities of 1ß-hydroxyeuscaphic acid from the seeds of Butyrospermum parkii," *Natural Product Sciences*, vol. 15, pp. 76–82, 2009.

[19] D. L. F. Tsamo, J. D. D. Tamokou, I. C. Kengne et al., "Antimicrobial and antioxidant secondary metabolites from Trifolium baccarinii Chiov. (Fabaceae) and their mechanisms of antibacterial action," *BioMed Research International*, vol. 2021, Article ID 3099428, 15 pages, 2021.

[20] I. C. Kengne, L. D. T. Feugap, A. J. Njouendou et al., "Antibacterial, antifungal and antioxidant activities of whole plant chemical constituents of Rumex abyssinicus," *BMC Complementary Medicine and Therapies*, vol. 21, no. 1, p. 164, 2021.

[21] P. Chumkaew, S. Kato, and K. Chantrapromma, "New cytotoxic steroids from the fruits of Syzygium siamense," *Journal of Asian Natural Products Research*, vol. 12, no. 5, pp. 424–428, 2010.

[22] J. F. Carvalho, M. M. C. Silva, M. L. Sá e Melo, and M. L. S. Silva, "Efficient trans-diaxial hydroxylation of Δ5-steroids," *Tetrahedron*, vol. 66, no. 13, pp. 2455–2462, 2010.

[23] K. W. Woo, J. Y. Han, and S. U. Choi, "Triterpenes from perilla frutescens var. acuta and their cytotoxic activity," *Natural Product Sciences*, vol. 20, pp. 71–75, 2014.

[24] M. K. Elhaj, S. M. Yagi, A. A. Qahtan et al., "Screening of phytochemicals and bioactivities of different parts of Ludwigia erecta (L.) H. Hara," *Biotechnology & Biotechnological Equipment*, vol. 35, no. 1, pp. 283–289, 2021.

[25] M. G. Djouossi, J. D. D. Tamokou, D. Ngnokam et al., "Antimicrobial and antioxidant flavonoids from the leaves of Oncoba spinosa Forssk (Salicaceae)," *BMC Complementary and Alternative Medicine*, vol. 15, no. 1, p. 134, 2015.

[26] R. T. Nzogong, F. S. T. Ndjateu, S. E. Ekom et al., "Antimicrobial and antioxidant activities of triterpenoid and phenolic derivatives from two Cameroonian Melastomataceae plants: dissotis senegambiensis and Amphiblemma monticola," *BMC Complementary and Alternative Medicine*, vol. 18, no. 1, p. 159, 2018.

[27] J. D. D. Tamokou, T. A. Mbaveng, and V. Kuete, "Antimicrobial activities of African medicinal spices and vegetables," in *Medicinal Spices and Vegetables from Africa: Therapeutic Potential against Metabolic Inflammatory Infectious and Systemic Diseases*, pp. 207–237, Elsevier, Amsterdam, Netherlands, 18 edition, 2017.

[28] S. Acharyya, P. Sarkar, D. R. Saha, A. Patra, T. Ramamurthy, and P. K. Bag, "Intracellular and membrane-damaging activities of methyl gallate isolated from Terminalia chebula against multidrug-resistant Shigella spp.," *Journal of Medical Microbiology*, vol. 64, no. 8, pp. 901–909, 2015.

[29] J. D. D. Tamokou, J. A. Fosso, A. J. Njouendou, S. Ekom, and I. Kengne, "Antibacterial activity of three Cameroonian medicinal plants traditionally used for the treatment of urinary tract infections and their synergistic effects with amoxicillin and serum," *Journal of Advances in Medical and Pharmaceutical Sciences*, vol. 15, no. 2, pp. 1–14, 2017.

[30] M. Puzari, M. Sharma, and P. Chetia, "Emergence of antibiotic resistant Shigella species: a matter of concern," *Journal of Infection and Public Health*, vol. 11, no. 4, pp. 451–454, 2018.

[31] D. F. Sahm, C. Thornsberry, D. C. Mayfield, M. E. Jones, and J. A. Karlowsky, "Multidrug-resistant urinary tract isolates of *Escherichia coli*: p," no. 5, pp. 1402–1406, 2001.

[32] J. Oteo, E. Lázaro, F. J. de Abajo, F. Baquero, and J. Campos, "Antimicrobial-resistant i," no. 4, pp. 546–553, 2005.

[33] A. Bartoloni, L. Pallecchi, M. Benedetti et al., "Multidrug-resistant commensal *Escherichia coli* in children, Peru and Bolivia," *Emerging Infectious Diseases*, vol. 12, no. 6, pp. 907–913, 2006.

[34] E. Sieniawska, M. Swatko-Ossor, R. Sawicki, K. Skalicka-Wozniak, and G. Ginalska, "Natural terpenes influence the activity of antibiotics against isolated *Mycobacterium tuberculosis*," *Medical Principles and Practice*, vol. 26, no. 2, pp. 108–112, 2017.

[35] D. J. Weber, N. E. Tolkoff-Rubin, and R. H. Rubin, "Amoxicillin and potassium clavulanate: an antibiotic combination. mechanism of action, pharmacokinetics, antimicrobial spectrum, clinical efficacy and adverse effects," *Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy*, vol. 4, no. 3, pp. 122–133, 1984.

[36] M. T. Pasko, S. C. Piscitelli, and A. D. Van Slooten, "Fluconazole: a new triazole antifungal agent," *The Annals of Pharmacotherapy*, vol. 24, no. 9, pp. 860–867, 1990.

[37] L. Zhou, Y. Ding, W. Chen, P. Zhang, Y. Chen, and X. Lv, "The in vitro study of ursolic acid and oleanolic acid inhibiting cariogenic microorganisms as well as biofilm," *Oral Diseases*, vol. 19, no. 5, pp. 494–500, 2013.