# Parts, Period, and Extraction Solvents as Parameters InfluencingHarungana madagascariensis Lam. ex Poir. (Hypericaceae) Antibacterial Activity

Authors: D. Deutou Tégaboué; C. L. Cidjeu Pouaha; R. M. Ebelle Etame; Sikam K. G.; D. Ngo Hagbe; Issa Moussa; Raphael Tchientcheu; R. S. Mouokeu; R. A. Ngono Ngane
Journal: Evidence-Based Complementary and Alternative Medicine
 (2021)

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## Abstract

The study of the conditions which affect the pharmacological activity of medicinal plants is the basis for the development of phytomedicine. This study aimed at identifying some optimal conditions for antibacterial activity of extracts ofHarungana madagascariensis. The leaves, bark, and roots were harvested in Douala, Littoral Region of Cameroon, in August 2019 at three times of the day: early in the morning (6 a.m.), at midday (12 p.m.), and in the afternoon (6 p.m.). They were dried at room temperature and ground to give a fine powder. Each powder was macerated in methanol for 72 h and boiled in distilled water and palm wine. The obtained extracts were evaluated for their antibacterial activity by broth microdilution method on 31 clinical and 4 reference bacterial strains. The results show that the best extraction yield was obtained using the bark extracted from palm wine by decoction. The methanol bark extract was found to be more active than other solvents (8 and 512 µg/mL). In fact, the activity of the 6 a.m. extract was significant on 4 of the 35 strains tested (MIC< 100 µg/mL) and moderate on 27 of the 35 strains tested (100≤ MIC≤ 625 μg/mL). The activity of the noon extract was significant on 8 strains and moderate on 23 strains and, finally, the activity of the extract of 6 p.m. was significant on 9 strains and moderate on 25 strains. The extract harvested at 6 p.m. inhibited the growth of the 35 strains tested and revealed significant activity (MIC <100 μg/mL) on Staphylococcus aureus, Escherichia coli, Citrobacter freundii, Serratia marcescens, Salmonella enterica serovar typhi, and Yersinia enterocolitica, making this hour the best harvesting time. This study further confirms that the bark of H. madagascariensis is a potent antibacterial agent, being better still when harvested at 6 p.m. and extracted with methanol.


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## Body

## 1. Introduction

Infectious diseases are one of the leading causes of death worldwide [1]. According to the World Health Organisation, these diseases are responsible for 17 million deaths per year worldwide [2]. To overcome these infectious diseases, many antibacterials are available, including tetracycline, amoxicillin, amoxicillin + clavulanic acid, doxycycline cotrimoxazol. penicillin, imipenem, and amikacin. However, probabilistic prescriptions and the nonrational use of antibiotics have led to the development of bacterial resistance. In addition, the high cost of these antibiotics, the duration of the treatment, and poverty accentuate this phenomenon in developing countries. These justify the constant search for antimicrobial substances. The use of medicinal plants is an alternative, due to their accessibility, availability, and great diversity in bioactive molecules [3]. Indeed, the antimicrobial properties of many plants have been investigated and they are also used extensively in daily clinical practice. Their active principle have led to the development of some modern available medicine such as aspirin, digitoxin, morphine, quinine, and pilocarpine [4].Cameroon, Africa in miniature, is home to a set of important and varying plant species, includingH. madagascariensis commonly called Nketto by the Bamileke tribe. It is a tropical plant, abundant in the savana and the forest region [5]. Its antibacterial and antifungal properties have been established [6]. Many studies reveal the antibacterial properties of different parts of this plant, extracted with several solvents, including methanol, water and palm wine. The antibacterial activity of the leaf extract was established by Moulari et al., [6] and Tankeo et al., [7]. The antibacterial properties of methanol extract from stem bark have been highlighted by Hopogap [8] and Ebelle Etame et al. [9]. In addition, works on the antibacterial activity of methanol extracts, fractions, and compounds of H. madagascariensis were investigated [7]. This antibacterial activity was associated with the presence of betulinic acid, ferruginin, and kaempferol-3-0-β- D-glucopyranoside [7]. Following the work of Magne [10] on the influence of parts and the harvest period on the antifungal activity of H. madagascariensis, it was clearly established that the roots of this plant harvested at noon had the best activity. Based on Jaradat et al.’s work [11], it was found that the geographical location and ecological conditions affect the chemical constituents and the antibacterial activity of the essential oil of Ruta chalepensis leaves in Palestine. It, therefore, appears that the antimicrobial properties of H. madagascariensis are well documented. However, plant antimicrobial activity may vary according to the part [10], extraction solvent [5], harvest period [10], and harvest location [12]. With regard to the antibacterial activity of H. madagascariensis, it seems important to seek the best conditions for the expression of the antibacterial activity. It is in this context that this work falls, the objective of which is to identify the optimal conditions for antibacterial activity of extracts of H. madagascariensis, more specifically, (1) to assess the influence of the plant’s part on the yield and antibacterial activity of H. madagascariensis, (2) to determine the effect of the harvest time on the yield and antibacterial activity of H. madagascariensis, (3) and to assess the effect of the extraction solvent on the yield and antibacterial activity of H. madagascariensis.


## 2. Materials and Methods

### 2.1. Materials

#### 2.1.1. Plant Materials

The parts used in this study consisted of the leaves, barks, and roots ofH. madagascariensis. They were harvested in Douala in the Littoral Region (Cameroon), more precisely at PK17. The different parts were harvested in August 2019 at three times of the day: early in the morning (6 a.m.), at midday (12 p.m.), and in the afternoon (6 p.m.). The identification of the plant was confirmed at the National Herbarium of Cameroon by comparison with the reference sample No 4224 HNC.


#### 2.1.2. Microorganisms

31 clinical strains and 4 reference strains of enteric bacteria were used in the study. Clinical strains were isolated from stool of patients with gastrointestinal problems. The reference strains were a kind gift of Professor Kuete Victor, Department of Biochemistry, University of Dschang, Cameroon. The sensibility of all the strains to commonly used antibiotics is summarised in Table1.Table 1
Sensitivity and resistance profile of the bacterial strains used for the experiment.
Bacteria characteristicsEscherichia coliATCC 10536 reference strainATCC 8739 reference strainE.C 99 clinical isolate: IPMS, AXI, NORS, CFMS, CROS, CIPS, ANI, CHLSE.C 136 clinical isolate: IPMS, AXR, NORS, CFMS, CROS, CIPS, ANS, CHLSE.C 137 clinical isolate: IPMS, AXS, NORS, CFMS, CROS, CIPS, ANI, CHLSE.C 2 clinical isolate: TZPS, CTXS, CAZS, ES, IPMS, FURS, AMPR, TICR, GNR, TOBR, ANR, NALR, SIPR, OFLRE.C 4 clinical isolate: CTXS, CAZS, ES, IPMS, GNS, TOBS, FURS, AMPR, TICR, TZPR, NAR, CIPR, OFXRE.C 5 clinical isolate: TEPS, CTXS, CAZS, ES, IPMS, ANS, GNS, TOBS, CIPSCitrobacter freundiiCIT B 80 clinical isolate: STXR, DOR, GNR, AMCR, TER, CIPR, CHLR, AKS, CTS, AXR, CROR, CAZR, IPMS, NORR, ANSCIT B 81 clinical isolate: STXS, DOI, GNS, AMCR, TER, CIPS, CHLS, AKS, CTS, AXR, CROR, CAZS, IPMS, NORS, ANSSerratia marcescensSERB 115 clinical isolate: STXR, DOR, GNS, AMCS, TER, CIPS, CHLS, AKS, CTS, AXR, CROS, CAZS, IPMS, NORS, ANSYersinia enterocoliticaYERB 121 clinical isolate: STXS, DOR, GNS, AMCS, TER, CIPS, CHLS, ANS, CTR, AXR, CROI, CAZI, IPMS, NORS, ANSYERBI clinical isolate: STXS, DOR, GNS, AMCR, TER, CIPS, CHLR, AKS, CTR, AXR, CROI, CAZI, IPMS, NORI, ANSEnterobacter aerogenesENT 51 clinical isolate: STXR, DOR, GNS, AMCI, TER, CIPS, CHLS, AKS, CTR, AXR, CROS, CAZI, IPMS, NORS, ANIENT 119 clinical isolate: IPMS, AXI, NORS, CFMS, CROS, CIPS, ANI, CHLSENT 167 clinical isolate: STXR, DOR, GNS, AMCR, TER, CIPI, CHLS, AKS, CTR, AXR, CROI, CAZI, IPMS, NORR, ANRKlebsiella pneumoniaeKL 111 clinical isolate: IPMS, AXI, NORS, CFMS, CROI, CIPS, ANI, CHLSKLPB101 clinical isolate: STXS, DOS, GNS, AMCI, TES, CIPS, CHLS, AKS, CTS, AXI, CROI, CAZS, IPMS, NORS, ANSSalmonella enterica serovar typhiATCC 6539 reference strainSAL 9 clinical isolate: IPMS, AXR, NORS, CFMS, CROR, CIPS, ANI, CHLISal clinical isolate: AMI, IPMS, AXS, NORS, ANI, CFMS, CROR, CIPS, ANI, CHLSSalmonella paratyphi B clinical isolate: AMR, TER, SXTR, ANR, CIPS, CHLSSalmonella typhimurium clinical isolate: AMR, TER, SXTR, NAR, CIPS, CHLSPseudomonas aeruginosaATCC 27853 reference strainProvidencia stuartiiPS NEA 16 clinical isolate MDR: protein synthesis inhibitor OmpF of OmpCBacteria Gram-positiveStaphylococcus aureusSTAPH 1 clinical isolate: IPMS, DOS, AMCS, ES, VASMRSA 9 resistant clinical isolate: OFXR, FLXR, KR, ER, CHLR, IMP/CSRMRSA 11 resistant clinical isolate: OFXR, KR, ER, CIPR, IM/CSRMRSA 12 resistant clinical isolate: OFXR, FLXR, KR, ER, IM/CSRMRSA 3 resistant clinical isolate: OFXR, KR, ER, TER3 clinical isolate: IPMS, AMCS, AMS, DOS, VAS, ESST 120 clinical isolate: IPMS, AXS, AMS, DOS, VAS, ESSA 3 clinical isolate: CCR, QUIS/DALS, LINS, VAR, TER, TIGS, NITS, RIFR, TRIR/SULRStreptococcus sp.STR 7 clinical isolate: CCS, QUIS, LINs, VAS, TER, TIGS, NITS, STXRAK: amikacin; AM: ampicillin; CHL: chloramphenicol; CFM: cefixime; CIP: ciprofloxacin; CRO: ceftriaxone; DO: doxycycline; E: erythromycin; FLX: flomoxef; IM/CS: imipenem/sodium cilastatin; K: kanamycin; NA: nalidixic acid; NOR: norfloxacin; OFX: ofloxacin; TE: tetracycline; STX: cotrimoxazole; AMC: amoxicillin + clavulanic acid; AX: amoxicillin; AZM: azithromycin; CAZ: ceftazidim; CC: clindamycin; CN: cefalexin; CT: ceftolozane + tazobactam; IPM: imipenem; L: lincomycin; NOR: norfloxacin; TE: tetracycline; QUI: quinupristin; LIN: linezolid; TIG: tigecycline; NIT: nitrofurantoin; RIF: rifampicin; VA: vancomycin; GN: gentamicin; TOB: tobramycin; TIC: ticarcillin; TZP: piperacillin/tazobactam; CTX: cefotaxime; R: resistant; S: sensitive; I: intermediate.


### 2.2. Methods

#### 2.2.1. Extract Preparation

The bark and roots ofH. madagascariensis were cut into small pieces and dried in the shade (24 ± 2°C) for approximately 30 days. The leaves were directly dried under the same conditions. Each of these was ground at ambient temperature, using a mechanical grinder to obtain a fine powder.


#### 2.2.2. Methanol Extracts

The powders obtained were extracted as reported by Foutse et al. [13]. For this purpose, 300 g of each powder was mixed with 1000 mL methanol and maintained at room temperature for 72 h with twice daily homogenization. The obtained solution was then filtered using Whatman No. 1 filter paper. The filtrate was concentrated using a “RE52AA” rotary evaporator at 55°C. These extracts were dried in an oven for 48 h at 40°C to remove residual solvent.


#### 2.2.3. Aqueous Extracts

The powders obtained were extracted according to Atsafack et al.’s protocol [14]. For this purpose, 50 g of each powder was added to 300 mL of distilled water and then boiled at 100°C for 15 min. The mixture was then left to stand for 6 h in the dark and further filtered using Whatman No. 1 filter paper. The filtrate was dried in an oven at 40°C for 48 h to obtain an aqueous crude extract.


#### 2.2.4. Hydroalcoholic Extracts

The extracts were obtained by following the protocol reported by Etame Loé et al. [15]. For this purpose, 100 g powder was added to 300 mL of palm wine and boiled at 100°C for 15 min. After 6 h in the dark at room temperature, the mixture was filtered using Whatman No. 1 filter paper. The filtrate was dried in an oven at 45°C to obtain a crude hydroalcoholic extract.From each plant material and extraction solvent, the extract was weighed and the yield (%) calculated based on the mass of the sample of plant material. Those extracts were frozen during antibacterial evaluation.


#### 2.2.5. Antibacterial Activity of Plant Extracts

The minimum inhibitory concentrations (MICs) of the extracts were determined by the broth microdilution method [16]. In each of the 96 wells of a microtiter plate, 100 μL of Mueller–Hinton broth was introduced. Subsequently, 100 μL extract prepared at 4096 μg/mL was added to the upper wells. A series of twofold dilutions was carried out to obtain concentrations of extracts ranging from 8 to 1024 μg/mL. Finally, 100 μL of bacterial inoculum at 1.5 × 106 CFU/mL was added to each well. The inoculum was prepared from overnight bacterial cell culture. For that purpose, three to four colonies of each strain were introduced into an assay tube containing 10 mL of sterilised distilled water and adjusted to 0.5 McFarland turbidity scale, that is, approximately 1.5 × 108 CFU/mL. The purity of each strain was verified using specific culture medium prior to antibacterial testing. Ciprofloxacin was used as a positive control at concentrations ranging from 2 to 256 μg/mL, while 2.5% DMSO was used as negative control. The plates were sealed and incubated at 37°C for 24 h. Each test was performed in triplicate and repeated thrice. At the end of the incubation time, the bacterial growth was monitored using p-iodonitrotetrazolium chloride (INT) 2%. For this purpose, 40 μL of the INT solution was introduced into each well. The plates were incubated at 37°C for 30 min, and the MIC value was determined as the smallest concentration of the substance for which no change in INT colour was observed [13]. Based on the MIC values, the activity of the extracts was considered as significant (MIC<100 μg/mL), moderate (100≤ MIC≤ 625 μg/mL), and weak (MIC> 625 μg/mL) [17].Following MIC determination, minimum bactericidal concentrations (MBCs) were determined as reported by Foutse et al. [13]. In brief, 10 μL aliquots of the medium drawn from wells which did not show any growth during MIC assay were subcultured on Mueller–Hinton agar and incubated for 18 hours at 35°C. The lowest concentration of the substance from which negative growth was recorded was considered as MBC. The MBC/MIC ratio made it possible to distinguish the bactericidal effect (MBC/MIC≤4) and the bacteriostatic effect (MBC/MIC> 4).


## 2.1. Materials

### 2.1.1. Plant Materials

The parts used in this study consisted of the leaves, barks, and roots ofH. madagascariensis. They were harvested in Douala in the Littoral Region (Cameroon), more precisely at PK17. The different parts were harvested in August 2019 at three times of the day: early in the morning (6 a.m.), at midday (12 p.m.), and in the afternoon (6 p.m.). The identification of the plant was confirmed at the National Herbarium of Cameroon by comparison with the reference sample No 4224 HNC.


### 2.1.2. Microorganisms

31 clinical strains and 4 reference strains of enteric bacteria were used in the study. Clinical strains were isolated from stool of patients with gastrointestinal problems. The reference strains were a kind gift of Professor Kuete Victor, Department of Biochemistry, University of Dschang, Cameroon. The sensibility of all the strains to commonly used antibiotics is summarised in Table1.Table 1
Sensitivity and resistance profile of the bacterial strains used for the experiment.
Bacteria characteristicsEscherichia coliATCC 10536 reference strainATCC 8739 reference strainE.C 99 clinical isolate: IPMS, AXI, NORS, CFMS, CROS, CIPS, ANI, CHLSE.C 136 clinical isolate: IPMS, AXR, NORS, CFMS, CROS, CIPS, ANS, CHLSE.C 137 clinical isolate: IPMS, AXS, NORS, CFMS, CROS, CIPS, ANI, CHLSE.C 2 clinical isolate: TZPS, CTXS, CAZS, ES, IPMS, FURS, AMPR, TICR, GNR, TOBR, ANR, NALR, SIPR, OFLRE.C 4 clinical isolate: CTXS, CAZS, ES, IPMS, GNS, TOBS, FURS, AMPR, TICR, TZPR, NAR, CIPR, OFXRE.C 5 clinical isolate: TEPS, CTXS, CAZS, ES, IPMS, ANS, GNS, TOBS, CIPSCitrobacter freundiiCIT B 80 clinical isolate: STXR, DOR, GNR, AMCR, TER, CIPR, CHLR, AKS, CTS, AXR, CROR, CAZR, IPMS, NORR, ANSCIT B 81 clinical isolate: STXS, DOI, GNS, AMCR, TER, CIPS, CHLS, AKS, CTS, AXR, CROR, CAZS, IPMS, NORS, ANSSerratia marcescensSERB 115 clinical isolate: STXR, DOR, GNS, AMCS, TER, CIPS, CHLS, AKS, CTS, AXR, CROS, CAZS, IPMS, NORS, ANSYersinia enterocoliticaYERB 121 clinical isolate: STXS, DOR, GNS, AMCS, TER, CIPS, CHLS, ANS, CTR, AXR, CROI, CAZI, IPMS, NORS, ANSYERBI clinical isolate: STXS, DOR, GNS, AMCR, TER, CIPS, CHLR, AKS, CTR, AXR, CROI, CAZI, IPMS, NORI, ANSEnterobacter aerogenesENT 51 clinical isolate: STXR, DOR, GNS, AMCI, TER, CIPS, CHLS, AKS, CTR, AXR, CROS, CAZI, IPMS, NORS, ANIENT 119 clinical isolate: IPMS, AXI, NORS, CFMS, CROS, CIPS, ANI, CHLSENT 167 clinical isolate: STXR, DOR, GNS, AMCR, TER, CIPI, CHLS, AKS, CTR, AXR, CROI, CAZI, IPMS, NORR, ANRKlebsiella pneumoniaeKL 111 clinical isolate: IPMS, AXI, NORS, CFMS, CROI, CIPS, ANI, CHLSKLPB101 clinical isolate: STXS, DOS, GNS, AMCI, TES, CIPS, CHLS, AKS, CTS, AXI, CROI, CAZS, IPMS, NORS, ANSSalmonella enterica serovar typhiATCC 6539 reference strainSAL 9 clinical isolate: IPMS, AXR, NORS, CFMS, CROR, CIPS, ANI, CHLISal clinical isolate: AMI, IPMS, AXS, NORS, ANI, CFMS, CROR, CIPS, ANI, CHLSSalmonella paratyphi B clinical isolate: AMR, TER, SXTR, ANR, CIPS, CHLSSalmonella typhimurium clinical isolate: AMR, TER, SXTR, NAR, CIPS, CHLSPseudomonas aeruginosaATCC 27853 reference strainProvidencia stuartiiPS NEA 16 clinical isolate MDR: protein synthesis inhibitor OmpF of OmpCBacteria Gram-positiveStaphylococcus aureusSTAPH 1 clinical isolate: IPMS, DOS, AMCS, ES, VASMRSA 9 resistant clinical isolate: OFXR, FLXR, KR, ER, CHLR, IMP/CSRMRSA 11 resistant clinical isolate: OFXR, KR, ER, CIPR, IM/CSRMRSA 12 resistant clinical isolate: OFXR, FLXR, KR, ER, IM/CSRMRSA 3 resistant clinical isolate: OFXR, KR, ER, TER3 clinical isolate: IPMS, AMCS, AMS, DOS, VAS, ESST 120 clinical isolate: IPMS, AXS, AMS, DOS, VAS, ESSA 3 clinical isolate: CCR, QUIS/DALS, LINS, VAR, TER, TIGS, NITS, RIFR, TRIR/SULRStreptococcus sp.STR 7 clinical isolate: CCS, QUIS, LINs, VAS, TER, TIGS, NITS, STXRAK: amikacin; AM: ampicillin; CHL: chloramphenicol; CFM: cefixime; CIP: ciprofloxacin; CRO: ceftriaxone; DO: doxycycline; E: erythromycin; FLX: flomoxef; IM/CS: imipenem/sodium cilastatin; K: kanamycin; NA: nalidixic acid; NOR: norfloxacin; OFX: ofloxacin; TE: tetracycline; STX: cotrimoxazole; AMC: amoxicillin + clavulanic acid; AX: amoxicillin; AZM: azithromycin; CAZ: ceftazidim; CC: clindamycin; CN: cefalexin; CT: ceftolozane + tazobactam; IPM: imipenem; L: lincomycin; NOR: norfloxacin; TE: tetracycline; QUI: quinupristin; LIN: linezolid; TIG: tigecycline; NIT: nitrofurantoin; RIF: rifampicin; VA: vancomycin; GN: gentamicin; TOB: tobramycin; TIC: ticarcillin; TZP: piperacillin/tazobactam; CTX: cefotaxime; R: resistant; S: sensitive; I: intermediate.


## 2.1.1. Plant Materials

The parts used in this study consisted of the leaves, barks, and roots ofH. madagascariensis. They were harvested in Douala in the Littoral Region (Cameroon), more precisely at PK17. The different parts were harvested in August 2019 at three times of the day: early in the morning (6 a.m.), at midday (12 p.m.), and in the afternoon (6 p.m.). The identification of the plant was confirmed at the National Herbarium of Cameroon by comparison with the reference sample No 4224 HNC.


## 2.1.2. Microorganisms

31 clinical strains and 4 reference strains of enteric bacteria were used in the study. Clinical strains were isolated from stool of patients with gastrointestinal problems. The reference strains were a kind gift of Professor Kuete Victor, Department of Biochemistry, University of Dschang, Cameroon. The sensibility of all the strains to commonly used antibiotics is summarised in Table1.Table 1
Sensitivity and resistance profile of the bacterial strains used for the experiment.
Bacteria characteristicsEscherichia coliATCC 10536 reference strainATCC 8739 reference strainE.C 99 clinical isolate: IPMS, AXI, NORS, CFMS, CROS, CIPS, ANI, CHLSE.C 136 clinical isolate: IPMS, AXR, NORS, CFMS, CROS, CIPS, ANS, CHLSE.C 137 clinical isolate: IPMS, AXS, NORS, CFMS, CROS, CIPS, ANI, CHLSE.C 2 clinical isolate: TZPS, CTXS, CAZS, ES, IPMS, FURS, AMPR, TICR, GNR, TOBR, ANR, NALR, SIPR, OFLRE.C 4 clinical isolate: CTXS, CAZS, ES, IPMS, GNS, TOBS, FURS, AMPR, TICR, TZPR, NAR, CIPR, OFXRE.C 5 clinical isolate: TEPS, CTXS, CAZS, ES, IPMS, ANS, GNS, TOBS, CIPSCitrobacter freundiiCIT B 80 clinical isolate: STXR, DOR, GNR, AMCR, TER, CIPR, CHLR, AKS, CTS, AXR, CROR, CAZR, IPMS, NORR, ANSCIT B 81 clinical isolate: STXS, DOI, GNS, AMCR, TER, CIPS, CHLS, AKS, CTS, AXR, CROR, CAZS, IPMS, NORS, ANSSerratia marcescensSERB 115 clinical isolate: STXR, DOR, GNS, AMCS, TER, CIPS, CHLS, AKS, CTS, AXR, CROS, CAZS, IPMS, NORS, ANSYersinia enterocoliticaYERB 121 clinical isolate: STXS, DOR, GNS, AMCS, TER, CIPS, CHLS, ANS, CTR, AXR, CROI, CAZI, IPMS, NORS, ANSYERBI clinical isolate: STXS, DOR, GNS, AMCR, TER, CIPS, CHLR, AKS, CTR, AXR, CROI, CAZI, IPMS, NORI, ANSEnterobacter aerogenesENT 51 clinical isolate: STXR, DOR, GNS, AMCI, TER, CIPS, CHLS, AKS, CTR, AXR, CROS, CAZI, IPMS, NORS, ANIENT 119 clinical isolate: IPMS, AXI, NORS, CFMS, CROS, CIPS, ANI, CHLSENT 167 clinical isolate: STXR, DOR, GNS, AMCR, TER, CIPI, CHLS, AKS, CTR, AXR, CROI, CAZI, IPMS, NORR, ANRKlebsiella pneumoniaeKL 111 clinical isolate: IPMS, AXI, NORS, CFMS, CROI, CIPS, ANI, CHLSKLPB101 clinical isolate: STXS, DOS, GNS, AMCI, TES, CIPS, CHLS, AKS, CTS, AXI, CROI, CAZS, IPMS, NORS, ANSSalmonella enterica serovar typhiATCC 6539 reference strainSAL 9 clinical isolate: IPMS, AXR, NORS, CFMS, CROR, CIPS, ANI, CHLISal clinical isolate: AMI, IPMS, AXS, NORS, ANI, CFMS, CROR, CIPS, ANI, CHLSSalmonella paratyphi B clinical isolate: AMR, TER, SXTR, ANR, CIPS, CHLSSalmonella typhimurium clinical isolate: AMR, TER, SXTR, NAR, CIPS, CHLSPseudomonas aeruginosaATCC 27853 reference strainProvidencia stuartiiPS NEA 16 clinical isolate MDR: protein synthesis inhibitor OmpF of OmpCBacteria Gram-positiveStaphylococcus aureusSTAPH 1 clinical isolate: IPMS, DOS, AMCS, ES, VASMRSA 9 resistant clinical isolate: OFXR, FLXR, KR, ER, CHLR, IMP/CSRMRSA 11 resistant clinical isolate: OFXR, KR, ER, CIPR, IM/CSRMRSA 12 resistant clinical isolate: OFXR, FLXR, KR, ER, IM/CSRMRSA 3 resistant clinical isolate: OFXR, KR, ER, TER3 clinical isolate: IPMS, AMCS, AMS, DOS, VAS, ESST 120 clinical isolate: IPMS, AXS, AMS, DOS, VAS, ESSA 3 clinical isolate: CCR, QUIS/DALS, LINS, VAR, TER, TIGS, NITS, RIFR, TRIR/SULRStreptococcus sp.STR 7 clinical isolate: CCS, QUIS, LINs, VAS, TER, TIGS, NITS, STXRAK: amikacin; AM: ampicillin; CHL: chloramphenicol; CFM: cefixime; CIP: ciprofloxacin; CRO: ceftriaxone; DO: doxycycline; E: erythromycin; FLX: flomoxef; IM/CS: imipenem/sodium cilastatin; K: kanamycin; NA: nalidixic acid; NOR: norfloxacin; OFX: ofloxacin; TE: tetracycline; STX: cotrimoxazole; AMC: amoxicillin + clavulanic acid; AX: amoxicillin; AZM: azithromycin; CAZ: ceftazidim; CC: clindamycin; CN: cefalexin; CT: ceftolozane + tazobactam; IPM: imipenem; L: lincomycin; NOR: norfloxacin; TE: tetracycline; QUI: quinupristin; LIN: linezolid; TIG: tigecycline; NIT: nitrofurantoin; RIF: rifampicin; VA: vancomycin; GN: gentamicin; TOB: tobramycin; TIC: ticarcillin; TZP: piperacillin/tazobactam; CTX: cefotaxime; R: resistant; S: sensitive; I: intermediate.


## 2.2. Methods

### 2.2.1. Extract Preparation

The bark and roots ofH. madagascariensis were cut into small pieces and dried in the shade (24 ± 2°C) for approximately 30 days. The leaves were directly dried under the same conditions. Each of these was ground at ambient temperature, using a mechanical grinder to obtain a fine powder.


### 2.2.2. Methanol Extracts

The powders obtained were extracted as reported by Foutse et al. [13]. For this purpose, 300 g of each powder was mixed with 1000 mL methanol and maintained at room temperature for 72 h with twice daily homogenization. The obtained solution was then filtered using Whatman No. 1 filter paper. The filtrate was concentrated using a “RE52AA” rotary evaporator at 55°C. These extracts were dried in an oven for 48 h at 40°C to remove residual solvent.


### 2.2.3. Aqueous Extracts

The powders obtained were extracted according to Atsafack et al.’s protocol [14]. For this purpose, 50 g of each powder was added to 300 mL of distilled water and then boiled at 100°C for 15 min. The mixture was then left to stand for 6 h in the dark and further filtered using Whatman No. 1 filter paper. The filtrate was dried in an oven at 40°C for 48 h to obtain an aqueous crude extract.


### 2.2.4. Hydroalcoholic Extracts

The extracts were obtained by following the protocol reported by Etame Loé et al. [15]. For this purpose, 100 g powder was added to 300 mL of palm wine and boiled at 100°C for 15 min. After 6 h in the dark at room temperature, the mixture was filtered using Whatman No. 1 filter paper. The filtrate was dried in an oven at 45°C to obtain a crude hydroalcoholic extract.From each plant material and extraction solvent, the extract was weighed and the yield (%) calculated based on the mass of the sample of plant material. Those extracts were frozen during antibacterial evaluation.


### 2.2.5. Antibacterial Activity of Plant Extracts

The minimum inhibitory concentrations (MICs) of the extracts were determined by the broth microdilution method [16]. In each of the 96 wells of a microtiter plate, 100 μL of Mueller–Hinton broth was introduced. Subsequently, 100 μL extract prepared at 4096 μg/mL was added to the upper wells. A series of twofold dilutions was carried out to obtain concentrations of extracts ranging from 8 to 1024 μg/mL. Finally, 100 μL of bacterial inoculum at 1.5 × 106 CFU/mL was added to each well. The inoculum was prepared from overnight bacterial cell culture. For that purpose, three to four colonies of each strain were introduced into an assay tube containing 10 mL of sterilised distilled water and adjusted to 0.5 McFarland turbidity scale, that is, approximately 1.5 × 108 CFU/mL. The purity of each strain was verified using specific culture medium prior to antibacterial testing. Ciprofloxacin was used as a positive control at concentrations ranging from 2 to 256 μg/mL, while 2.5% DMSO was used as negative control. The plates were sealed and incubated at 37°C for 24 h. Each test was performed in triplicate and repeated thrice. At the end of the incubation time, the bacterial growth was monitored using p-iodonitrotetrazolium chloride (INT) 2%. For this purpose, 40 μL of the INT solution was introduced into each well. The plates were incubated at 37°C for 30 min, and the MIC value was determined as the smallest concentration of the substance for which no change in INT colour was observed [13]. Based on the MIC values, the activity of the extracts was considered as significant (MIC<100 μg/mL), moderate (100≤ MIC≤ 625 μg/mL), and weak (MIC> 625 μg/mL) [17].Following MIC determination, minimum bactericidal concentrations (MBCs) were determined as reported by Foutse et al. [13]. In brief, 10 μL aliquots of the medium drawn from wells which did not show any growth during MIC assay were subcultured on Mueller–Hinton agar and incubated for 18 hours at 35°C. The lowest concentration of the substance from which negative growth was recorded was considered as MBC. The MBC/MIC ratio made it possible to distinguish the bactericidal effect (MBC/MIC≤4) and the bacteriostatic effect (MBC/MIC> 4).


## 2.2.1. Extract Preparation

The bark and roots ofH. madagascariensis were cut into small pieces and dried in the shade (24 ± 2°C) for approximately 30 days. The leaves were directly dried under the same conditions. Each of these was ground at ambient temperature, using a mechanical grinder to obtain a fine powder.


## 2.2.2. Methanol Extracts

The powders obtained were extracted as reported by Foutse et al. [13]. For this purpose, 300 g of each powder was mixed with 1000 mL methanol and maintained at room temperature for 72 h with twice daily homogenization. The obtained solution was then filtered using Whatman No. 1 filter paper. The filtrate was concentrated using a “RE52AA” rotary evaporator at 55°C. These extracts were dried in an oven for 48 h at 40°C to remove residual solvent.


## 2.2.3. Aqueous Extracts

The powders obtained were extracted according to Atsafack et al.’s protocol [14]. For this purpose, 50 g of each powder was added to 300 mL of distilled water and then boiled at 100°C for 15 min. The mixture was then left to stand for 6 h in the dark and further filtered using Whatman No. 1 filter paper. The filtrate was dried in an oven at 40°C for 48 h to obtain an aqueous crude extract.


## 2.2.4. Hydroalcoholic Extracts

The extracts were obtained by following the protocol reported by Etame Loé et al. [15]. For this purpose, 100 g powder was added to 300 mL of palm wine and boiled at 100°C for 15 min. After 6 h in the dark at room temperature, the mixture was filtered using Whatman No. 1 filter paper. The filtrate was dried in an oven at 45°C to obtain a crude hydroalcoholic extract.From each plant material and extraction solvent, the extract was weighed and the yield (%) calculated based on the mass of the sample of plant material. Those extracts were frozen during antibacterial evaluation.


## 2.2.5. Antibacterial Activity of Plant Extracts

The minimum inhibitory concentrations (MICs) of the extracts were determined by the broth microdilution method [16]. In each of the 96 wells of a microtiter plate, 100 μL of Mueller–Hinton broth was introduced. Subsequently, 100 μL extract prepared at 4096 μg/mL was added to the upper wells. A series of twofold dilutions was carried out to obtain concentrations of extracts ranging from 8 to 1024 μg/mL. Finally, 100 μL of bacterial inoculum at 1.5 × 106 CFU/mL was added to each well. The inoculum was prepared from overnight bacterial cell culture. For that purpose, three to four colonies of each strain were introduced into an assay tube containing 10 mL of sterilised distilled water and adjusted to 0.5 McFarland turbidity scale, that is, approximately 1.5 × 108 CFU/mL. The purity of each strain was verified using specific culture medium prior to antibacterial testing. Ciprofloxacin was used as a positive control at concentrations ranging from 2 to 256 μg/mL, while 2.5% DMSO was used as negative control. The plates were sealed and incubated at 37°C for 24 h. Each test was performed in triplicate and repeated thrice. At the end of the incubation time, the bacterial growth was monitored using p-iodonitrotetrazolium chloride (INT) 2%. For this purpose, 40 μL of the INT solution was introduced into each well. The plates were incubated at 37°C for 30 min, and the MIC value was determined as the smallest concentration of the substance for which no change in INT colour was observed [13]. Based on the MIC values, the activity of the extracts was considered as significant (MIC<100 μg/mL), moderate (100≤ MIC≤ 625 μg/mL), and weak (MIC> 625 μg/mL) [17].Following MIC determination, minimum bactericidal concentrations (MBCs) were determined as reported by Foutse et al. [13]. In brief, 10 μL aliquots of the medium drawn from wells which did not show any growth during MIC assay were subcultured on Mueller–Hinton agar and incubated for 18 hours at 35°C. The lowest concentration of the substance from which negative growth was recorded was considered as MBC. The MBC/MIC ratio made it possible to distinguish the bactericidal effect (MBC/MIC≤4) and the bacteriostatic effect (MBC/MIC> 4).


## 3. Results and Discussion

### 3.1. Results

#### 3.1.1. Extraction Yields

The extraction yields varied with the part of the plant, the harvest period, and the extraction method/solvent (Table2). The highest yields were obtained using the bark regardless of the harvest period and extraction solvent. In contrast, the lowest extraction yields were obtained using the roots. Furthermore, methanol extracts from the leaves and bark had the highest extraction yields when harvested in the morning. These yields decreased during the day relatively to leaves and barks. The hydroalcoholic extract had the best yield whatever the plant part and the harvest period.Table 2
Extraction yield (%) of methanol, aqueous, and palm wine decoctions fromH. madagascariensis at different harvest times/periods.
PartsSolventsMethanolPalm wineWater6 a.m.12 p.m.6 p.m.6 a.m.12 p.m.6 p.m.6 a.m.12 p.m.6 p.m.Leaves11.694.777.3926.5724.5324.487.480.940.94Bark15.4715.0213.3628.3624.0526.9526.3425.22.02Roots4.36.599.5522.4326.7224.824.51.71.98


#### 3.1.2. Antibacterial Activities of Methanol Extracts

(1) Leaf Extract. The methanol extracts of the leaves of H. madagascariensis were active on 34 out of the 35 strains tested with MICs values ranging between 64 and 1024 µg/mL (Table 3). Their activity was significant on 3 out of the 35 strains tested. The activity of the 6 a.m. extract was significant on 1 strain, Serratia marcescens, out of the 35 strains tested and moderate on 18 strains. The activity of the midday extract was significant on 2 out of the 35 strains tested and moderate on 22 strains. The activity of the evening extract was significant on 2 strains and moderate on 29 strains.Table 3
Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of methanol extracts from the leaves ofH. madagascariensis (µg/mL).
LeavesCIP6 a.m.12 p.m.6 p.m.MICMBCRMICMBCRMICMBCRMICMBCRGram-negative bacteriaEscherichia coliATCC 8739256—Nd256—Nd25610244≤0.5816ATCC 10536512—Nd512—Nd256102442648EC 5128—Nd64—Nd64102416482EC 4512—Nd25651221285124≤0.564128EC 2512—Nd51210242512—Nd4328EC 1361024—Nd1024—Nd256—Nd46416EC 137———1024—Nd512—Nd4328Citrobacter freundiiCITB 80——————256—Nd13232CITB 81———256102446410241616464Serratia marcescensSERB 1156412826412821281024813232Yersinia enterocoliticaYERB 1211024—Nd12810248128—Nd≤0.5/48YERBI———51210242128—Nd122Salmonella enterica serovar typhiATCC 65391281024812810248128—Nd482SAL 9———256—Nd256—Nd482S AL128—Nd128—Nd128—Nd0.564128Salmonella paratyphi B256—Nd256—Nd———2168Salmonella typhimurium256—Nd25610244512—Nd4328Pseudomonas aeruginosaATCC 27853———51210242256—Nd16322PA 16512—Nd1024—Nd512—Nd23216Providencia stuartiiPS NEA 16—————————2168Klebsiella pneumoniaeKL 1111024—Nd———256—Nd32642KLPB101—————————8324Enterobacter aerogenesENT 119———1281024825610244144ENT 1672561024451210242256—Nd16322ENT 51———1024—Nd512—Nd8162Gram-positive bacteriaStaphylococcus aureusSTAPH 1256—Nd256—Nd128102481632231024—Nd256—Nd256—Nd188MRSA 32561024451210242512102420.524MRSA 925610244128—Nd128—Nd0.53264MRSA 125121024251210242512102428324MRSA 13512—Nd512—Nd512—Nd11616ST 120——————512—Nd23216ST 113102410241——————4328SA 312810248128—Nd12810248188Streptococcus sp.STR 7512—Nd1024—Nd2561024416464MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; R: MBC/MIC; CIP: ciprofloxacin; Nd: not determined; —: no activity.These results show that the leaves have better activity when they are harvested in the evening with MICs between 64 and 512µg/mL. The calculation of the MBC/MIC ratio reveals that the extracts had a bactericidal effect on 14 of the 35 strains tested and a bacteriostatic one on 21 of the 35 strains tested.Escherichia coli and Staphylococcus aureus strains were more sensitive to methanol extracts from the leaves. Among the 7 Escherichia coli strains tested, one strain presented a significant sensitivity and 5 strains presented a moderate sensitivity. S. aureus strains tested showed moderate sensitivity in 6 of the 9 strains tested, including methicillin-resistant strains.(2) Bark Extracts. The methanol extracts of the barks of H. madagascariensis, regardless of the time of harvest, were active on all 35 strains tested with MICs between 8 and 1024 µg/mL (Table 4). Their activity was significant on 9 out of the 35 tested strains.Table 4
Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of methanol extracts from the bark ofH. madagascariensis (µg/mL).
BarkCIP6 a.m.12 p.m.6 p.m.MICMBCRMICMBCRMICMBCRMICMBCRGram-negative bacteriaEscherichia coliATCC 87391281024864—Nd64102416≤0.5816ATCC 1053625610244128—Nd12851242648EC 5256—Nd25610244128—Nd482EC 41282562256—Nd12810248≤0.564128EC 2512—Nd1024—Nd512—Nd4328EC 136512—Nd512—Nd5121024246416EC 137256512225610244256102444328Citrobacter freundiiCITB 8012810248128—Nd64512813232CITB 81256512225610244256512216464Serratia marcescensSERB 11564512864512864256413232Yersinia enterocoliticaYERB 121256—Nd32—Nd32102432≤0.5/48YERBI512102421024—Nd25610244122Salmonella enterica serovar typhiATCC 6539128—Nd32—Nd32102432482SAL 9256—Nd128—Nd128—Nd482S AL128—Nd12810248641024160.564128Salmonella paratyphi B12812812561024425651222168Salmonella typhimurium512—Nd512—Nd128—Nd4328Pseudomonas aeruginosaATCC 27853512—Nd51210242256512216322PA 16512—Nd512—Nd256—Nd23216Providencia stuartiiPS NEA 16512—Nd256—Nd256—Nd2168Klebsiella pneumoniaeKL 111256—Nd512—Nd2561024432642KLPB101——————256102448324Enterobacter aerogenesENT 119128—Nd128—Nd128—Nd144ENT 167256—Nd128102482561024416322ENT 511024—Nd512—Nd1024—Nd8162Gram-positive bacteriaStaphylococcus aureusSTAPH 16425646451264512816322364—Nd≤8—Nd32—Nd188MRSA 3512—Nd5121024225651220.524MRSA 964—Nd810241288—Nd0.53264MRSA 122561024412810248128102488324MRSA 13512—Nd512—Nd256—Nd11616ST 120———1024—Nd256—Nd23216ST 113———512—Nd512—Nd4328SA 31281024864641128—Nd188Streptococcus sp.STR 751210242256—Nd128512416464MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; R: MBC/MIC; CIP: ciprofloxacin; Nd: not determined; —: no activity.The activity of the morning extract was significant on 4 strains and moderate on 27 strains. The activity of the midday extract was significant on 8 and moderate on 23 strains and, finally, the activity of the evening extract was significant on 9 and moderate on 25 strains. These results show that the barks have better activity when they are harvested in the evening with MICs between 8 and 512µg/mL.The MBC/MIC ratio revealed that the extracts had a bactericidal effect on 13 of the 35 strains tested and a bacteriostatic one on 22 strains.The strains ofS. aureus and E. coli appear to be more sensitive to the methanol extract of barks. Among the 9 strains of S. aureus, 6 strains showed a moderate activity and 3 strains a weak activity.(3). Root Extract. The methanol extracts of the roots of H. madagascariensis, regardless of the harvesting time, were active on 24 of the 35 strains tested with MICs between 128 and 1024 µg/mL (Table 5). The activity of morning extract was moderate on 15 strains, that of midday on 16, and that in the evening on 20 strains. The MBC/MIC ratio showed that the extracts had a bactericidal effect on 8 strains and a bacteriostatic effect on 27 strains. S. aureus strains were more sensitive to extracts from the roots. Indeed, among the 9 strains of S. aureus, including the methicillin-resistant strains, 8 strains showed moderate activity.Table 5
Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of methanol extracts from the roots ofH. madagascariensis (µg/mL).
RootsCIP6 a.m.12 p.m.6 p.m.MICMBCRMICMBCRMICMBCRMICMBCRGram-negative bacteriaEscherichia coliATCC 8739512—Nd512—Nd256—Nd≤0.5816ATCC 10536—————————2648EC 5512—Nd512—Nd256—Nd482EC 4—————————≤0.564128EC 2—————————4328EC 136————————46416EC 137—————————4328Citrobacter freundiiCITB 80——————1024—Nd13232CITB 81—————————16464Serratia marcescensSERB 11512810248256—Nd128—Nd13232Yersinia enterocoliticaYERB 1211024—Nd128—Nd5125121≤0.5/48YERBI—————————122Salmonella enterica serovar typhiATCC 653925610244512—Nd25610244482SAL 91024—Nd1024—Nd1024—Nd482S AL12825622561024425610244≤0.564128Salmonella paratyphi B128—Nd512—Nd256—Nd2168Salmonella typhimurium——————512102424328Pseudomonas aeruginosaATCC 278531024—Nd——————16322PA 16128—Nd———128—Nd23216Providencia stuartiiPS NEA 16——————1024—Nd2168Klebsiella pneumoniaeKL 111—————————32642KLPB101————————8324Enterobacter aerogenesENT 119128—Nd512—Nd512—Nd144ENT 1671024102411024102415121024216322ENT 51—————————8162Gram-positive bacteriaStaphylococcus aureusSTAPH 15121024512—Nd512—Nd1632231024—Nd512—Nd512—Nd188MRSA 3512—Nd51210242512102420.524MRSA 9256—Nd256—Nd128102480.53264MRSA 1212810248256—Nd256102448324MRSA 13128—Nd512—Nd512—Nd11616ST 120—————————23216ST 113512—Nd512——512102424328SA 3256—Nd51210242256—Nd188Streptococcus sp.STR 7——————512—Nd16464MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; R: MBC/IMC; CIP: ciprofloxacin; Nd: not determined; —: no activity.


#### 3.1.3. Antibacterial Activity of Aqueous Extracts

(1) Leaf Extracts. The aqueous extracts of the leaves of H. madagascariensis, regardless of the time of harvest, were active on 28 out of the 35 strains tested with MICs between 128 and 1024 µg/mL (Table 6). The activity was moderate on 22 strains when harvested in the morning, 4 at midday, and one in the evening. The MBC/MIC ratio showed that the extracts had a bactericidal effect on one strain and a bacteriostatic effect on the other 34.Table 6
Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the aqueous extracts of the leaves ofH. madagascariensis (µg/mL).
LeavesCIP6 a.m.12 p.m.6 p.m.MICMBCRMICMBCRMICMBCRMICMBCRGram-negative bacteriaEscherichia coliATCC 8739256—Nd128—Nd512—Nd11616ATCC 10536256—Nd——Nd———26432EC 5256—Nd——Nd1024102414328EC 4512—Nd1024—Nd———16464EC 2512—Nd——Nd1024—Nd4328EC 136512—Nd1024—Nd1024—Nd46416EC 137512—Nd1024—Nd———4328Citrobacter freundiiCITB 80512—Nd1024—Nd———13232CITB 81512—Nd1024—Nd———16464Serratia marcescensSERB 115—————————13232Yersinia enterocoliticaYERB 121—————————144YERBI512—Nd512—Nd1024—Nd122Salmonella enterica serovar typhiATCC 6539—————————482SAL 9512—Nd——————482S AL2562561———1024102410.564128Salmonella paratyphi B102410241——————2168Salmonella typhimurium512————————441Pseudomonas aeruginosaATCC 27853512—Nd1024—Nd1024—Nd16322PA 16256—Nd1024—Nd———23216Providencia stuartiiPS NEA 16—————————2168Klebsiella pneumoniaeKL 111512—Nd512—Nd1024—Nd32642KLPB101512—Nd———1024—Nd8324Enterobacter aerogenesENT 119512—Nd——————144ENT 167512—Nd——————16322ENT 511024—Nd1024—Nd1024—Nd8162Gram-positive bacteriaStaphylococcus aureusSTAPH 1102410241——————163223—————————188MRSA 3—————————0.524MRSA 912810248256—Nd1024102410.53264MRSA 12102410241——————8324MRSA 13—————————11616ST 120512—Nd——————23216ST 113102410241102410241———4328SA 3102410241——————188Streptococcus sp.STR 7512————————16464MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration: R: MBC/MIC; CIP: ciprofloxacin; Nd: not determined; —: no activity.(2) Bark Extracts. The aqueous extracts of the barks of H. madagascariensis were active on all the strains tested (35) with MICs between 64 and 1024 µg/mL (Table 7). Their activity was significant on at least 4 strains. The activity was moderate on 21 strains with the morning extract and 8 strains with the midday extract. The activity of the evening extracts was significant on 4 and moderate on 30 strains. The MBC/MIC ratio revealed that the extracts had a bactericidal effect on 6 of the 35 strains tested.Table 7
Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the aqueous extracts of the bark ofH. madagascariensis (µg/mL).
BarkCIP6 a.m.12 p.m.6 p.m.MICMBCRMICMBCRMICMBCRMICMBCRGram-negative bacteriaEscherichia coliATCC 8739——————64—Nd11616ATCC 10536256—Nd———512—Nd26432EC 5——————512—Nd4328EC 41024—Nd———512—Nd16464EC 21024—Nd———512—Nd4328EC 136512—Nd256—Nd512—Nd46416EC 137512—Nd1024—Nd256—Nd4328Citrobacter freundiiCITB 80512—Nd1024—Nd256—Nd13232CITB 81——————512—Nd16464Serratia marcescensSERB 115512—Nd512—Nd6410241613232Yersinia enterocoliticaYERB 121512—Nd1024102411285124144YERBI512—Nd———256—Nd122Salmonella enterica serovar typhiATCC 65391024102411024102412565122482SAL 9102410241102410241512—Nd482S AL512—Nd102410241128102480.564128Salmonella paratyphi B512—Nd102410241512102412168Salmonella typhimurium——————512—Nd441Pseudomonas aeruginosaATCC 27853512—Nd———1024—Nd16322PA 16256—Nd1024—Nd512—Nd23216Providencia stuartiiPS NEA 16512—Nd512—Nd641024162168Klebsiella pneumoniaeKL 111256—Nd256—Nd256—Nd32642KLPB1011024—Nd———512—Nd8324Enterobacter aerogenesENT 119512512151210242128—Nd144ENT 1671024102411024102412561024416322ENT 51256—Nd———512—Nd8162Gram-positive bacteriaStaphylococcus aureusSTAPH 1512—Nd10241024112851241632231024102415121024225610244188MRSA 3102410241102410241512—Nd0.524MRSA 9——————641024160.53264MRSA 12256—Nd512—Nd128102488324MRSA 1351251211024102411281024811616ST 120256—Nd256—Nd256—Nd23216ST 113——————512—Nd4328SA 3512——102410241256—Nd188Streptococcus sp.STR 7256—Nd———256—Nd16464MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; R: MBC/MIC; CIP: ciprofloxacin; Nd: not determined; —: no activity.(3) Roots Extract. The aqueous extract of the roots of H. madagascariensis was found to be almost inactive on the bacterial strains tested.


#### 3.1.4. Antibacterial Activity of Hydroalcoholic Extracts

The palm wine extracts from leaves, bark, and roots ofH. madagascariensis were found to be inactive on the bacterial strains tested.


## 3.1. Results

### 3.1.1. Extraction Yields

The extraction yields varied with the part of the plant, the harvest period, and the extraction method/solvent (Table2). The highest yields were obtained using the bark regardless of the harvest period and extraction solvent. In contrast, the lowest extraction yields were obtained using the roots. Furthermore, methanol extracts from the leaves and bark had the highest extraction yields when harvested in the morning. These yields decreased during the day relatively to leaves and barks. The hydroalcoholic extract had the best yield whatever the plant part and the harvest period.Table 2
Extraction yield (%) of methanol, aqueous, and palm wine decoctions fromH. madagascariensis at different harvest times/periods.
PartsSolventsMethanolPalm wineWater6 a.m.12 p.m.6 p.m.6 a.m.12 p.m.6 p.m.6 a.m.12 p.m.6 p.m.Leaves11.694.777.3926.5724.5324.487.480.940.94Bark15.4715.0213.3628.3624.0526.9526.3425.22.02Roots4.36.599.5522.4326.7224.824.51.71.98


### 3.1.2. Antibacterial Activities of Methanol Extracts

(1) Leaf Extract. The methanol extracts of the leaves of H. madagascariensis were active on 34 out of the 35 strains tested with MICs values ranging between 64 and 1024 µg/mL (Table 3). Their activity was significant on 3 out of the 35 strains tested. The activity of the 6 a.m. extract was significant on 1 strain, Serratia marcescens, out of the 35 strains tested and moderate on 18 strains. The activity of the midday extract was significant on 2 out of the 35 strains tested and moderate on 22 strains. The activity of the evening extract was significant on 2 strains and moderate on 29 strains.Table 3
Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of methanol extracts from the leaves ofH. madagascariensis (µg/mL).
LeavesCIP6 a.m.12 p.m.6 p.m.MICMBCRMICMBCRMICMBCRMICMBCRGram-negative bacteriaEscherichia coliATCC 8739256—Nd256—Nd25610244≤0.5816ATCC 10536512—Nd512—Nd256102442648EC 5128—Nd64—Nd64102416482EC 4512—Nd25651221285124≤0.564128EC 2512—Nd51210242512—Nd4328EC 1361024—Nd1024—Nd256—Nd46416EC 137———1024—Nd512—Nd4328Citrobacter freundiiCITB 80——————256—Nd13232CITB 81———256102446410241616464Serratia marcescensSERB 1156412826412821281024813232Yersinia enterocoliticaYERB 1211024—Nd12810248128—Nd≤0.5/48YERBI———51210242128—Nd122Salmonella enterica serovar typhiATCC 65391281024812810248128—Nd482SAL 9———256—Nd256—Nd482S AL128—Nd128—Nd128—Nd0.564128Salmonella paratyphi B256—Nd256—Nd———2168Salmonella typhimurium256—Nd25610244512—Nd4328Pseudomonas aeruginosaATCC 27853———51210242256—Nd16322PA 16512—Nd1024—Nd512—Nd23216Providencia stuartiiPS NEA 16—————————2168Klebsiella pneumoniaeKL 1111024—Nd———256—Nd32642KLPB101—————————8324Enterobacter aerogenesENT 119———1281024825610244144ENT 1672561024451210242256—Nd16322ENT 51———1024—Nd512—Nd8162Gram-positive bacteriaStaphylococcus aureusSTAPH 1256—Nd256—Nd128102481632231024—Nd256—Nd256—Nd188MRSA 32561024451210242512102420.524MRSA 925610244128—Nd128—Nd0.53264MRSA 125121024251210242512102428324MRSA 13512—Nd512—Nd512—Nd11616ST 120——————512—Nd23216ST 113102410241——————4328SA 312810248128—Nd12810248188Streptococcus sp.STR 7512—Nd1024—Nd2561024416464MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; R: MBC/MIC; CIP: ciprofloxacin; Nd: not determined; —: no activity.These results show that the leaves have better activity when they are harvested in the evening with MICs between 64 and 512µg/mL. The calculation of the MBC/MIC ratio reveals that the extracts had a bactericidal effect on 14 of the 35 strains tested and a bacteriostatic one on 21 of the 35 strains tested.Escherichia coli and Staphylococcus aureus strains were more sensitive to methanol extracts from the leaves. Among the 7 Escherichia coli strains tested, one strain presented a significant sensitivity and 5 strains presented a moderate sensitivity. S. aureus strains tested showed moderate sensitivity in 6 of the 9 strains tested, including methicillin-resistant strains.(2) Bark Extracts. The methanol extracts of the barks of H. madagascariensis, regardless of the time of harvest, were active on all 35 strains tested with MICs between 8 and 1024 µg/mL (Table 4). Their activity was significant on 9 out of the 35 tested strains.Table 4
Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of methanol extracts from the bark ofH. madagascariensis (µg/mL).
BarkCIP6 a.m.12 p.m.6 p.m.MICMBCRMICMBCRMICMBCRMICMBCRGram-negative bacteriaEscherichia coliATCC 87391281024864—Nd64102416≤0.5816ATCC 1053625610244128—Nd12851242648EC 5256—Nd25610244128—Nd482EC 41282562256—Nd12810248≤0.564128EC 2512—Nd1024—Nd512—Nd4328EC 136512—Nd512—Nd5121024246416EC 137256512225610244256102444328Citrobacter freundiiCITB 8012810248128—Nd64512813232CITB 81256512225610244256512216464Serratia marcescensSERB 11564512864512864256413232Yersinia enterocoliticaYERB 121256—Nd32—Nd32102432≤0.5/48YERBI512102421024—Nd25610244122Salmonella enterica serovar typhiATCC 6539128—Nd32—Nd32102432482SAL 9256—Nd128—Nd128—Nd482S AL128—Nd12810248641024160.564128Salmonella paratyphi B12812812561024425651222168Salmonella typhimurium512—Nd512—Nd128—Nd4328Pseudomonas aeruginosaATCC 27853512—Nd51210242256512216322PA 16512—Nd512—Nd256—Nd23216Providencia stuartiiPS NEA 16512—Nd256—Nd256—Nd2168Klebsiella pneumoniaeKL 111256—Nd512—Nd2561024432642KLPB101——————256102448324Enterobacter aerogenesENT 119128—Nd128—Nd128—Nd144ENT 167256—Nd128102482561024416322ENT 511024—Nd512—Nd1024—Nd8162Gram-positive bacteriaStaphylococcus aureusSTAPH 16425646451264512816322364—Nd≤8—Nd32—Nd188MRSA 3512—Nd5121024225651220.524MRSA 964—Nd810241288—Nd0.53264MRSA 122561024412810248128102488324MRSA 13512—Nd512—Nd256—Nd11616ST 120———1024—Nd256—Nd23216ST 113———512—Nd512—Nd4328SA 31281024864641128—Nd188Streptococcus sp.STR 751210242256—Nd128512416464MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; R: MBC/MIC; CIP: ciprofloxacin; Nd: not determined; —: no activity.The activity of the morning extract was significant on 4 strains and moderate on 27 strains. The activity of the midday extract was significant on 8 and moderate on 23 strains and, finally, the activity of the evening extract was significant on 9 and moderate on 25 strains. These results show that the barks have better activity when they are harvested in the evening with MICs between 8 and 512µg/mL.The MBC/MIC ratio revealed that the extracts had a bactericidal effect on 13 of the 35 strains tested and a bacteriostatic one on 22 strains.The strains ofS. aureus and E. coli appear to be more sensitive to the methanol extract of barks. Among the 9 strains of S. aureus, 6 strains showed a moderate activity and 3 strains a weak activity.(3). Root Extract. The methanol extracts of the roots of H. madagascariensis, regardless of the harvesting time, were active on 24 of the 35 strains tested with MICs between 128 and 1024 µg/mL (Table 5). The activity of morning extract was moderate on 15 strains, that of midday on 16, and that in the evening on 20 strains. The MBC/MIC ratio showed that the extracts had a bactericidal effect on 8 strains and a bacteriostatic effect on 27 strains. S. aureus strains were more sensitive to extracts from the roots. Indeed, among the 9 strains of S. aureus, including the methicillin-resistant strains, 8 strains showed moderate activity.Table 5
Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of methanol extracts from the roots ofH. madagascariensis (µg/mL).
RootsCIP6 a.m.12 p.m.6 p.m.MICMBCRMICMBCRMICMBCRMICMBCRGram-negative bacteriaEscherichia coliATCC 8739512—Nd512—Nd256—Nd≤0.5816ATCC 10536—————————2648EC 5512—Nd512—Nd256—Nd482EC 4—————————≤0.564128EC 2—————————4328EC 136————————46416EC 137—————————4328Citrobacter freundiiCITB 80——————1024—Nd13232CITB 81—————————16464Serratia marcescensSERB 11512810248256—Nd128—Nd13232Yersinia enterocoliticaYERB 1211024—Nd128—Nd5125121≤0.5/48YERBI—————————122Salmonella enterica serovar typhiATCC 653925610244512—Nd25610244482SAL 91024—Nd1024—Nd1024—Nd482S AL12825622561024425610244≤0.564128Salmonella paratyphi B128—Nd512—Nd256—Nd2168Salmonella typhimurium——————512102424328Pseudomonas aeruginosaATCC 278531024—Nd——————16322PA 16128—Nd———128—Nd23216Providencia stuartiiPS NEA 16——————1024—Nd2168Klebsiella pneumoniaeKL 111—————————32642KLPB101————————8324Enterobacter aerogenesENT 119128—Nd512—Nd512—Nd144ENT 1671024102411024102415121024216322ENT 51—————————8162Gram-positive bacteriaStaphylococcus aureusSTAPH 15121024512—Nd512—Nd1632231024—Nd512—Nd512—Nd188MRSA 3512—Nd51210242512102420.524MRSA 9256—Nd256—Nd128102480.53264MRSA 1212810248256—Nd256102448324MRSA 13128—Nd512—Nd512—Nd11616ST 120—————————23216ST 113512—Nd512——512102424328SA 3256—Nd51210242256—Nd188Streptococcus sp.STR 7——————512—Nd16464MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; R: MBC/IMC; CIP: ciprofloxacin; Nd: not determined; —: no activity.


### 3.1.3. Antibacterial Activity of Aqueous Extracts

(1) Leaf Extracts. The aqueous extracts of the leaves of H. madagascariensis, regardless of the time of harvest, were active on 28 out of the 35 strains tested with MICs between 128 and 1024 µg/mL (Table 6). The activity was moderate on 22 strains when harvested in the morning, 4 at midday, and one in the evening. The MBC/MIC ratio showed that the extracts had a bactericidal effect on one strain and a bacteriostatic effect on the other 34.Table 6
Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the aqueous extracts of the leaves ofH. madagascariensis (µg/mL).
LeavesCIP6 a.m.12 p.m.6 p.m.MICMBCRMICMBCRMICMBCRMICMBCRGram-negative bacteriaEscherichia coliATCC 8739256—Nd128—Nd512—Nd11616ATCC 10536256—Nd——Nd———26432EC 5256—Nd——Nd1024102414328EC 4512—Nd1024—Nd———16464EC 2512—Nd——Nd1024—Nd4328EC 136512—Nd1024—Nd1024—Nd46416EC 137512—Nd1024—Nd———4328Citrobacter freundiiCITB 80512—Nd1024—Nd———13232CITB 81512—Nd1024—Nd———16464Serratia marcescensSERB 115—————————13232Yersinia enterocoliticaYERB 121—————————144YERBI512—Nd512—Nd1024—Nd122Salmonella enterica serovar typhiATCC 6539—————————482SAL 9512—Nd——————482S AL2562561———1024102410.564128Salmonella paratyphi B102410241——————2168Salmonella typhimurium512————————441Pseudomonas aeruginosaATCC 27853512—Nd1024—Nd1024—Nd16322PA 16256—Nd1024—Nd———23216Providencia stuartiiPS NEA 16—————————2168Klebsiella pneumoniaeKL 111512—Nd512—Nd1024—Nd32642KLPB101512—Nd———1024—Nd8324Enterobacter aerogenesENT 119512—Nd——————144ENT 167512—Nd——————16322ENT 511024—Nd1024—Nd1024—Nd8162Gram-positive bacteriaStaphylococcus aureusSTAPH 1102410241——————163223—————————188MRSA 3—————————0.524MRSA 912810248256—Nd1024102410.53264MRSA 12102410241——————8324MRSA 13—————————11616ST 120512—Nd——————23216ST 113102410241102410241———4328SA 3102410241——————188Streptococcus sp.STR 7512————————16464MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration: R: MBC/MIC; CIP: ciprofloxacin; Nd: not determined; —: no activity.(2) Bark Extracts. The aqueous extracts of the barks of H. madagascariensis were active on all the strains tested (35) with MICs between 64 and 1024 µg/mL (Table 7). Their activity was significant on at least 4 strains. The activity was moderate on 21 strains with the morning extract and 8 strains with the midday extract. The activity of the evening extracts was significant on 4 and moderate on 30 strains. The MBC/MIC ratio revealed that the extracts had a bactericidal effect on 6 of the 35 strains tested.Table 7
Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the aqueous extracts of the bark ofH. madagascariensis (µg/mL).
BarkCIP6 a.m.12 p.m.6 p.m.MICMBCRMICMBCRMICMBCRMICMBCRGram-negative bacteriaEscherichia coliATCC 8739——————64—Nd11616ATCC 10536256—Nd———512—Nd26432EC 5——————512—Nd4328EC 41024—Nd———512—Nd16464EC 21024—Nd———512—Nd4328EC 136512—Nd256—Nd512—Nd46416EC 137512—Nd1024—Nd256—Nd4328Citrobacter freundiiCITB 80512—Nd1024—Nd256—Nd13232CITB 81——————512—Nd16464Serratia marcescensSERB 115512—Nd512—Nd6410241613232Yersinia enterocoliticaYERB 121512—Nd1024102411285124144YERBI512—Nd———256—Nd122Salmonella enterica serovar typhiATCC 65391024102411024102412565122482SAL 9102410241102410241512—Nd482S AL512—Nd102410241128102480.564128Salmonella paratyphi B512—Nd102410241512102412168Salmonella typhimurium——————512—Nd441Pseudomonas aeruginosaATCC 27853512—Nd———1024—Nd16322PA 16256—Nd1024—Nd512—Nd23216Providencia stuartiiPS NEA 16512—Nd512—Nd641024162168Klebsiella pneumoniaeKL 111256—Nd256—Nd256—Nd32642KLPB1011024—Nd———512—Nd8324Enterobacter aerogenesENT 119512512151210242128—Nd144ENT 1671024102411024102412561024416322ENT 51256—Nd———512—Nd8162Gram-positive bacteriaStaphylococcus aureusSTAPH 1512—Nd10241024112851241632231024102415121024225610244188MRSA 3102410241102410241512—Nd0.524MRSA 9——————641024160.53264MRSA 12256—Nd512—Nd128102488324MRSA 1351251211024102411281024811616ST 120256—Nd256—Nd256—Nd23216ST 113——————512—Nd4328SA 3512——102410241256—Nd188Streptococcus sp.STR 7256—Nd———256—Nd16464MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; R: MBC/MIC; CIP: ciprofloxacin; Nd: not determined; —: no activity.(3) Roots Extract. The aqueous extract of the roots of H. madagascariensis was found to be almost inactive on the bacterial strains tested.


### 3.1.4. Antibacterial Activity of Hydroalcoholic Extracts

The palm wine extracts from leaves, bark, and roots ofH. madagascariensis were found to be inactive on the bacterial strains tested.


## 3.1.1. Extraction Yields

The extraction yields varied with the part of the plant, the harvest period, and the extraction method/solvent (Table2). The highest yields were obtained using the bark regardless of the harvest period and extraction solvent. In contrast, the lowest extraction yields were obtained using the roots. Furthermore, methanol extracts from the leaves and bark had the highest extraction yields when harvested in the morning. These yields decreased during the day relatively to leaves and barks. The hydroalcoholic extract had the best yield whatever the plant part and the harvest period.Table 2
Extraction yield (%) of methanol, aqueous, and palm wine decoctions fromH. madagascariensis at different harvest times/periods.
PartsSolventsMethanolPalm wineWater6 a.m.12 p.m.6 p.m.6 a.m.12 p.m.6 p.m.6 a.m.12 p.m.6 p.m.Leaves11.694.777.3926.5724.5324.487.480.940.94Bark15.4715.0213.3628.3624.0526.9526.3425.22.02Roots4.36.599.5522.4326.7224.824.51.71.98


## 3.1.2. Antibacterial Activities of Methanol Extracts

(1) Leaf Extract. The methanol extracts of the leaves of H. madagascariensis were active on 34 out of the 35 strains tested with MICs values ranging between 64 and 1024 µg/mL (Table 3). Their activity was significant on 3 out of the 35 strains tested. The activity of the 6 a.m. extract was significant on 1 strain, Serratia marcescens, out of the 35 strains tested and moderate on 18 strains. The activity of the midday extract was significant on 2 out of the 35 strains tested and moderate on 22 strains. The activity of the evening extract was significant on 2 strains and moderate on 29 strains.Table 3
Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of methanol extracts from the leaves ofH. madagascariensis (µg/mL).
LeavesCIP6 a.m.12 p.m.6 p.m.MICMBCRMICMBCRMICMBCRMICMBCRGram-negative bacteriaEscherichia coliATCC 8739256—Nd256—Nd25610244≤0.5816ATCC 10536512—Nd512—Nd256102442648EC 5128—Nd64—Nd64102416482EC 4512—Nd25651221285124≤0.564128EC 2512—Nd51210242512—Nd4328EC 1361024—Nd1024—Nd256—Nd46416EC 137———1024—Nd512—Nd4328Citrobacter freundiiCITB 80——————256—Nd13232CITB 81———256102446410241616464Serratia marcescensSERB 1156412826412821281024813232Yersinia enterocoliticaYERB 1211024—Nd12810248128—Nd≤0.5/48YERBI———51210242128—Nd122Salmonella enterica serovar typhiATCC 65391281024812810248128—Nd482SAL 9———256—Nd256—Nd482S AL128—Nd128—Nd128—Nd0.564128Salmonella paratyphi B256—Nd256—Nd———2168Salmonella typhimurium256—Nd25610244512—Nd4328Pseudomonas aeruginosaATCC 27853———51210242256—Nd16322PA 16512—Nd1024—Nd512—Nd23216Providencia stuartiiPS NEA 16—————————2168Klebsiella pneumoniaeKL 1111024—Nd———256—Nd32642KLPB101—————————8324Enterobacter aerogenesENT 119———1281024825610244144ENT 1672561024451210242256—Nd16322ENT 51———1024—Nd512—Nd8162Gram-positive bacteriaStaphylococcus aureusSTAPH 1256—Nd256—Nd128102481632231024—Nd256—Nd256—Nd188MRSA 32561024451210242512102420.524MRSA 925610244128—Nd128—Nd0.53264MRSA 125121024251210242512102428324MRSA 13512—Nd512—Nd512—Nd11616ST 120——————512—Nd23216ST 113102410241——————4328SA 312810248128—Nd12810248188Streptococcus sp.STR 7512—Nd1024—Nd2561024416464MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; R: MBC/MIC; CIP: ciprofloxacin; Nd: not determined; —: no activity.These results show that the leaves have better activity when they are harvested in the evening with MICs between 64 and 512µg/mL. The calculation of the MBC/MIC ratio reveals that the extracts had a bactericidal effect on 14 of the 35 strains tested and a bacteriostatic one on 21 of the 35 strains tested.Escherichia coli and Staphylococcus aureus strains were more sensitive to methanol extracts from the leaves. Among the 7 Escherichia coli strains tested, one strain presented a significant sensitivity and 5 strains presented a moderate sensitivity. S. aureus strains tested showed moderate sensitivity in 6 of the 9 strains tested, including methicillin-resistant strains.(2) Bark Extracts. The methanol extracts of the barks of H. madagascariensis, regardless of the time of harvest, were active on all 35 strains tested with MICs between 8 and 1024 µg/mL (Table 4). Their activity was significant on 9 out of the 35 tested strains.Table 4
Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of methanol extracts from the bark ofH. madagascariensis (µg/mL).
BarkCIP6 a.m.12 p.m.6 p.m.MICMBCRMICMBCRMICMBCRMICMBCRGram-negative bacteriaEscherichia coliATCC 87391281024864—Nd64102416≤0.5816ATCC 1053625610244128—Nd12851242648EC 5256—Nd25610244128—Nd482EC 41282562256—Nd12810248≤0.564128EC 2512—Nd1024—Nd512—Nd4328EC 136512—Nd512—Nd5121024246416EC 137256512225610244256102444328Citrobacter freundiiCITB 8012810248128—Nd64512813232CITB 81256512225610244256512216464Serratia marcescensSERB 11564512864512864256413232Yersinia enterocoliticaYERB 121256—Nd32—Nd32102432≤0.5/48YERBI512102421024—Nd25610244122Salmonella enterica serovar typhiATCC 6539128—Nd32—Nd32102432482SAL 9256—Nd128—Nd128—Nd482S AL128—Nd12810248641024160.564128Salmonella paratyphi B12812812561024425651222168Salmonella typhimurium512—Nd512—Nd128—Nd4328Pseudomonas aeruginosaATCC 27853512—Nd51210242256512216322PA 16512—Nd512—Nd256—Nd23216Providencia stuartiiPS NEA 16512—Nd256—Nd256—Nd2168Klebsiella pneumoniaeKL 111256—Nd512—Nd2561024432642KLPB101——————256102448324Enterobacter aerogenesENT 119128—Nd128—Nd128—Nd144ENT 167256—Nd128102482561024416322ENT 511024—Nd512—Nd1024—Nd8162Gram-positive bacteriaStaphylococcus aureusSTAPH 16425646451264512816322364—Nd≤8—Nd32—Nd188MRSA 3512—Nd5121024225651220.524MRSA 964—Nd810241288—Nd0.53264MRSA 122561024412810248128102488324MRSA 13512—Nd512—Nd256—Nd11616ST 120———1024—Nd256—Nd23216ST 113———512—Nd512—Nd4328SA 31281024864641128—Nd188Streptococcus sp.STR 751210242256—Nd128512416464MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; R: MBC/MIC; CIP: ciprofloxacin; Nd: not determined; —: no activity.The activity of the morning extract was significant on 4 strains and moderate on 27 strains. The activity of the midday extract was significant on 8 and moderate on 23 strains and, finally, the activity of the evening extract was significant on 9 and moderate on 25 strains. These results show that the barks have better activity when they are harvested in the evening with MICs between 8 and 512µg/mL.The MBC/MIC ratio revealed that the extracts had a bactericidal effect on 13 of the 35 strains tested and a bacteriostatic one on 22 strains.The strains ofS. aureus and E. coli appear to be more sensitive to the methanol extract of barks. Among the 9 strains of S. aureus, 6 strains showed a moderate activity and 3 strains a weak activity.(3). Root Extract. The methanol extracts of the roots of H. madagascariensis, regardless of the harvesting time, were active on 24 of the 35 strains tested with MICs between 128 and 1024 µg/mL (Table 5). The activity of morning extract was moderate on 15 strains, that of midday on 16, and that in the evening on 20 strains. The MBC/MIC ratio showed that the extracts had a bactericidal effect on 8 strains and a bacteriostatic effect on 27 strains. S. aureus strains were more sensitive to extracts from the roots. Indeed, among the 9 strains of S. aureus, including the methicillin-resistant strains, 8 strains showed moderate activity.Table 5
Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of methanol extracts from the roots ofH. madagascariensis (µg/mL).
RootsCIP6 a.m.12 p.m.6 p.m.MICMBCRMICMBCRMICMBCRMICMBCRGram-negative bacteriaEscherichia coliATCC 8739512—Nd512—Nd256—Nd≤0.5816ATCC 10536—————————2648EC 5512—Nd512—Nd256—Nd482EC 4—————————≤0.564128EC 2—————————4328EC 136————————46416EC 137—————————4328Citrobacter freundiiCITB 80——————1024—Nd13232CITB 81—————————16464Serratia marcescensSERB 11512810248256—Nd128—Nd13232Yersinia enterocoliticaYERB 1211024—Nd128—Nd5125121≤0.5/48YERBI—————————122Salmonella enterica serovar typhiATCC 653925610244512—Nd25610244482SAL 91024—Nd1024—Nd1024—Nd482S AL12825622561024425610244≤0.564128Salmonella paratyphi B128—Nd512—Nd256—Nd2168Salmonella typhimurium——————512102424328Pseudomonas aeruginosaATCC 278531024—Nd——————16322PA 16128—Nd———128—Nd23216Providencia stuartiiPS NEA 16——————1024—Nd2168Klebsiella pneumoniaeKL 111—————————32642KLPB101————————8324Enterobacter aerogenesENT 119128—Nd512—Nd512—Nd144ENT 1671024102411024102415121024216322ENT 51—————————8162Gram-positive bacteriaStaphylococcus aureusSTAPH 15121024512—Nd512—Nd1632231024—Nd512—Nd512—Nd188MRSA 3512—Nd51210242512102420.524MRSA 9256—Nd256—Nd128102480.53264MRSA 1212810248256—Nd256102448324MRSA 13128—Nd512—Nd512—Nd11616ST 120—————————23216ST 113512—Nd512——512102424328SA 3256—Nd51210242256—Nd188Streptococcus sp.STR 7——————512—Nd16464MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; R: MBC/IMC; CIP: ciprofloxacin; Nd: not determined; —: no activity.


## 3.1.3. Antibacterial Activity of Aqueous Extracts

(1) Leaf Extracts. The aqueous extracts of the leaves of H. madagascariensis, regardless of the time of harvest, were active on 28 out of the 35 strains tested with MICs between 128 and 1024 µg/mL (Table 6). The activity was moderate on 22 strains when harvested in the morning, 4 at midday, and one in the evening. The MBC/MIC ratio showed that the extracts had a bactericidal effect on one strain and a bacteriostatic effect on the other 34.Table 6
Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the aqueous extracts of the leaves ofH. madagascariensis (µg/mL).
LeavesCIP6 a.m.12 p.m.6 p.m.MICMBCRMICMBCRMICMBCRMICMBCRGram-negative bacteriaEscherichia coliATCC 8739256—Nd128—Nd512—Nd11616ATCC 10536256—Nd——Nd———26432EC 5256—Nd——Nd1024102414328EC 4512—Nd1024—Nd———16464EC 2512—Nd——Nd1024—Nd4328EC 136512—Nd1024—Nd1024—Nd46416EC 137512—Nd1024—Nd———4328Citrobacter freundiiCITB 80512—Nd1024—Nd———13232CITB 81512—Nd1024—Nd———16464Serratia marcescensSERB 115—————————13232Yersinia enterocoliticaYERB 121—————————144YERBI512—Nd512—Nd1024—Nd122Salmonella enterica serovar typhiATCC 6539—————————482SAL 9512—Nd——————482S AL2562561———1024102410.564128Salmonella paratyphi B102410241——————2168Salmonella typhimurium512————————441Pseudomonas aeruginosaATCC 27853512—Nd1024—Nd1024—Nd16322PA 16256—Nd1024—Nd———23216Providencia stuartiiPS NEA 16—————————2168Klebsiella pneumoniaeKL 111512—Nd512—Nd1024—Nd32642KLPB101512—Nd———1024—Nd8324Enterobacter aerogenesENT 119512—Nd——————144ENT 167512—Nd——————16322ENT 511024—Nd1024—Nd1024—Nd8162Gram-positive bacteriaStaphylococcus aureusSTAPH 1102410241——————163223—————————188MRSA 3—————————0.524MRSA 912810248256—Nd1024102410.53264MRSA 12102410241——————8324MRSA 13—————————11616ST 120512—Nd——————23216ST 113102410241102410241———4328SA 3102410241——————188Streptococcus sp.STR 7512————————16464MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration: R: MBC/MIC; CIP: ciprofloxacin; Nd: not determined; —: no activity.(2) Bark Extracts. The aqueous extracts of the barks of H. madagascariensis were active on all the strains tested (35) with MICs between 64 and 1024 µg/mL (Table 7). Their activity was significant on at least 4 strains. The activity was moderate on 21 strains with the morning extract and 8 strains with the midday extract. The activity of the evening extracts was significant on 4 and moderate on 30 strains. The MBC/MIC ratio revealed that the extracts had a bactericidal effect on 6 of the 35 strains tested.Table 7
Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the aqueous extracts of the bark ofH. madagascariensis (µg/mL).
BarkCIP6 a.m.12 p.m.6 p.m.MICMBCRMICMBCRMICMBCRMICMBCRGram-negative bacteriaEscherichia coliATCC 8739——————64—Nd11616ATCC 10536256—Nd———512—Nd26432EC 5——————512—Nd4328EC 41024—Nd———512—Nd16464EC 21024—Nd———512—Nd4328EC 136512—Nd256—Nd512—Nd46416EC 137512—Nd1024—Nd256—Nd4328Citrobacter freundiiCITB 80512—Nd1024—Nd256—Nd13232CITB 81——————512—Nd16464Serratia marcescensSERB 115512—Nd512—Nd6410241613232Yersinia enterocoliticaYERB 121512—Nd1024102411285124144YERBI512—Nd———256—Nd122Salmonella enterica serovar typhiATCC 65391024102411024102412565122482SAL 9102410241102410241512—Nd482S AL512—Nd102410241128102480.564128Salmonella paratyphi B512—Nd102410241512102412168Salmonella typhimurium——————512—Nd441Pseudomonas aeruginosaATCC 27853512—Nd———1024—Nd16322PA 16256—Nd1024—Nd512—Nd23216Providencia stuartiiPS NEA 16512—Nd512—Nd641024162168Klebsiella pneumoniaeKL 111256—Nd256—Nd256—Nd32642KLPB1011024—Nd———512—Nd8324Enterobacter aerogenesENT 119512512151210242128—Nd144ENT 1671024102411024102412561024416322ENT 51256—Nd———512—Nd8162Gram-positive bacteriaStaphylococcus aureusSTAPH 1512—Nd10241024112851241632231024102415121024225610244188MRSA 3102410241102410241512—Nd0.524MRSA 9——————641024160.53264MRSA 12256—Nd512—Nd128102488324MRSA 1351251211024102411281024811616ST 120256—Nd256—Nd256—Nd23216ST 113——————512—Nd4328SA 3512——102410241256—Nd188Streptococcus sp.STR 7256—Nd———256—Nd16464MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; R: MBC/MIC; CIP: ciprofloxacin; Nd: not determined; —: no activity.(3) Roots Extract. The aqueous extract of the roots of H. madagascariensis was found to be almost inactive on the bacterial strains tested.


## 3.1.4. Antibacterial Activity of Hydroalcoholic Extracts

The palm wine extracts from leaves, bark, and roots ofH. madagascariensis were found to be inactive on the bacterial strains tested.


## 4. Discussion

Plants are an undeniable source of active substances, many of which are effective at the level of traditional pharmacopoeia as well as at the level of scientific research [17]. Among the multitude of active plants well established all over the word, the unanimity about the part used and the extraction solvents is a permanent quest. Moreover, few studies are interested in the contributions of the agroecological zone, the age, and the daily metabolic variation within the plant. If the standardisation of the active principles of plants is a prerequisite for their efficient use for the pharmacological purpose, it seems certain that it inevitably involves the mastery of the conditions which could affect the variability of the principles at both qualitative and quantitative levels. It is in this context that this work falls, with the general objective being to identify the optimal conditions for the antibacterial activity of H. madagascariensis extract.The extraction yields ofH. madagascariensis obtained vary from one part of the plant to another and according to the harvest period and extraction solvent used. For methanol extracts, the best yields were obtained using the bark harvested in the morning and at midday. Similarly, the lowest yield was obtained using the roots harvested in the morning (6 a.m.). The variation in extraction yield reflects that of potent antimicrobial compounds whose high concentrations could be obtained at certain times of the day. Similar results have been reported by several authors. Indeed, Ramezani et al. [18] reported a maximum essential oils extract with Eucalyptus nicholii and Rosmarinus officinalis in the central region of Iran at midday and in the afternoon (6 p.m.). Similarly, Magne [10] reported that the extraction yields of H. madagascariensis varied with the plant part and the collection period. These results, therefore, highlight the importance of the plants harvesting period for their pharmacological properties since the yield is an essential component for the pharmacological study of the active substances from plants.The barks well concentrated the phytochemical constituents ofH. madagascariensis, whatever the time of harvest. The variation in extraction yield reflects that of potentially antimicrobial compounds whose high concentrations could be obtained with the barks. Thus, the barks of H. madagascariensis could be privileged parts of harvest for the pharmacological use of this plant. Similar results were reported by Wang and Liu [19] who found large variation in the composition among oils from roots, fruits, stems, leaves, and flowers of Litsea cubeba. These results highlight the importance of plant part for the exploration or exploitation of pharmacological properties.The different yields of methanol and aqueous extracts of the bark ofH. madagascariensis obtained at certain times of the day approximate to those obtained by Ebelle Etame et al. [9] and Magne [10], i.e., 14.5% and 19.79%, respectively. These high yields constitute an important factor in the bioavailability of the extracts for later use as a basic principle of phytomedicine.To make a juxtaposition between the ethnobotanical and scientific means, on the one hand and, and to raise the interest of the solvent, on the other hand, the methanol, aqueous, and hydroalcoholic extracts of the leaves, bark, and roots ofH. madagascariensis were prepared. From the antibacterial activity of different extracts, as a function of solvent, part, and harvesting period, it appears that the barks are the most active part of the plant, with methanol followed by water being the best extraction solvent. The barks of H. madagascariensis could be the best part concentrating compounds responsible for the antibacterial activity. The results are in line with earlier mentioned data where the bark was found to be the suitable plant part for antifungal substances. The antimicrobial activity of plant extracts is due to the presence of secondary metabolites, and the activity of some classes of phytochemicals has been demonstrated [20]. According to previous results on this plant, the activity observed could be associated with the presence of harunganin [21, 22], Malagasy anthrone [23], harongin anthrone [22], harunmadagascarins A and B, kengaquinone, physcion [24], madagascol [25], vismiaquinone [24, 25], betulinic acid, kaempferol-3-O-β-D-glucopyranoside and madagascin, ferruginin A [7].The results of the antibacterial activity corroborate those of Krief [8] who showed that the methanol extract of bark of H. madagascariensis had antibacterial properties with MIC values varying between 32 and 1024 µg/mL. Moreover, the result brings to light the need for complementarity between ethnobotanical knowledge and scientific research for more effective development of medicinal plants. In addition, under traditional conditions, it highlights the use of water rather than palm wine in the preparation of the decoction for antibacterial use.It appeared that extracts are more active when the parts of the plant are harvested in the evening (6 p.m.). The plant uses solar energy for the synthesis of primary metabolites, which are further converted into secondary metabolites, with the concentration of each depending on the plant and several conditions [26]. The accumulation of these compounds responsible for antibacterial activity could be maximum in H. madagascariensis (around 6 p.m.). These results corroborate those of Da Silva et al. [27] who found a maximum of bioactive compounds in some plants when harvested in the afternoon. They also corroborate Ramezani et al.’s findings [18]. This result highlights once more the interest in considering endogenous and exogenous factors on which the concentration of the active ingredients in the plant depends. According to the World Health Organisation [28], medicinal plants should be harvested in the optimal season or time to ensure the production of the highest possible quality and quantity of bioproducts.By comparing the MIC values obtained from the different extraction solvents, regardless of the time of the day and the harvested parts, it appears that methanol is the best extraction solvent with MICs between 8 and 1024µg/mL. According to Osama [29], during the extraction with a solvent, phytomolecules dissolve in the extraction solvent according to their polarity. Thus, the active compounds of this plant could be a mixture of polar and semipolar compounds best extracted with methanol. These results are in line with those of Benbrinis [30] who found that, compared to water, the methanol extract of the aerial part of Santolina chamaecyparissus, a medicinal plant from the pharmacopoeia tradition of Algeria, had a considerable antibacterial effect on the strains tested.The methanol extracts of bark fromH. madagascariensis were more active on E. coli and S. aureus strains which are resistant to some commonly used antibiotics in Cameroon including ampicillin, piperacillin/tazobactam, nalidixic acid, ciprofloxacin, ofloxacin, kanamycin, erythromycin, tetracycline, flomoxef, and chloramphenicol. The efficiency of the methanol extracts of the barks of H. madagascariensis, thus, proves that the bark of this plant is a potential source of antibacterial molecules capable of fighting against diseases mostly caused by these microorganisms, including those caused by their multiresistant forms. The results are complementary to previous works, which reported the antibacterial activity of the aqueous extract of the leaves and bark of H. madagascariensis on S. paratyphi, S. aureus, and E. coli. [7].


## 5. Conclusion

This study focused on the evaluation of the influence of the plant’s parts, the harvest period, and the solvent on the yield and antibacterial activity ofH. madagascariensis. The aim was to highlight the optimal conditions for antibacterial activity of H. madagascariensis extracts. It appeared that the highest yields were obtained with the bark of H. madagascariensis regardless of the harvest period and the extraction solvent. It also appeared that bark extracts had the best antibacterial activity. The ideal time for harvesting the barks for good antibacterial activity was the evening (6 p.m.), and the solvent that made it possible to concentrate the maximum of the antibacterial compound was methanol. This study further confirms the use of the bark of H. madagascariensis as an antibacterial agent and, better still, the use of the barks harvested at 6 p.m. and extracted with methanol.



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*Source: 6615596-2021-03-11.xml*