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Assessment of antibacterial activity of some extracts of Securinega virosa (Roxb. ex Willd.) Baill on pathogens bacteria

Mani Adrien KOUANGBE^1*, Messou TCHUMOU¹, Monon KONE², Karamoko OUATTARA¹, Jean David N’GUESSAN³

¹ Department of Microbiology and Molecular Biology, Training and Research Unit of Agriculture, fisheries resources and agro-industry, University of San Pedro, San Pedro, Côte d’Ivoire

² Department of Biochemistry-Genetics, Training and Research Unit of Biological Sciences, Peleforo Gon Coulibaly University of Korhogo, Korhogo, Côte d'Ivoire.

³ Department of Biology and Health, Training and Research Unit of Bioscience, University of Félix Houphouët-Boigny, Abidjan, Côte d’Ivoire

Article Info:

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Article History:

Received 12 Nov 2022

Reviewed 19 Dec 2022

Accepted 01 Jan 2023

Published 15 Jan 2023

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Cite this article as:

Kouangbe MA, Tchumou M, Kone M, Ouattara K, N’Guessan JD, Assessment of antibacterial activity of some extracts of Securinega virosa (Roxb. ex Willd.) Baill on pathogens bacteria, Journal of Drug Delivery and Therapeutics. 2023; 13(1):116-122

DOI: http://dx.doi.org/10.22270/jddt.v13i1.5726 _______________________________________________*Address for Correspondence:

KOUANGBE Mani Adrien, Department of Microbiology and Molecular Biology, Training and Research Unit of Agriculture, fisheries resources and agro-industry, University of San Pedro, San Pedro, Côte d’Ivoire. BP 1800 San Pedro

Abstract

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Today, faced with the emergence of microbial resistance to antibiotics, the renewal of the arsenal of anti-infective drugs is acutely posed. In order to explore other sources of anti-infective drugs, this study therefore proposed to evaluate the antibacterial activity in vitro of several extracts of Securinega virosa, a well-known combretacea of populations in northern Côte d'Ivoire. To achieve this objective, the method of determining the diameters of the zones of inhibition on wells in an agar medium as well as that of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration were carried out. The results showed that the aqueous extract was not active on all the bacteria tested. The dichloromethane fraction at 500 mg/mL inhibited growth of Shigella Typhi (12.5 mm), followed by Streptococcus sp. (12.33 0.25 mm) and Staphylococcus aureus Meti-R (11.75 1.25 mm). The ethyl acetate fraction inhibited primarily Gram-positive bacteria with average diameters of 12 mm to 500 mg/mL. The ethanol fraction was most active on all bacteria with inhibition diameters ranging from 9 to 13.33 mm to 500 mg/mL. It showed the lowest MIC (3.12 mg/mL) on gram-positive and large Gram-negative levels ranging from 12.5 to 50 mg/mL. This study through its results provides data in favor of the traditional use of Securinega virosa in therapy.

Keywords : Securinega virosa, in vitro, antibacterial, activity

Both in industrialized countries and in countries around the tropics, infectious diseases continue to rank among the causes, the most common causes of human mortality in the world¹. Yet, the chemotherapy of bacterial infections that began in the early 1940s with Flemming's discovery of penicillin followed by the advent of new molecules in therapeutics had the important benefit of increasing human life expectancy. After less than half a century of existence, this brilliant picture is darkened by the progressive appearance of pathogen resistance to antimicrobials. This phenomenon, which is making the drugs used to treat infections less effective, has emerged as one of the major public health threats of the 21st century². According to the British government, antimicrobial resistance could kill 10 million people a year by 2050³

Faced with this emerging public health problem, the renewal of the arsenal of anti-infective molecules has become a priority. This leads to the search for new antimicrobial agents mainly among plant extracts with the aim of discovering new chemical structures that are effective and free of toxicity⁴. Indeed, plants have been used for centuries to treat infectious diseases and are considered an important source of new antimicrobial agents⁵. In this perspective, more and more researchers are directing their work in the evaluation of antimicrobial effects of plant extracts namely root, stem, leaf or flower extracts⁶

Like these colleagues, the present study focused on the root barks of Securinega virosa (Roxb. ex Willd) Baill. In Côte d'Ivoire, ethnobotanical studies have shown that this plant is used in the traditional treatment of infectious and metabolic diseases⁷. Pharmacological studies attribute anti-diabetic⁸, anti-diarrheal⁹, anti-oxidant¹⁰ and anti-malarial¹¹ properties to it.

This study plans to explore the antibacterial potential of different extracts of Securinega virosa (Roxb. ex Willd) Baill on pathogenic bacteria (Gram negative and Gram positive) to humans.

Müller-Hinton agar (Liofilchem^®, Italy) for the study of bacterial susceptibility to different plant extracts, ordinary agar (Liofilchem^®, Italy) and Methylene Blue Eosin agar (Cultimed^®, USA) for the isolation and maintenance of bacterial strains were used.

Cefotaxime (Himedia^®, India) and Gentamycin (Himedia^®, India) disks were used as reference antibiotics.

The following equipment was used for this study: an oven (Med Center Venticell®), a refrigerator (XPer®), a rotary evaporator (Buchi®), a magnetic stirrer (IKAMAG-RCT®), a grinder (IKAMAG-RCT®), a centrifuge (Rotina 380, HETTICH®), an autoclave (Autotester E, DRY-PV®).

Agar well diffusion method was used to screen the antibacterial and antifungal activities of different solvent extracts as displayed by Daoud et al.¹⁴ Cefotaxime (CTX 30 μg) for enterobacteria and gentamycin (GEN 10 μg) for anothers bacteria served as positive controls.

Broth dilution technique in Mueller Hinton were used according to Black and Black¹⁵. Nine experimental tubes whose concentration varies to double dilution from 50 to 0.195 mg/ml and 2 control tubes, the growth control tube (TC) and the sterility control tube (TS) are prepared. The slope of the experimental tubes and that of the TC tube was seeded. The tubes were incubated at 37 °C for 24 hours. The MIC was the concentration of the first tube from which no microbial visible growth¹⁶.

MBC is the lowest concentration of substance that leaves at most 0.01 % of surviving germs.

Using a loop calibrated at 2 μL the contents of the tubes in which no haze was observed were seeded on MH (Box B) in parallel streaks 5 cm in length at the surface, starting with by the MIC tube. After 24 hours incubation in an oven at 37 °C, the numbers of colonies on the streaks of box B with those of box A were compared. In practice, the CMB corresponds to the concentration of the experimental tube whose number of colonies present on the streak is less than or equal to the number of colonies present on the streak of the dilution 10^-4.

III-1 Results of sensitivity testing of bacteria to aqueous extract and organic fractions Securinega virosa

Table 1 shows the diameters of the bacterial growth inhibition zones with respect to the total aqueous extract of Securinega virosa. For all bacteria tested, the diameters of the growth inhibition zones are all less than 8 mm. However, a slight sensitivity is observed on the growth of Gram + bacteria (S. aureus Meti-R and Streptococcus sp) for which, the average diameter of the inhibition zone is 8 mm at the concentration of 250 mg/mL.

Table 1: Diamètre d’inhibition de l’extrait aqueux de Securinega virosa

00.00±00 : diameter of the inhibition zone ˂ 8 mm. Values are means of three replicates affected by the standard error of the mean (m±esm). CTX: Cefotaxime; GEN: Gentamycin; ESBL: Extended spectrum beta-lactamase; Meti-R: Meticillin resistant; ND: not determined

III-1-2- Diameters of growth inhibition zones obtained with the dichloromethanic fraction of Securinega virosa

The diameters of the growth inhibition zones obtained with the dichloromethanic fraction of Securinega virosa are presented by Table 2. The results show that only the high concentrations (250 and 500 mg/mL) inhibit the in vitro growth of bacteria to varying degrees. The largest inhibition zone diameters of 12.58±0.23 and 12.58±0.23 are obtained at the concentration of 500 mg/mL on the growth of E. coli ESBL and S. dysenteriae 1079PI15 respectively.

Table 2 : Diameters of growth inhibition zones obtained with the dichloromethanic fraction of Securinega virosa

III-1-3- Diameters of growth inhibition zones obtained with the acetate fraction of Securinega virosa

The results of the diameters of growth inhibition zones are shown in Table 3. The acetate fraction did not inhibit the in vitro growth of E. coli ATCC 25922, E. coli ESBL and P. aeruginosa 131813. For these bacteria, the inhibition diameters were all less than 8 mm. The greatest sensitivity to this fraction was observed on the growth of Streptococcus sp. (12.33±0.25 mm) followed by S. aureus Meti-R (11.75±1.25 mm) at the concentration of 500 mg/mL.

Table 3 : Diameters of growth inhibition zones obtained with the acetate fraction of Securinega virosa

III-1-4- Diameters of growth inhibition zones obtained with the ethanolic fraction of Securinega virosa

The results of the diameters of growth inhibition zones are reported in the Table 4.

With respect to the ethanolic fraction, the highest sensitivity was observed with S. aureus Meti-R 1532C/10 (13.66 mm) and Streptococcus sp. (13.33 mm) strains, followed by S. dysenteriae 1079PI15 (12.93 mm) at 500 mg/mL. The E. coli ESBL strain was the least sensitive with 9.46 mm at 500 mg/mL.

Table 4 : Diameters of growth inhibition zones obtained with the ethanolic fraction of Securinega virosa

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) The antibacterial parameters obtained with the organic fractions and the total aqueous extract are presented in Table 5. Only for acetatic and ethanolic fractions, MIC and MBC could be determined. For these two fractions, the MBC /MIC ratio is less than 2, implying that they are bactericidal.

Moreover, ethanolic fraction showed the lowest MIC (3.12 mg/mL) observed with S. aureus Meti-R 1532C/10 and Streptococcus sp strains, while the highest MIC was 50 mg/mL obtained with S. Typhi 43PI16 strain.

Table 6 : Antibacterial parameters (Minimum Inhibitory Concentration and Minimum Bactericidal Concentration) of aqueous extract and organic fractions of Securinega virosa

ND: not determined; MIC: Minimum Inhibitory Concentration; MBC: Minimum Bactericidal Concentration; ETHS: Ethanolic fraction of S. virosa;

ETAS: total aqueous extract of S. virosa; EDMS: dichloromethane fraction of S. virosa; EAS: acetate fraction of S. virosa; Bcid: Bactericidal

This study was intended to evaluate in vitro the antibacterial potential of Securinega virosa on some pathogenic strains of enterobacteria commonly involved in diarrheal diseases and on strains of Gram positive bacteria including Staphylococcus aureus Meti-R and Streptococcus sp.

As presented by the results in Table 1, the total aqueous extract was inactive on all bacteria tested despite the high concentrations. Similar findings were made in Benin by Onzo et al.¹⁷ with extracts from four leaves (Thalia geniculota, Musa spp, Manihot esculenta, and Daniella oliveri) used as food packaging, then in India by Patel et al.¹⁸ with methanol and acetonic extract from some medicinal plants and in China by Sukesh et al.¹⁹with hexanic and chloroformic extracts from two plants (Gymnema sylvestre and Andrographis paniculata). These results can be explained by the concentration of active ingredients and their solubilization in water used for extraction¹⁷. Moreover, the explanation can also be found in the inefficiency of the active molecules in these plants in relation to the membrane structure and the origin of the strains.

Yala et al.²⁰ explain this lack of antibacterial activity by the fact that some strains have developed resistance mechanisms to the antibacterial molecules present in the aqueous extract.

In addition, for Yala et al.²⁰ it is also possible that the solvents used in the extraction are responsible for the lack of activity of the plant extracts. Undoubtedly, the solvent used may not have been able to retain the molecules sought because of its polarity.

Dichloromethane extract inhibited growth of bacterial strains tested at higher concentrations (250 and 500 mg/mL). This study confirms previous findings in the literature that antimicrobial activities have a direct relationship to increased extract concentrations²¹. Indeed, in their work on the antimicrobial activity of plant extracts, several authors have suggested in most cases the use of high concentrations of extracts to obtain proven effects²².

However, the diameters of the growth inhibition zones were low. This could be explained by the presence in this extract of very few bioactive molecules. Indeed, the phytochemical screening performed by Kouangbé et al.²³ only revealed the presence of polyterpenes more precisely sterols. Although the antibacterial activity of these substances is demonstrated by several authors²⁴, their low activity here, would be related either to their low concentration in the extract or they did not cross bacterial barriers.

The ethyl acetate fraction of Securinega virosa was more active on the Shigella Typhi strain (10 mm), and gram-positive bacteria such as Streptococcus sp. (12.33±0.25 mm) and S. aureus Meti-R (11.75±1.25 mm) at 500 mg/mL with MICs ranging from 6.25 to 12.5 mg/mL. No susceptibility to this fraction was observed with Escherichia coli strains. These results could be explained by the absence of anti-Escherichia compounds. A plant extract may contain several phytomolecules. However, they may have targeted antibacterial activities that take into account their polarity, their concentration in the extract and the phenotype of the target bacteria.

The ethanolic fraction was active on all bacteria tested in this study. The smallest MICs (3.12 mg/mL) were recorded with Gram-positive bacteria in contrast to Gram-negative bacteria, with which MICs are higher (12.5 to 25 mg/mL). In the same direction, this fraction induced inhibition diameters ranging from 9 to 13 mm. These results could be explained by the choice of solvent, the methods of preparation of extracts and the part of the plant used.

Ethanol would concentrate much better the bioactive compounds responsibles for antibacterial activity. Similar results were previously obtained by Dickson et al.²⁵ and later confirmed by Amenu et al.²⁶. These authors showed that of all the root extracts of Securinega virosa tested, only the ethanol extract was active on all the bacteria used in their study. Yéo et al.²⁷ also reported that among the ethanolic fractions, acetatic, dichloromethanic and acetatic obtained by exhaustion of the total aqueous extract of the roots of Cochlospermum planchonii and tested on the in vitro growth of strains of Salmonella. Typhi, Vibrio. cholerea, Staphylococcus. aureus ATCC, Staphylococcus aureus Méti-R, Pseudomonas aeruginosa Imip-I, Pseudomonas aeruginosa ATCC, Salmonella Typhi ESBL, Escherichia coli ATCC and Escherichia coli ESBL, only the ethanolic and acetatic fractions showed a proven antibacterial activity. The conclusion of this study thus corroborates that proposed by Yéo et al.²⁷.

The active ingredients would therefore be intermediate polarity compounds, better concentrated in ethanol and making ethanol as the best extraction solvent.

In short, the inefficiency of aqueous extracts and the low activity of dichloromethane fraction could be explained by the extraction method used to concentrate the active ingredients in the solvents with intermediate polarity (ethyl acetate and ethanol). However, other work has shown better antimicrobial activity with chloroformic fractions²⁸ and petroleum ether²⁹.

The highest inhibition diameters were obtained with Staphylococcus aureus Meti-R (13.66 0.30 mm) and Streptococcus sp (13.33 0.52 mm) showing the high sensitivity of Gram-positive bacteria to Gram-negative bacteria. The high sensitivity of Staphylococcus aureus Meti-R to the alcoholic fractions of Securinega virosa was confirmed by Enwa et al.³⁰. The high sensitivity of Gram-positive bacteria to plant extracts compared to Gram-negative bacteria has been reported by several authors³¹. This difference in sensitivity between Gram-negative and Gram-positive bacteria is believed to be due to the variation in parietal structure of both cell types. In fact, the cell wall of Gram-positive bacteria consists of 70 to 90 % peptidoglycan unlike Gram-negative bacteria whose wall has only 20 % and an external membrane with two lipid layers. These structural differences between Gram-positive and Gram-negative bacteria would result in variation in the penetration of antimicrobial substances³². The inhibitory effect of the extracts on the synthesis of the bacterial cell wall (reticulation of peptidoglycan), which is less concentrated in gram-negative bacteria, may also be responsible for their reduced sensitivity to aqueous extract and organic fractions compared to Gram-positive bacteria³³

The results of this study would argue in favour of a real antibacterial profile of Securinega virosa. This property was also highlighted by Anarado et al.³⁴ then Ezeabara et al.³⁵ during their work.

Securinega virosa is a plant well known by rural populations for its antimicrobial properties. The results of this study provide scientific arguments supporting its properties. Antibacterial tests carried out in vitro should be supplemented by in vivo tests in order to consolidate the results obtained.

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	Diameters of growth inhibition zones (mm)
Tested strains	Concentrations (mg/mL)			Antibiotics (µg)
Tested strains	C₁ = 500	C₂ = 250	C₃ = 125	CTX (30)	GEN (10)
E. coli ATCC 25922	00.00±0.00	00.00±0.00	00.00±0.00	30	ND
E. coli ESBL	00.00±0.00	00.00±0.00	00.00±0.00	10	ND
P. aeruginosa 131813	00.00±0.00	00.00±0.00	00.00±0.00	ND	21
S. Typhi 43PI16	10.14±0.80	08.09±0.33	00.00±0.00	25	23
S. Typhi 1586	10.50±0.44	10.16±0.84	00.00±0.00	28	30
S. dysenteriae 1079PI15	09.21±0.81	00.00±0.00	00.00±0.00	ND	ND
K. oxytoca	10.50±0.40	08.61±0.10	00.00±0.00	ND	ND
S. aureus Meti-R	11.75±1.25	09.80±0.58	00.00±0.00	ND	ND
Streptococcus sp	12.33±0.25	10.91±0.22	08.25±0.84	ND	15

	Diameters of growth inhibition zones (mm)
Tested strains	Concentrations (mg/mL)			Antibiotics (µg)
Tested strains	C₁ = 500	C₂ = 250	C₃ = 125	CTX (30)	GEN (10)
E. coli ATCC 25922	10,25±0,33	08.08±0.69	00.00±0.00	30	ND
E. coli ESBL	09.46±0.55	00.00±0.00	00.00±0.00	10	ND
P. aeruginosa 131813	11.44±0.08	00.00±0.00	00.00±0.00	ND	21
S. Typhi 43PI16	10.18±0.55	10.30±0.00	00.00±0.00	25	23
S. Typhi 1586	10.01±0.22	08.66±0.23	00.00±0.00	28	30
S. dysenteriae 1079PI15	12.93±0.11	10.60±0.20	08.81±0.80	ND	ND
K. oxytoca	12.03±0.76	09.50±0.86	08.36±0.55	ND	ND
S. aureus Meti-R	13.66±0.30	11.06±0.69	09.08±0.74	ND	ND
Streptococcus sp	13.33±0.52	10.15±0.46	08.68±0.58	ND	15

Extracts	Antibacterial parameters (mg/mL)	Gram-negative bacteria							Gram-positive bacteria
Extracts	Antibacterial parameters (mg/mL)	E. coli ATCC	E. coli BLSE	S. Typhi 43PI16	S. Typhi 1586	P. aeruginosa 131813	S. dysenteriae 1079PI15	K. oxytoca	S. aureus Meti-R	Streptococcus sp
EDMS	CMI	> 50	> 50	> 50	> 50	> 50	> 50	> 50	> 50	> 50
	CMB	> 50	> 50	> 50	> 50	> 50	> 50	> 50	> 50	> 50
	CMB/CMI	ND	ND	ND	ND	ND	ND	ND	ND	ND
	Effet	ND	ND	ND	ND	ND	ND	ND	ND	ND
EAS	CMI	> 50	> 50	6,25	6,25	> 50	25	12,5	12,5	6,25
	CMB	> 50	> 50	6,25	6,25	> 50	25	50	12,5	12,5
	CMB/CMI	ND	ND	1	1	ND	1	2	1	2
	Effet	ND	ND	Bcid	Bcid	ND	Bcid	Bcid	Bcid	Bcid
ETHS	CMI	25	25	50	25	12,5	12,5	12,5	3,12	3,12
	CMB	25	50	50	50	25	25	25	6,25	6,25
	CMB/CMI	1	2	1	2	2	2	2	2	2
	Effet	Bcid	Bcid	Bcid	Bcid	Bcid	Bcid	Bcid	Bcid	Bcid
ETAS	CMI	> 50	> 50	> 50	> 50	> 50	> 50	> 50	> 50	> 50
	CMB	> 50	> 50	> 50	> 50	> 50	> 50	> 50	> 50	> 50
	CMB/CMI	ND	ND	ND	ND	ND	ND	ND	ND	ND
	Effet	ND	ND	ND	ND	ND	ND	ND	ND	ND