literature review on khaya senegalensis

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• Published: 08 June 2021

Assessment of the antibacterial effect of Khaya senegalensis on some Gram-negative bacteria

• Victorien Dougnon   ORCID: orcid.org/0000-0001-9047-7299 1 ,
• Edna Hounsa 1 ,
• Hornel Koudokpon 1 ,
• Eric Agbodjento 1 ,
• Anny Afaton 1 ,
• Kevin Sintondji 1 ,
• Jean Robert Klotoe 1 ,
• Julien Segbo 1 &
• Lamine Baba-Moussa 2  

Bulletin of the National Research Centre volume  45 , Article number:  107 ( 2021 ) Cite this article

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The matter of antimicrobial resistance evokes the urgency to explore alternatives to the antibiotics traditionally used for microbial infections. This study aimed to elucidate the mechanism of action of the antibacterial effect of Khaya senegalensis liable for bacterial strains responsible for diarrheal infections.

The data collected indicate that the bacterial strains tested ( Salmonella Typhimurium ATCC 14028, Escherichia coli ATCC 25922, Shigella spp. and Salmonella spp.) were sensitive to the extracts of Khaya senegalensis (Desr.) A.Juss. (aqueous and hydro-ethanol) to varying degrees. The hydro-ethanolic extract was active on all strains with a MIC of 25 mg/mL coupled with a bactericidal effect. The aqueous extract was only active on the Salmonella spp. strain. Membrane permeability test data show that Khaya senegalensis extracts affect the bacterial strains tested by attacking the stability of their outer membrane. This potential indicated by the high percentage of membrane destabilization of the bacteria is significantly ( p  < 0.05) better than that of cefixime used as a reference.

This study revealed that Khaya senegalensis destroys Gram-bacteria by attacking the stability of their cytoplasmic membrane. These data provide for the first time the mode of action of Khaya senegalensis extracts concerning their antibacterial activity.

Infectious diseases constitute a serious public ill health thanks to their frequency and gravity especially in developing countries (Bourgeois et al. 2016 ). Among these diseases, diarrheal diseases are the foremost fatal, especially in children from West Africa. In fact, these diarrheal diseases are liable for 1.8 million deaths annually worldwide where 90% are children under the age of five living in developing countries (Bryce et al. 2005 ). Diarrheal diseases represent the third reason of death from infectious diseases of all ages and the 5th cause of premature death worldwide (WHO 2014 ). According to WHO, in Benin, diarrheal diseases are one of the main causes of morbidity. Indeed, they have a direct impact on the costs associated with health care, including several factors such as consultation, medication and in some cases, hospitalization, which represents a burden on household spending (WSP-ESI-Benin 2012 ). The pathogens of diarrheal diseases are mainly bacteria (Djague et al. 2020 ).

Medical therapy is based on the use of conventional antibiotics and antidiarrheal drugs. The use of antimicrobials should not be done routinely. In fact, a clinical distinction is made between diarrheal episodes caused by enterotoxigenic E. coli and those caused by rotavirus or Cryptosporidium against which antimicrobial drugs are ineffective (WHO 2005 ). Moreover, this use of antimicrobials sometimes presents undesirable effects and favors the development of resistant bacteria. Nowadays, antimicrobial resistance constitutes a real public health problem for the effective management of infectious diseases (Nahrgang et al. 2018 ). The most of the bacteria responsible for diarrheal episodes develop resistance to the antibiotics used in modern therapy (Ahmed et al. 2000 ; Qu et al. 2016 ). In addition, the anti-diarrheal drugs used until now are often ineffective and do not succeed in preventing dehydration and improving nutritional status, which should be the main objectives of treatment (WHO 2005 ). Concerning drug treatments that have shown some effectiveness, namely the use of oral rehydration solutions (ORS) to reduce fluid and electrolyte losses in diarrheic patients, it should be noted that in rural and semi-urban areas, the lack of knowledge of patients regarding their application limits their use (WHO 2005 ). In addition, the lack of qualified health personnel in most rural African communities and the high cost of therapies in case of severity do not contribute to a better management of diarrheal disease (WHO 2005 ). In the search for alternative solutions, World Health Organization (WHO) had initiated a diarrheal disease control program based on traditional medicine practices and prevention approaches (WHO 2011 ). This approach is even more justified given that about 80% of the population of developing countries like Benin continue to use traditional medicine based on the use of medicinal plants for their primary health needs (WHO 2002 ).

Khaya senegalensis (Desr.) A.Juss. ( K. senegalensis ) is a medicinal plant from the Beninese flora widely used in traditional African medicine (Akoègninou et al. 2006 ). In West Africa it is a plant that benefits from a multiple use value mainly used for bacterial infections (Issa et al. 2018 ). In southern Benin, the practitioners of traditional medicine used K. senegalensis for the treatment of bacterial infections, particularly diarrheal infectious (Akoègninou et al. 2006 ). Literature data confirmed the antibacterial potential of the stem bark of the K. senegalensis on bacterial strains such as Salmonella Typhi , Salmonella Paratyphi and Salmonella Typhimurium (Abdallah et al. 2016 ; Katawa et al. 2018 ; Koudoro Yaya et al. 2018 ; Ugoh et al. 2014 ). These data suggest the antibacterial potential of the plant and justify its use in traditional medicine in the treatment of bacterial infections. However, despite the plurality of data available on the biological activities of this plant, particularly on its antimicrobial properties, it is clear that no study has so far explored the mechanism of action of its antibacterial activity. This study aimed to elucidate the mode of action of the antibacterial effect of aqueous and hydro-ethanolic extracts of K. senegalensis on Gram-negative bacteria involved in diarrheal infections.

Plant material

The plant material used in this study consists of fresh stem bark of Khaya senegalensis (Desr.) A.Juss. These samples were collected in Abomey-Calavi in July 2020 and certified at the National Herbarium of Benin at the University of Abomey Calavi under the identification number YH435/HNB.

Biological material

The strains of Gram-negative bacteria involved in the occurrence of diarrheal infections, obtained from the Research Unit in Applied Microbiology and Pharmacology of natural substances, University of Abomey-Calavi, Benin were the biological material used. The characteristics of these strains are summarized in Table 1 .

Study methods

Phytochemical screening of the plant.

This part of the study consisted of the qualitative identification of eleven major phytochemical classes in the powder of the stem bark of K. senegalensis according to the method described by Houghton and Raman ( 1998 ). These are tannins, gallic tannins, flavonoids, anthocyanins, leuco-anthocyanins, alkaloids, mucilages, reducing compounds, sterols, terpenes and saponosides.

Preparation of plant extracts

The bark of the plant stems collected, cleaned and dried at room temperature at the Research Unit of Applied Microbiology and Pharmacology of natural substances was ground to powder using the Retsch electric mill. Two types of extraction (aqueous and hydro-ethanolic) were obtained following the methodology used by Klotoé et al. ( 2020 ). Briefly, fifty (50) grams of powder were macerated in 500 ml of distilled water for the aqueous extract and in 500 ml of the mixture of water and ethanol at 50% v/v for the hydro-ethanolic extract. After 72 h of agitation at room temperature, the homogenate obtained was filtered three times on cotton wool and once on Wattman paper N ° 1. Crude extract was obtained after evaporation at a temperature of 40 °C in an oven (oven).

Determination of bioactive molecules of the extracts

Determination of total polyphenols

The method of Singleton et al. ( 1999 ) using the commercial Folin Ciocalteu Reagent (FCR) was adopted. Briefly, 50 µL of the extract was mixed with 250 μL of the FCR (10 times diluted in distilled water) and 750 μL of an aqueous solution of sodium carbonate Na 2 CO 3 (7.5%). After 8 min of incubation, 950 µL of distilled water was added and mixed with the vortex and incubated for 2 h. Optical densities (OD) were read at 760 nm using a CECIL CE 2041 spectrophotometer. The reading was made against a blank consisting of a mixture of 250 µL of FCR, 750 µL of Na 2 CO 3 and 1 mL of distilled water. Samples were prepared in triplicates. Gallic acid (0–200 µg/mL) was used as standard reference. The total polyphenols content was determined as mg of gallic acid equivalent/g of extract (mg GAE/g) from the equation of the linear calibration curve ( y  = 0.0012 x −  0.0388 with R 2  = 0.9988).

Determination of total flavonoids

Flavonoids contents were measured by the method using aluminium trichloride (AlCl 3 ) described by Zhishen et al. ( 1999 ) and used by Klotoé et al. ( 2020 ). 500 μL μL of AlCl 3 (2%), 500 μL of the extract and 3 mL of methanol were mixed thoroughly. The blank consists of 500 μL of AlCl 3 and 3.5 mL of methanol. Absorbance reading was done at the spectrophotometer at 415 nm after 10 min of incubation. Samples were prepared in triplicates. Rutin (0–1 mg/mL) was used as a reference standard. Total flavonoids content was determined as mg of rutin equivalent/g of extract (mgRuE/g) from the equation of the linear calibration curve ( y  = 44.135 x  − 0.1893 with R 2  = 0.9909).

In vitro Antibacterial activity of plant extracts

Three steps were followed for performing this antibacterial test.

Preparation of extracts and sterility test

100 mg/mL of extract solution (aqueous and hydro-ethanolic) were prepared in distilled water. To verify the sterility of these prepared extract solutions an inoculation of aliquots of each solution was applied on Mueller Hinton medium according to the methodology described by Agbankpe et al. ( 2016 ).

Sensitivity test of bacterial strains to extracts

A portion of pure 24-h colony from Mueller Hinton's medium from each strain was emulsified in 5 mL of physiological water to obtain a turbidity of 0.5 on the MAC Farland scale. Each inoculum was inoculated by swabbing onto plates containing Mueller Hinton agar (CA-SFM 2020 ). 50 μL of each extract (100 mg/mL) was placed in the 6 mm diameter wells. A negative control was prepared with sterile distilled water. Standard antibiotic discs (Ciprofloxacin, Cefixime and Gentamicin) were used as positive controls. For an hour, the Petri dishes were pre-incubated at room temperature for the pre-diffusion of the substances and then incubated at 37 °C in an oven for 18 h. The test was repeated three times. The incubated plates were examined to measure the zones of inhibition. The standard used for reading the results of the antibiogram tests is presented in Table 2 (Tsirinirindravo and Andrianarisoa 2010 ; WHO 2002 ).

Determination of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of active extracts

This part of the study following the method applied by Lègba et al. ( 2018 ). It consisted of the 96-well plate method. 100 μL of the initial extract solution at the concentration of 100 mg/mL, were added to 100 μL of Mueller–Hinton broth. A series of dilution from well to well was carried out and then 100 μL of various bacterial suspensions were added. Positive (100 μL of MH broth added to 100 μL of bacterial suspension) and negative controls (100 μL of MH broth added to 100 μL of the extracts) were prepared. The microplates were incubated at 37 °C for 24 h. All the wells of the MIC at the higher concentrations were then inoculated on a Mueller Hinton agar then the Petri box were placed at 37 °C for 24 h for the determination of the MBC. The antibiotic power (AP) of each extract was then calculated with the formula MBC/MIC. The antibacterial effect or power is judged to be bactericidal or bacteriostatic based on the AP = MBC/MIC. If 1 ≤ AP ≤ 2, the effect is bactericidal and if 4 ≤ AP ≥ 16, the effect is bacteriostatic.

Permeability test outer membrane of Gram-negative bacteria

This test is based on the evaluation of the destabilizing power of the membrane of bacteria by plant extracts. It was determined according to the method employed by Djague et al. ( 2020 ). In a 96-well microplate, the MIC and 2 MIC of the extract in triplicate were prepared by serial dilution. 100 μL of the suspension of the tested bacteria (1.5 × 10 6  CFU/mL) was added to all wells and the plate was incubated at 37˚C for 24 h. Cefixime was used as a positive control. Muller Hinton broth and bacterial suspension served as negative control. The optical densities were read at 405 nm. The percentage of destabilization was calculated using the formula below:

% D : Percentage of destabilization; A o : Absorbance of the negative control; A s : Absorbance of test samples.

Data analysis

The in vitro antibacterial test was repeated three times and the results were analyzed using Graph Pad 7 software. The quantitative variables were then presented as mean ± standard deviation. ANOVA analysis of variance was used to compare the percentage of bacterial membrane destabilization between samples. The Student's t -test was used to analyze the quantitative composition in total polyphenols and flavonoids of the extracts. The significance level was set at 5%.

Phytochemical composition

Data collected for the phytochemical screening revealed the presence of tannins, flavonoids, leuco-anthocyanins, alkaloid, mucilage, reducing compounds and saponosides in the powder of stem bark of K. senegalensis.

Determination of total polyphenols and flavonoids content

The extracts present interesting content of polyphenols and flavonoids. Hydro-ethanolic extract exhibited significantly ( p  < 0.05) higher total polyphenol and flavonoid content compared to aqueous extract (Table 3 ).

Sensitivity test

Aqueous and Hydro-ethanolic extracts of K. senegalensis were tested against E. coli ATCC 25922, Salmonella Typhimirium ATCC 14028, Shigella spp. and Salmonella spp. Results of antibiogram showed variable sensitivity depending on the type of extract. Indeed, on all bacterial strains tested, K. senegalensis aqueous extract was active only on Salmonella spp. with an inhibition diameter of 14 ± 1 mm. The hydro-ethanolic extract was active on all strains tested with variable activity (Fig.  1 ). The best antibacterial activity for this hydro-ethanolic extract was obtained on Salmonella spp. (17 ± 2.51 mm as inhibition diameter).

Diameter of inhibition of the hydro-ethanolic extract of khaya senegalensis on the bacterial strains

MIC, MBC and AP of the extracts studied on the tested strains

The collected data related to MIC, MBC and AP are presented in Table 4 . Analysis of these data indicates that the MIC was 25 mg/ml for all strains sensitive to both hydro-ethanolic and aqueous extracts. The same observation is made for the MBC. These observations were used to determine the antibiotic power of these active extracts (AP = 1).

Effects of extracts on outer membrane permeability of bacterial strains

Figure  2 presents the results of the permeability test of the outer membrane of bacteria strains. The analysis of the data shows that the extracts studied complexed more divalent ions (increase of the destabilization percentage) than the positive control (Cefixime) at the concentrations tested (MIC and 2 MIC). However, no significant difference is to be reported between the effect obtained (percentage of destabilization) for MIC and 2 MIC ( p  > 0.05). The same trend is obtained regardless of the microorganism.

Effect of destabilization of outer membranes of various bacteria by Khaya senegalensis . Legend: For same microorganism, bars carrying same letter (a, b) are not significantly different while for same concentration, bars carrying the same Greek alphabet (α, β) are not significantly different ( p  > 0.05), Waller Duncan. KSa: Aqueous extract; KSb: hydro-ethanolic extract

This study aimed at providing scientific data on the mechanism of action of the antibacterial effect of K. senegalensis extracts. Data from the qualitative screening of K. senegalensis stem bark powder indicate the presence of tannins, flavonoids, leuco-anthocyanins, alkaloid, mucilage, reducing compounds and saponosides. In addition the extracts studied showed richness in total polyphenols and flavonoids. Similar observations were reported by Ugoh et al. ( 2014 ) in a study conducted in Nigeria. These observations are in agreement with the richness in polyphenolic compounds of plants of the same botanical family (Meliaceae) as K. senegalensis (John et al. 2014 ; Olatunji et al. 2021 ; Saleem et al. 2018 ). These data translating a richness in bioactive molecules of K. senegalensis could justified the use of the plant in the traditional treatment of diseases in several African pharmacopoeias (Abdel-Wareth et al. 2014 ). Furthermore, the difference observed between the polyphenolic compound content of the two extracts studied could be explained by the influence of the extraction solvent. Indeed, it is documented in the scientific literature that the extraction solvents have an influence on the quantity of bioactive molecules concentrated in the plant extracts (Babbar et al. 2014 ; Dirar et al. 2019 ; Do et al. 2014 ). In this study, the hydro-ethanol extract showed the best content of total polyphenols and flavonoids. This reflects that the mixed solvent (water–ethanol) concentrated more bioactive molecules than water. These data corroborate those reported by several authors who underlined the strong capacity of mixed solvents in the extraction of total polyphenols and flavonoids (Bourgou et al. 2016 ; Do et al. 2014 ; Mohammedi and Atik 2011 ; Venkatesan et al. 2019 ; Vieito et al. 2018 ). Trabelsi et al. ( 2012 ) justify this extraction capacity of mixed solvents by the increased solubility of bioactive compounds in this type of solvent.

Data obtained from the in vitro antibacterial test indicate that K. senegalensis bark is endowed with antibacterial properties on the strains involved in the diarrheal infections tested in this study. From the two extracts tested, the hydro-ethanolic extract was active on all bacterial strains with a Minimum Inhibitory Concentration (MIC) of 25 mg/mL coupled with a bactericidal effect. This antibacterial potential of the K. senegalensis could be justify by the existence of bioactive compounds such as flavonoids, tannins, alkaloids identified in the stem bark of K. senegalensis. Indeed, these bioactive molecules are known for their antimicrobial power referring to several reports in the literature (Djague et al. 2020 ; Kabir et al. 2015 ; Scalbert 1991 ). Specifically, some data in the literature attribute the antimicrobial activity of medicinal plants mainly to their polyphenolic compounds (Ghimire et al. 2017 ; Miklasińska-Majdanik et al. 2018 ). Indeed, these secondary metabolites are known to be responsible for a variety of biological activities of medicinal plants. Among them, flavonoids are one of the most studied polyphenolic compounds for their antibacterial properties (Cushnie and Lamb 2005 ; Farhadi et al. 2019 ). Similarly, tannins are a group of polyphenolic compounds that are very active in antimicrobial activities (Maisetta et al. 2019 ). Other data in the literature report the antimicrobial potential of alkaloids (Cushnie et al. 2014 ; Othman et al. 2019 ). These data justify the correlation, reported by several authors, between the antibacterial activity of K. senegalensis and these bioactive compounds (Abdallah et al. 2016 ; Kubmarawa et al. 2009 ; Ugoh et al. 2014 ). However, the difference of the antibacterial activity of the extracts observed in this study could be explained by the difference of their polyphenolic compounds content. Indeed, the hydro-ethanolic extract (the most active on the inhibition of the studied bacterial strains) is richer in polyphenolic compounds than the aqueous extract. These data show that the intensity of the biological activity of medicinal plant extracts is proportional to their content of bioactive compounds (Wang et al. 2016 ).

However, the anti-salmonella power of K. senegalensis obtained in the present study is similar to that reported by Ugoh et al. ( 2014 ) on Salmonella enterica subsp. enterica serovar Typhi strain. On the other hand, the comparison of the data of the present study with those obtained by Abdallah et al. ( 2016 ) and Katawa et al. ( 2018 ) shows opposite results. Except for Salmonella spp., the aqueous extract was inactive on the other three tested strains ( E. coli , Salmonella Typhymirium and Shigella spp.) while the study of Abdallah et al. ( 2016 ) inform, for the same type of extract (aqueous extract), an inhibition of E. coli , Salmonella spp. and Shigella spp. with an MIC varying between 12.5 and 25 mg/mL. Katawa et al. ( 2018 ), on the other hand, showed contrary to our data that the hydro-ethanolic extract of de K. senegalensis was not active on Salmonella Typhimirium strain but active on other Salmonella strains ( Salmonella Typhi, Salmonella enterica and Salmonella Paratyphi). This discrepancy in results observed could be attributed to the influence of environmental factors on the composition of secondary metabolites in medicinal plants (Borges et al. 2017 ; Liu et al. 2016 ).

Several models were used to study the mode of action of antibacterial agents in relation to different bacterial targets (Pinto et al. 2017 ). In this study the permeability test of the outer membrane of bacteria was adopted to evaluate the mode of action of aqueous and hydro-ethanolic extracts of the stem bark of K. senegalensis against Salmonella Typhimurium ATCC 14028, Escherichia coli ATCC 25922, Shigella spp. and Salmonella spp ; four strains involved in diarrheal infections. The data obtained highlight a remarkable potential for destabilizing the membrane of the bacterial strains of the extracts tested with a better effect compared to Cefixime used as a reference. These observations reflect that the extracts have a more enhanced mode of action on the destabilization of the outer membrane of the bacterial strains tested than Cefixime. Similar results were reported by Djague et al. ( 2020 ) for different extracts of Garcinia kola and Alchornea cordifolia and Polymyxin B used as a reference in their study and which has the same mode of action as cefixime. Moreover, with regard to their phytochemical composition, the mode of action of the antibacterial activity of the extracts tested on strains responsible for diarrheal infections can be attributed to phenolic compounds (flavonoids, tannins etc.). Indeed, these phenolic compounds are able to destabilize the external membrane of bacteria by complexing the divalent cations that stabilize them (Frirdich and Whitfield 2005 ; Vaara 1992 ). This antibacterial action promotes the bursting of the cytoplasmic membrane and alterations in Ionic homeostasis between the intracellular and extracellular compartments of Gram-negative bacteria (Trombetta et al. 2005 ). Thus, the membrane antigens responsible for the virulence of bacteria will be affected. These observations explain the destabilizing effect of K. senegalensis extracts for the bacterial membrane of the strains tested.

This study allowed to elucidate for the first time the mode of action of the antibacterial effect of K. senegalensis regarding its antibacterial activity, compared to the scientific literature currently available on the antibacterial properties of this plant. Such data evoke prospects for precision-oriented research on the mechanism of action of antibacterial effects of medicinal plants. This would optimize the search for new bioactive molecules for the fight against antimicrobial resistance.

This study revealed a variation in the antibacterial activity of aqueous and hydro-ethanolic extracts of K. senegalensis on strains of Salmonella Typhimurium ATCC 14028, Escherichia coli ATCC 25922, Shigella spp. and Salmonella spp. involved in diarrheal infections. The hydro-ethanolic extract showed better activity compared to the aqueous extract. These tested extracts exert their antibacterial effects on the tested bacteria by attacking the stability of their cytoplasmic membrane. This study provided for the first time data on the mode of action of K. senegalensis extracts concerning their antibacterial activity.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Abbreviations

Aluminium trichloride

Absorbance of the negative control

Antibiotic Potency

American Type Culture Collection

Absorbance of test samples

Antibiotic Committee of the French Society of Microbiology

Colony format unit

Folin ciocalteu reagent

Gallic acid equivalent

National Herbarium of Benin

Minimum Bactericidal Concentration

Minimum inhibitory concentration

Sodium carbonate

Optical densities

Rutin equivalent

Statistical Package for the Social Sciences

Yedomonhan Hounnankpon (ID of the name of the curator of the National Herbarium of Benin)

World Health Organization

Water Sanitation Program- Economic and Social Impacts

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Acknowledgements

The authors are very grateful to the World Academy of Sciences (TWAS) and the United Nations Educational, Scientific and Cultural Organization (UNESCO). These two institutions reviewed this research project and provided technical advice on its implementation steps. Finally, they financed this research with funds allocated to the research team under the TWAS Research Grant Award_20-254 RG/BIO/AF/AC_G. They are also grateful to SEEDING LABS who provided useful equipments to the first author’s institution, Dr Victorien DOUGNON. They thank Dr Paul LUNGA from Cameroon, who provided great support for performing these analyses.

This study was funded by The World Academy of Sciences (TWAS) and the United Nations Educational, Scientific and Cultural Organization (UNESCO) under the TWAS Research Grant Award _20-254 RG/BIO/AF/AC_G.

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HE, DV, AE, AA, SK, SJ, B-ML wrote the protocol. HE, AE, AA, SK processed the samples. DV, HE, AE did the statistical analyses. HE, DV, AE wrote the draft of the manuscript. DV, SJ, B-ML reviewed the manuscript. All the authors have read and approved the manuscript.

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Dougnon, V., Hounsa, E., Koudokpon, H. et al. Assessment of the antibacterial effect of Khaya senegalensis on some Gram-negative bacteria. Bull Natl Res Cent 45 , 107 (2021). https://doi.org/10.1186/s42269-021-00568-0

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Fulani Knowledge of the Ecological Impacts of Khaya senegalensis (Meliaceae) Foliage Harvest in Benin and its Implications for Sustainable Harvest

• Published: 25 July 2009
• Volume 63 , pages 256–270, ( 2009 )

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Fulani Knowledge of the Ecological Impacts of Khaya senegalensis (Meliaceae) Foliage Harvest in Benin and Its Implications for Sustainable Harvest. An improved understanding of how local people view their impacts on the resources they exploit and how they perceive that their resources are affected by other factors can provide insight into reasons some resources are overexploited and into strategies to conserve them. In West Africa, various tree species are heavily harvested by indigenous herders for foliage to feed their cattle. The reported declines in populations of several of these species have both biological and cultural implications, as cattle are an integral part of indigenous cultures and livelihoods. In this study we investigated Fulani herders’ practices, knowledge, and perceptions of the ecological impacts of harvesting foliage of African mahogany, Khaya senegalensis, in Benin, and we tested some of the factors that may influence them. Fulani herders have detailed ecological knowledge of their impacts on the resources they depend on, and this is finely tuned to local ecological conditions. This knowledge is also widely spread across different sectors of Fulani communities and is highly congruent with scientific findings. However, due to the open-access context of K. senegalensis populations, detailed knowledge of sustainability does not translate into sustainable practices. Fulani perceptions of threats to populations differ significantly between ecological regions and provide key insights for locally relevant resource management plans. Traditional Fulani practices such as the sopoodu provide a basis for sustainable management of proposed Fulani-owned K. senegalensis plantations. This study illustrates how the assessment of local ecological knowledge, practices, and perceptions can play a key role in the design of culturally-appropriate conservation plans.

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Acknowledgments

This work was supported by grants from the Beatrice Krauss Fellowship and the Ecology, Evolution and Conservation Biology Program at the University of Hawaii at Manoa. We are grateful to the Fulani communities who participated in this research, to Yacoubou Boni, Samba Tobou Adam, and Rachidi Saliou for field assistance, and Ismail Moumouni, Florent Okry, Shimona Quazi, Nina Etkin, Kim Bridges, Andy Taylor, and Don Drake for useful discussion.

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Orou G. Gaoue

Present address: Department of Biology, Institute of Theoretical and Mathematical Ecology, University of Miami, 1301 Memorial Dr., Coral Gables, FL, 33146, USA

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Botany Department, University of Hawaii at Manoa, 3190 Maile Way, Honolulu, HI, 96822, USA

Orou G. Gaoue & Tamara Ticktin

Laboratoire d’Ecologie Appliquee, Universite d’Abomey Calavi, 01BP 526, Cotonou, Benin

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Gaoue, O.G., Ticktin, T. Fulani Knowledge of the Ecological Impacts of Khaya senegalensis (Meliaceae) Foliage Harvest in Benin and its Implications for Sustainable Harvest. Econ Bot 63 , 256–270 (2009). https://doi.org/10.1007/s12231-009-9091-6

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Published : 25 July 2009

Issue Date : September 2009

DOI : https://doi.org/10.1007/s12231-009-9091-6

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Studies on the antibacterial activity of Khaya senegalensis [(Desr.) A. Juss)] stem bark extract on Salmonella enterica subsp. enterica serovar Typhi [(ex Kauffmann and Edwards) Le Minor and Popoff]

To study the phytochemical screening and antibacterial activity of the stem bark extracts of Khaya senegalensis ( K. senegalensis ) against Salmonella enterica subsp. enterica serovar Typhi.

The plant components were extracted using methanol, ethanol and water. The phytochemical screening of the stem bark extracts were carried out using a standard method. The antibacterial assay of the stem bark extracts against Salmonella Typhi ( S. Typhi) using the agar well diffusion method with different concentrations of 50, 100, 200, 400 and 500 mg/mL and the corresponding concentrations of the control was carried out and the result compared with a standard antibiotic, amoxicillin as the control.

The results obtained from the phytochemical screening of the three plant bark extracts of K. senegalensis showed 10 plant secondary metabolites including saponins, tannins, reducing sugars, aldehyde, phlobatannins, flavonoids, terpenoids, alkaloids, cardiac glycoside and anthroquinones. The ethanol and aqueous extracts showed antibacterial activities against S . Typhi at concentration of 50 mg/mL with the zone diameter of inhibition (ZDI) of 14 mm and 15 mm respectively. The ethanol and aqueous extracts also showed zone diameter of inhibition of 23 mm and 25 mm respectively at 250 mg/mL and 27 mm each at 500 mg/mL. The ethanol and aqueous stem bark extracts gave the highest ZDI at 500 mg/mL while 100 mg/mL gave the least ZDI for ethanol extract and 50 mg/mL for the aqueous extract. This was followed by 400 mg/mL that gave 24 mm ZDI of the aqueous extract and 27 mm of the ethanol extract. The methanol extract showed intermediate susceptibility evidenced by ZDI of 10 mm at 100 mg/mL concentration. The methanol extract also showed antibacterial activity of 24 mm ZDI against the test organism at a higher concentration of 250 mg/mL and 26 mm at 500 mg/mL concentration. The methanol, ethanol and aqueous extracts displayed antibacterial activities against S . Typhi with a statistical significant difference at ( P ≤0.05). The extracts compared favourably with the standard antibiotic, the control. The minimum inhibitory concentration of the extracts was 250, 200, 200 and 100 mg/mL for methanol, ethanol, aqueous extracts and amoxicillin (control) respectively. The minimum lethal concentration of the extracts was 250, 250, 400 and 200 mg/mL for methanol, ethanol, aqueous extracts and control respectively.

Conclusions

The antibacterial properties of K. senegalensis stem bark extract can be harnessed for the production of new antibiotics or the enhancement of already existing antibiotics.

1. Introduction

The increase of life threatening infections that are resistant to commonly used antibiotics has become a worldwide problem. It is becoming an important cause of morbidity in immune compromised patients in developing countries [1] . The increasing prevalence of multi-drug resistant strains of bacteria and the recent appearance of strains with reduced susceptibility to antibiotics has raised the specter of untreatable bacterial infections and adds urgency to the search for new infection-fighting strategies [2] , [3] .

Presently, there are global problems of emergence of multiple antibiotics resistance as well as emergence of new and resurrection of previously eradicated diseases. Reports on ethno botanical survey indicate a general consensus on the use of antimicrobial active medicinal plants to provide cheaper drugs [4] . There is a need to search for new and more potent antimicrobial compounds of natural origin to complement the existing synthetic antimicrobial drugs that are gradually becoming less potent against pathogenic microorganisms.

The continuous spread of multidrug-resistant pathogens has become a serious threat to public health and a major concern for infection control practitioners worldwide [5] . In addition to increasing the cost of drug regimens, this scenario has paved way for the re-emergence of previously controlled diseases and has contributed substantially to the high frequency of opportunistic and chronic infection cases in developing countries [6] , [7] .

For a long time, plants have been an important source of natural products for human health. The antimicrobial properties of plants have been investigated by a number of studies worldwide and many of them have been used as therapeutic alternatives because of their antimicrobial properties [8] . This study therefore focuses on the antibacterial potency of Khaya senegalensis (Desr.) A. Juss ( K. senegalensis ) stem bark extract on Salmonella enterica subsp. enterica serovar Typhi and its histopathological effects. K. senegalensis is highly priced in traditional medicine in West Africa. The main part used is the very bitter stem bark. Ethno-medically, the uses range from the treatment of fever, lumbago, cough, rheumatism and stomach/gastric pains [9] . In veterinary medicine, the bark is also used in the treatment of worm infestation, ulcer and mucous diarrhea in horses and camels [10] .

Typhoid fever is a worldwide bacterial disease that is caused by the ingestion of food and/or water contaminated by the feces and urine of infected persons or animals. The causative agent of the disease is Salmonella enterica ( S. enterica ) serovar Typhi and sometimes S. enterica serovar Paratyphi A, B, and C [11] . Symptoms usually develop 1-3 weeks after exposure, and may be mild or severe. They include high fever, malaise, headache, constipation or diarrhea, rose-colored spots on the chest, and enlarged spleen and liver. Healthy carrier state may follow acute illness. The bacteria responsible for typhoid fever are deposited in water or food by a human carrier and are then spread to other people in the area. Typhoid fever is rare in developed countries of the world, but continues to be a significant public-health issue in the developing countries like Nigeria. The disease has been given various names at different times in history, such as gastric fever, abdominal typhus, infantile remittent fever, slow fever, nervous fever or pathogenic fever.

2. Materials and methods

2.1. study area.

The study area for this research is Federal Capital Territory (FCT) Abuja. The territory is located just north of the confluence of the River Niger and River Benue. It is bordered by the states of Niger to the west and north, Kaduna to the northeast and south and Kogi to the southwest [12] . The Federal Capital lies between latitude 8.25 and 9.20 north of the equator and longitude 6.45 and 7.39 east of Greenwich Meridian. Abuja is geographically located in the Centre of the country. The FCT has a landmass of approximately 7.315 km 2 of which the actual city occupies 275.3 sq. km. It is situated within the savannah region with moderate climatic conditions [12] .

The population of FCT by the 2006 census is 1 405 201 and a population density of 192.1/km 2 , [12] . The territory is currently made of six local councils, comprising the city of Abuja and five local government areas namely Abuja, Gwagwalada, Kuje, Bwari, Kwali.

2.2. Preparation of stem bark extract

The stem bark of K. senegalensis was collected from within FCT Abuja, Nigeria. The plant material was air-dried to constant weight and milled into powder [13] . Twenty grams of powder was percolated in 200 mL of methanol, ethanol and distilled water [14] , [15] . The percolated mixture was filtered and evaporated at 45 °C using a water bath. An aqueous solution of the extract was made corresponding to different concentrations of 500, 250, 100 and 50 mg/mL.

2.3. Phytochemical analyses

Phytochemical analyses for qualitative detection of alkaloids, flavonoids, tannins and saponins were performed on the extracts [16] – [18] .

2.4. Test organism

The test organism was obtained from the stock culture of the Microbiology laboratory of Federal University of Technology, Minna, Nigeria and authenticated using cultural and morphological identification, microscopy after Gram's staining as well as biochemical characterization of test organism [5] , and maintained in a nutrient broth medium in a refrigerator for future use.

2.5. Standardization of inoculums

The density of suspension inoculated on to the media for susceptibility test was determined using the standard curve [19] . Cells corresponding to 1.0×10 8 CFU/mL were used [20] .

2.6. Preparation and sterilization of media

All the media used in this study were obtained in powdered form and constituted in distilled water according to the manufacturers' instructions. The various quantities and volumes of water depended on the particular medium. A weighed quantity of each medium was dissolved in specific volume of de-ionized water in a chemical flask, which was stoppered properly. It was sterilized by autoclaving at 121 °C and 15 pounds per square inch for 15 min and cooled to 45-50 °C before dispensing into pre-sterilized Petri dishes. These were left to gel on the workbenches. Glass materials used in this work were also sterilized by autoclaving at 121 °C and 15 pounds per square for 15 min. They were then brought out and allowed to cool down properly before use.

2.7. Membrane filtration

Membrane filters were used to sterilize the plant stem bark extracts.

2.8. Antibacterial assay

The sensitivity of the plant extract was determined using agar well diffusion technique with modifications [21] – [23] . Wells were bored into the already gelled nutrient agar medium which has been previous seeded with the test organism using the spread plate method. The 6 mm diameter wells were bored using a sterile cork borer. The wells were then filled with 0.2 mL of each of the extracts (500, 400, 250, 100 and 50 mg/mL) and care was taken not to allow the solution to spill to the surface of the medium. The plates were allowed to stand on the laboratory bench undisturbed for 1 h to allow proper absorption into the medium before the plates were incubated at 37 °C for 18 h. The plates were later observed for the zone diameter of inhibition (ZDI). The effects of the extracts on the test organism were compared with that of a standard antibiotic, amoxicillin as a control, using 12 mm as indicative of sensitivity according the guidelines of the Clinical and Laboratory Standards Institute of 2013 [24] .

2.9. Minimum inhibitory concentration (MIC) of the extract on the bacterial test organism

The MIC of the extract was determined using methods of Bukar et al. with modification [25] . Plant extracts of 500, 400, 250, 200, 100 and 50 mg/mL concentrations were prepared. One milliliter of the different concentrations of each extract was added to 9 mL of the nutrient broth in test tubes and 1 mL of the standardized inoculum of the test organism was also added. The control was also set up, but amoxicillin was used instead of the plant extracts. The activity was determined by visual method and increase in turbidity of the test tubes using spectrophotometer.

2.10. Minimum lethal concentration (MLC) of the extracts on the test organism

The MLC of the extracts was determined using the method of Ebomoyi et al. with modifications [4] . Samples were taken from tubes with no change in turbidity in the MIC assay and sub cultured onto freshly prepared nutrient agar plates and incubated at 37 °C for 18 h. The lowest concentration of the extract that did not allow any increase in number of viable cells or bacterial growth on the surface of the agar plates was taken as the MLC.

2.11. Statistical analyses

The results were expressed as mean±SD. The one-way ANOVA test was used to compare results among and within groups for any significant difference in antibacterial activity of the extracts and the control.

3.1. Phytochemical screening of extracts

The result of the phytochemical screening revealed the following metabolites as present in the methanol, ethanol and aqueous extracts of K. senegalensis ( Table 1 ). The methanol extract contains the 10 secondary metabolites identified in the stem bark extracts of K. senegalensis in this work, which included saponnins, tannins, reducing sugar, aldehyde, phlobatannins, flavonoids, terpenoids, alkaloids, cardiac glycoside and anthroquinones. The ethanol extract had all the metabolites present in the methanol extract except the reducing sugar and the flavonoids. The aqueous extract lacked reducing sugar, aldehyde, phlobatanins, cardiac glycoside and anthroquinones but had flavonoids.

+: Present, -: Absent.

The ZDI of the various extracts at different concentrations are shown in Table 2 . The highest ZDI of the extracts against Salmonella Typhi ( S . Typhi) were 26 mm, 27 mm and 27 mm at 500 mg/mL for methanol, ethanol and aqueous extracts respectively and 29 mm for the control (amoxicillin). This was followed by the ZDI of 24 mm, 23 mm and 25 mm at 250 mg/mL for methanol, ethanol and aqueous extracts respectively and 25 mm for the control at 250 mg/mL.

Values are expressed as mean±SD. Values with the same alphabets are significantly different ( P ≤0.05).

The results of the MIC of the different extracts against S . Typhi were 250, 200, 200 and 100 mg/mL for methanol, ethanol, aqueous extracts and amoxicillin (control) respectively.

The results of the MLC of the different extracts against S . Typhi were 250, 250, 400 and 200 mg/mL for methanol, ethanol, aqueous extracts and amoxicillin (control) respectively.

4. Discussions

The results obtained from the phytochemical screening of the three plant bark extracts of K. senegalensis showed 10 plant secondary metabolites including saponins, tannins, reducing sugars, aldehyde, phlobatannins, flavonoids, terpenoids, alkaloids, cardiac glycoside and anthroquinones.

The methanol extract contains the 10 secondary metabolites identified in the stem bark extracts of K. senegalensis in this work. This was followed by the ethanol and aqueous extracts. It is believed that the presence of these plant secondary metabolites may be responsible for the antibacterial activity exhibited by these stem bark extracts.

It can be seen that at low concentrations (10-200 mg/mL), the ZDI of the control [(≥20.0±1.5) mm] is significantly higher ( P ≤0.05) than the ZDI of the extracts [(10.0±1.5) mm]. However, at higher concentrations, the ZDI of control did not differ from those of the extracts ( P ≥0.05). According to Baker and Silverton [26] , an organism is considered sensitive to a chemical agent only when the ZDI is either equal to the control, more than or not more than 3 mm smaller than the control. The highest ZDI of the extracts against S . Typhi are 26 mm, 27 mm and 27 mm at 500 mg/mL for methanol, ethanol and aqueous extracts respectively and 29 mm for the control (amoxicillin). This is followed by the ZDI of 24 mm, 23 mm and 25 mm at 250 mg/mL for methanol, ethanol and aqueous extracts respectively and 25 mm for the control. These data are above the reference range of the Clinical Laboratory Standard Institute [24] , as such are considered sensitive in this work.

The MIC of the extracts is 250, 200, 200 and 100 mg/mL for methanol, ethanol, aqueous extracts and amoxicillin (control) respectively.

The MLC of the extracts is 250, 250, 400 and 200 mg/mL for methanol, ethanol, aqueous extracts and amoxicillin (control) respectively. The MLC of methanol and ethanol extracts competes favorably with that of the control. The MLC of the aqueous extract is higher which may probably be as a result of some of the active principles lacking in the extract.

The antibacterial properties of K. senegalensis stem bark extract can be harnessed for the production of new antibiotics or the enhancement of already existing antibiotics that are fast developing resistance to combat the problem of multi-drug resistance of S. Typhi.

Acknowledgments

The authors acknowledge the participating Laboratory Technologists at the Departments of Biological Sciences and Chemistry, University of Abuja, FCT-Abuja, Nigeria. This research was supported by the Faculty of Science, University of Abuja, FCT-Abuja, Nigeria.

This study focuses on the antibacterial potency of K. senegalensis stem bark extract on S. enterica subsp. enterica serovar Typhi.

Research frontiers

The present research work depicts the phytochemical screening and the antibacterial activity of aqueous and alcoholic extracts of K. senegalensis stem bark against Salmonella serovar Typhi.

Related reports

This report is related to some earlier report on the phytochemical screening and antibacterial activity of some other plant material extracts. It is related to the work of Ebomoyi and Okojie (2012) on the physiological mechanisms underlying the use of Garcinia kola Heckel in the treatment of asthma and Halilu et al . (2010) on the preliminary phytochemical screening, antibacterial activity and elemental analysis of the leaves and the root bark of Parinari curatellifolia .

Innovations and breakthroughs

K. senegalensis is a medicinal plant that can be used in the treatment of typhoid fever. In the present study, authors have demonstrated the antibacterial activity of K. senegalensis stem bark extract on S. enterica subsp. enterica serovar Typhi.

Applications

From the literature survey, it has been found that K. senegalensis stem bark is safe to humans. This scientific study supports and suggests that this plant may be used as an antibacterial agent.

Peer review

This is a valuable research work in which the authors have demonstrated the antibacterial activity of K. senegalensis stem bark extracts on S. enterica subsp. enterica serovar Typhi. The activity was assessed based on the MIC and the MLC of the stem bark extract. K. senegalensis was found to be a promising antibacterial agent against S. enterica subsp. enterica serovar Typhi.

Foundation Project: Supported by the Faculty of Science, University of Abuja, FCT- Abuja, Nigeria.

Conflict of interest statement: We declare that we have no conflict of interest.

A Literature Review—Khaya senegalensis, Anacardium ouest L., Cassia sieberiana DC., Pterocarpus erinaceus, Diospyros mespiliformis, Ocimum gratissimum, Manihot esculenta, Vernonia amygdalina Delile, Pseudocedrela kotschyi and Daniellia oliveri Possess Properties for Managing Infectious Diarrhea

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A Review of the Phytochemistry and Pharmacology of the Medicinal Plant: Khaya Senegalensis (Desr.) A. Juss

2022, International Journal of Research Publication and Reviews

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Khaya senegalensis, a member of the family maliaceae, is a plant commonly used by the local people of Nigeria for the treatment of dysentery, mucous, diarrhoea, vermifuge, taeniacide, depurative, Syphilis jaundice, dermatoses, scorpion bites, allergies infection of the gum, hookworm, and disinfection of the wounds. This work evaluates the phytochemical constituents of the plant's stem bark and the antioxidant properties of acetone, ethanol and methanol extracts of the plant part. The stem bark extracts of the plant were obtained by cold sequential extraction using the solvents, acetone, ethanol and methanol in that order. The three solvents show ability to extract some components of the plant's part, though and varied quantities. The result for the phytochemical screening shows that saponin, tannin, alkaloid, cardiac glycoside, flavonoids, phlobatannin some of the phytochemicals of the plant's part and could be responsible for its medicinal properties. The investigation of antioxidants properties of the extracts show acetone, ethanol and methanol to exhibit 66.282%, 84.838%, 85.921% scavenging ability respectively at 100µg/cm 3 compared with 97.437% of the standard, ascorbic acid. The extract therefore can be said to have good antioxidant property and hence can be applied in managing oxidative stress.

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The phytochemical constituents of stem bark extracts of Khaya senegalensis were isolated and analyzed using Gas Chromatography-Mass Spectrometry (GC-MS). A shade-dried stem bark of K. senegalensis was extracted using methanol and water as solvents. The main chemical compositions of the extracts were analyzed by GC-MS and preliminary phytochemical analysis was performed to confirm the various classes of active chemical. The chemical composition of methanolic stem bark extract of K. senegalensis included: 4-Hepten-3-one, 2, 6-Pyridinedicarboxylic acid, 3-O-methyl-d-glucose, myristic acid, pentadecanoic acid, n-Hexadecanoic acid, 9, 12-Octadecadienoic acid, and 11-Octadecenoic acid. Others are 9–Hexadecenoic acid, Stearic acid, I, E-11, Z-13-Octadecatriene, Cyclododecyne, Hexadecanoic acid, Ricinoleic acid, 13-Decosenoic acid, and 9-Hexadecenal. The Chemical composition of aqueous stem bark extract of K. senegalensis included 1, 2, 3-benzenetriol, n-Hexadecanoic acid, oleic acid, (Z)6,(Z)9-pentadecadeien-1-ol, 1,E-11,Z-13-octadecatriene, and 1-flourodecane. Other chemical constituents of the aqueous extract included 9-octadecanal, E-9-tetradecanal, and 2-methyl-Z, Z-3, 13-octadecadienol. The molecular weight of these compounds ranged from low to high with carbon skeleton of between C 7 and C 37. Both aromatic and aliphatic compounds were identified. K. senegalensis contains alkaloid, saponin, tannins and flavonoids. A good number of bioactive compounds were present in the stem bark of K. senegalensis.

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The aim of this work was to investigate and compare the phytochemical screening and antimicrobial activities of different crude extracts from leaves, barks and roots of Khaya senegalensis. Two different organic solvents including methanol and ethanol were used to prepare the crude extracts from the fresh and dry leaves, barks and roots. Antimicrobial activities of different crude extracts from dry and fresh leaves of Khaya senegalensis were determined by agar disc diffusion method with minor modification. In vitro phytochemical screening for all crude extracts from all leaves, barks and the roots were tested and shown positive result for all the compounds. The methanol crude extract and its derived fractions from leaves, barks and roots showed small and moderate antibacterial potential with one gram positive (Staphylococcus aureus) and three gram negative (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) bacteria in the range of 0–21%. In conclusion, all organic crude extracts from leaves, barks and roots could be used as potential sources of new antimicrobial properties.

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The antibacterial activity of the aqueous and ethanolic leaf and stem bark crude extracts of Khaya senegalensis against four bacteria species (Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumonia and Escherichia coli) was investigated using the agar well diffusion technique. At concentrations ranging from 400 to 1000 mg/ml the ethanol crude extracts showed activity against the four bacteria species, with mean zone of inhibition ranging from 7.67 ± 0.33 a to 19.66 ± 0.33 b . Similarly the aqueous crude extract at 400 to 1000 mg/ml recorded low activity with mean zone of inhibition ranging from 0.33 ± 0.33 a to 13.3 ± 0.33 c . Minimum inhibitory concentrations of the crude extracts were 200 and 400 mg/ml and the minimum bactericidal concentration was also 400 and 800 mg/ml. The lethal dose (LD50) of the crude extracts of K. senegalensis was found to be greater than 5000 mg/kg. The phytochemical components of the crude extracts include alkaloid, steroids, glycosides, ...

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