INTRODUCTION:

Antimicrobial agents are substances that eliminate or inhibit the growth of microorganisms¹. For centuries traditional healers in folk medicine have exploited plants and other natural sources of drugs for the treatment/management of infectious diseases. Modern medicine has been exploring these traditional drugs as possible sources of lead molecules in antimicrobial drug development and discovery^2,3,4,5. Searsia lancea (SL) also known as Rhus Lancea is an evergreen tree that can reach a height of seven meters and is known to thrive in temperate and tropical climates⁶. SL is a member of the Anacardiaceae family and is native to Southern Africa with four known species in Botswana⁶. This plant is locally known as "karee" (English), "moshabele" (setswana), and "nhlokotshane" (Kalanga). It is also known as the "Africa sumac" in North America.^7,8

The Basotho tribe in Lesetho traditionally use the fruits and leaves of SL to treat diabetes, herpes sores, high blood pressure, heart illnesses, and dizziness caused by anemia⁸. In Botswana the Kalanga tribe use the aqueous decoction of SL roots for the treatment of various microbial infections. Other traditional uses include the treatment of dermatological infections like tinea vesicolor and tinea capitis. It is also used for the treatment of diarrhoea, gonorrhoea, fever and common cold.

Essential oil extracted from the leaves of SL have reported antibacterial activity against several bacterial species namely Acinetobacter calcoaceticus , Bacillus subtilis, Citrobacter freundii , Clostridium perfringens, Clostridium sporogenes , Escherichia coli , Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhii, Staphylococcus aureus, and Yersinia enteroco litic⁹. The petroleum ether, dichloromethane, 80% ethanol, methanol and water extracts of SL leaves have also demonstrated significant antibacterial and antifungal potential against Bacillus subtilis; Escherichia coli; Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus Faecalis, multiple drug-resistant (MDR) E. coli, MDR K. pneumoniae, drug-sensitive Staphylococcus aureus, penicillin-resistant S. aureus and the fungi Candida Albicans ^10,11.In addition to the antibacterial activity, SL fruits and stem have demonstrated anticancer activity¹², antioxidant⁹, anti-inflammatory activity and anticholinesterase activity¹⁰.

Despite the extensive traditional use of this plant against popular infections there is limited literature on the scientific validity of its use in common diseases. Likewise, there is no evidence on the scientific use of SL roots for the treatment of common infectious diseases. Hence in view of the attributed folk medicinal significance of the plant and its availability as an indigenous resource the present work was undertaken to explore the antimicrobial activity of SL root extract against common infectious agents.

MATERIALS AND METHODS:

Plant Material:

Roots of SL were collected from the Amauwe riverbank in Semitwe village, Central district, Botswana. The plant roots were identified and verified by the department of biological sciences, University of Botswana.

Chemicals, reagents, organisms, and equipment:

The study required the use of chemicals like dichloromethane (DCM) (Ideal Chemicals, India), Dimethyl sulfoxide (DMSO) (Richest group, China), Fluconazole and ciprofloxacin disks (Mast group Ltd, United Kingdom). Muller Hinton (MH) [Merck, South Africa], Sarabound dextrose gar (SD) [HIMEDIA, India]. In house prepared normal saline and distilled water was used in this study. The organisms for the study i.e., E. coli (ATCC 31234), staph aureus (ATCC 31227), candida albicans (ATCC P360) and candida glabrata (ATCC 66032) were obtained from the National Health Laboratory, Gaborone Botswana. The equipment used in the study included analytical balance (Biobase Biodustry, Germany), rotary evaporator (Biobase, Germany), autoclave (MEDWISE, USA), incubator (Thermo scientific, USA), and hot plate (Thermo scientific, USA).

Study design and ethical considerations:

The study design followed was quantitative experimental research. The study was conducted in accordance with the existing ethical guidelines after necessary ethical approval (reference no: HPDME:13/18/1).

Macroscopic examination and preparation of crude extract:

SL root skin (700g) were examined for any foreign matter (contaminants such as insects, molds, soil and other undesirable particulate matter). The examined roots skins were then washed with clean water and air dried at room temperature (26-32℃) for 14 days in a clean room. The dried roots skins were then crushed to fine powder using clean mortar and pestle.

Preparation of crude extract:

The maceration extraction technique was adopted from literature⁹. Briefly, 100g of SL root powder was treated with 1000ml of DCM and distilled water separately. The mixture was left to macerate for 48 hours at room temperature (26-32℃). After 48 hours, the mixture was sieved using five sieves of different grades from course to extra-fine. The filtrate was then passed through a Whatman filter paper No.8. The DCM and DW was evaporated under reduced pressure using a rotary evaporator at 50℃ and 70℃ respectively. The dried crude extracts were stored in a refrigerator at 4℃ till analysis. The yield of extract (in percentages) was determined by the formula,

Yield of extract (%) = (weight of extract/ weight of sample taken) *100

Microbiological analysis:

Antibacterial activity evaluation:

The agar well diffusion method^13,14,15,16 was used to evaluate the antibacterial activity of SL root extracts against four reference strains (Escherichia coli ATCC 31234 and Staphylococcus aureus ATCC 31227). Separately, 2ml of sterile normal saline solutions were inoculated with isolated colonies of reference strains till the turbidity of the solutions matched that of 0.5 McFarland standard using visual inspection. Sterile cotton swabs were used to inoculate the organisms from the normal saline solutions onto the MH plates. After inoculation, the plates were allowed to dry for 5 minutes at room temperature. Post drying, a sterile cork borer was used to make wells measuring 9mm in diameter. 100µL of DCM (100mg/ml) and aqueous extracts (100mg/ml) were pipetted into the labelled wells. The plates were then incubated at 37℃ for 24 hours. Plant extracts with antibacterial properties were determined by the appearance of clear zones of bacterial growth inhibition around each disk after a 24h incubation period. The zone of inhibition is expressed as mean±SD (mm) of three replicate measurements for each treatment. Flucanazole (10mcg) and ciprofloxacin (1mcg) served as positive controls (10mcg). DMSO (99.9% ) and distilled water served as negative controls.

Antifungal activity evaluation:

The agar well diffusion method^13,14,15,16 was used to evaluate the antifungal activity of SL root extracts on reference strains (Candida albicans ATCC P360 and Candida glabrata ATCC 66032). Separately, 2ml of sterile normal saline solutions were inoculated with isolated colonies of reference strains till the turbidity of the solutions matched that of 0.5 McFarland standard based on visual inspection). Sterile cotton swabs were used to inoculate the organisms from the normal saline solutions onto the Sabouraud plates. After inoculation, the plates were allowed to dry for 5 minutes at room temperature. Post drying, sterile cork borer was used to make wells measuring 9mm in diameter. 100µL of DCM (100mg/ml) and aqueous extracts (100mg/ml) were pipetted into the labelled wells. The plates were then incubated at 37℃ for 24hours. Plant extracts with antibacterial properties were determined by the appearance of clear zones of fungal growth inhibition around each disk after a 24h incubation period. Fluconazole (10mcg) and ciprofloxacin (1mcg) served as positive controls (10mcg). DMSO (99.9%) and distilled water served as negative controls.

Determination of minimum inhibitory concentration (MIC):

The agar dilution method was used to determine the MIC of the plant extracts against two bacterial and two fungal reference strains¹⁷. The aqueous and the DCM SL crude root extract (100mg/ml) was serially diluted in MH and SD to prepare concentrations of 0.78-25mg/mL. The serially diluted solutions were then poured onto separate labelled Petri dishes. 2ml of sterile normal saline solutions were inoculated with isolated colonies of reference strains of each microorganism till the turbidity of each solution matched that of 0.5 McFarland standard. Each Petri dish was then inoculated separately with 100µL of reference organism and was incubated at 37℃ for 24 hours. The end point (MIC) was recorded visually as the lowest concentration of plant extract that completely inhibits the microorganism growth under the tested incubation conditions.

Statistical analysis:

The zone of inhibition is expressed as mean ±SD (mm) of three replicate measurements for each treatment. One way analysis of variance (ANOVA) was used to perform the statistical analysis. Statistically significant differences were considered for p≤0.05.

RESULTS:

Yield of crude extract:

The SL root on maceration with DW resulted in a 0.79 %w/w crude extract of brick red colour and on treatment with an organic solvent produced a light brown crude extract of 1.02% w/w.

Microbiological assays:

Antibacterial activity evaluation:

The antibacterial activity for the DW and DCM root extract of SL was evaluated against two bacterial strains, Escherichia coli (ATCC 31234) and Staphylococcus aureus (ATCC 31227). Ciprofloxacin and fluconazole discs were used as positive controls. The negative controls used in the study were DMSO and distilled water. The DW extracts achieved zones of inhibition of 23.67± 6.25mm and 30.50± 9.0mm for E.coli and Staph aureus respectively. The DCM extracts yielded zones of inhibition of 22.33±5.16 mm and 29.83±10.41 mm for E.coli and Staph aureus respectively. Fluconazole demonstrated no zone of inhibition against the tested reference bacteria strains. Ciprofloxacin produced zones of inhibition of 18.00±0.00mm and 38.67±0.57mm against E.coli and Staph aureus respectively. Negative controls produced zero zones of inhibition against both the organisms. The results are presented in Table 1.

Table 1: Antimicrobial activity of Distilled water and dichloromethane Sersia Lancea crude root extracts against reference microbial strains

	Microorganism Control strains	Zones of inhibition (mm)*
	Microorganism Control strains	Extract	Fluconazole ^a(10mcg/disc)	Ciprofloxacin^a (1mcg/disc)	DMSO^b	DW^b
DW extract (100mg/mL)	Candida albicans	24.50±2.16	26.33±1.15	00.00	00.00	00.00
	Candida glabrata	19.67±0.58	00.00	00.00	00.00	00.00
	Coli	23.67±6.25	00.00	18.00±0.00	00.00	00.00
	Staph aureus	30.50±9.00	00.00	38.67±0.57	00.00	00.00
DCM extract (100mg/mL)	Candida albicans	23.33±1.86	25±0.00	00.00	00.00	00.00
	Candida glabrata	18.00±0.01	00.00	00.00	00.00	00.00
	E.coli	22.33±5.16	00.00	17.36±0.57	00.00	00.00
	Staph aureus	29.83±10.41	00.00	39.33±0.57	00.00	00.00

*(n=3,mean±SD), a positive control, b negative control, DW distilled, DCM dichloromethane, DMSO dimethyl sulphoxide

Anti-fungal activity evaluation:

The antifungal activity of SL root extracts (DW and DCM), positive controls (ciprofloxacin and flucanazole) and negative controls (DMSO and DW) were studied against Candida albicans (ATCC P360) and Candida glabrata (ATCC 66032). The aqueous SL root extracts achieved zones of inhibition of 24.50±2.16mm and 19.67±0.58mm for Candida albicans and Candida glabrata respectively. The DCM extracts yielded the zones of inhibition of values 23.33±1.86 mm, 18±0.01mm for Candida albicans and Candida glabrata respectively. Fluconazole yielded a zone of inhibition of 25±0.00mm against Candida albicans and a zero zone of inhibition for Candida glabrata. Ciprofloxacin and the negative controls produced zero zones of inhibition against both the reference fungi. The results are presented in Table 1.

Minimum inhibitory concentration (MIC):

The results of the MIC experiment demonstrate that Candida albicans and E. coli were susceptible to the MIC of 3.12mg/mL for the SL distilled water root extract. The MIC of the aqueous extract against Candida glabrata and Staph aureus were 6.25 and 12.5mg/mL respectively. All the microorganisms were susceptible to MIC 6.25mg/mL for the DCM extract except for Candida glabrata with MIC of 12.5mg/mL. The results are presented in Table 2.

Table 2: Minimum inhibitory concentration of the aqueous and dichloromethane crude root extract of Sersia lancea

Organism	Minimum inhibitory concentration [MIC] (mg/ml)
Organism	Aqueous extract	Dichloromethane extract
Candida albicans	3.12	6.25
Candida glabrata	6.25	12.5
E.coli	3.12	6.25
Staph aureus	12.5	6.25

DISCUSSION:

World-wide, plant sources have been used as part of traditional systems of medicine for centuries^18,19,20. Botswana is known to harbour numerous plant species that have been used for their antimicrobial properties by the local people within the country²¹. The organisms used in this study are widely responsible for numerous bacterial and fungal skin infections. Research efforts on the Sersia species (syn: Rhus) indicate the promising potential of plants in this category to provide bio active compounds. Plants belonging to this species are distributed in Asia, Africa, Australia, Mediterranean, Central America and Mexico. The wide geographic distribution of these plant species makes it an appealing source to investigate antimicrobial molecules that can be obtained from source and reach the end consumer with minimal expenditure⁴.

The biological investigations in this study clearly demonstrate the antibacterial and antifungal potential of SL aqueous and organic extract against four common pathogens. The findings from the invitro antibacterial activity suggest that S.aureus was the most sensitive organism and the order of susceptibility of the pathogens to the tested extracts to be S. aureus> Candida albicans> E.coli> Candida glabrata. Our findings align parallel with previous studies that demonstrate the susceptibility of S.aureus, Candida albicans and E.coli to polar and non-polar extracts of SL^9,10,11. Higher zone of inhibition was observed for S.aureus than E.coli similar to previous studies^9,10,11. The difference in efficacy of plant extract against gram positive and gram negative bacteria could be attributed to the lipopolysaccharide outer cell wall membrane in the latter microorganism that act as a barrier to numerous substances¹⁸. This is the first study that highlights the susceptibility of Candida glabrata to SL root extract.

The MIC experiment in our study demonstrates that the low concentrations of SL aqueous extract show higher microbial inhibition for E.coli, Candida glabrata and Candida albicans in comparison to the DCM SL root extract. This is in support to previous studies that report the polar extract to demonstrate higher MIC. Isolation, purification and structure elucidation studies on the metahnolic extract of R.glabra branches and ethanolic extract of R.Coriaria fruits in the Sersia species ascribed the antimicrobial potential of the polar extract to compounds like methyl gallate, 4-methoxy-3,5-dihrodybenzoic acids, gallic acid and tannins¹⁸. Studies by Vambe et al 2021, identified 1-nonadecanol and 1-tetracosanol as antimicrobial compounds in methanolic extract of SL leaves²². Further studies are required to elucidate the compounds present in the water extract that are responsible for the antimicrobial effect. The observed MIC in our study for the aqueous and DCM root extract is similar to previous studies by for E.coli and Candida albicans respectively¹⁰. Literature studiesreport the MIC of the DCM and water SL leaf extract to be within 0.05 to 1.56 mg/mL for the tested bacteria^10,11. This difference in observation from the former studies could be attributed to the higher proportion of primary and secondary metabolites in leaves in comparison to the roots. Non uniformity in the composition of bioactive moieties can also affect the microbial activity resulting in plant extracts having varying activity when compared to the synthetic pharmaceuticals that are manufactured under controlled conditions²³.

The higher extraction potential of water validates the local antimicrobial use of the SL roots in traditional medicine. Additionally, water is a easy to handle solvent that is inexpensive, safe and readily available. The observed MIC values for the plant extracts are lower than the reported MIC for SL essential oil⁹. Additionally, this is the first study that reports the MIC of the SL root extract for Candida glabrata.

CONCLUSION:

The present study demonstrated the potential of the aqueous and DCM crude root extract of SL in inhibiting the multiplication of selected bacterial and fungal species. Additionally, this is the first report to demonstrate the antifungal property of SL root extract on Candida glabrata. The results in this work provide scope for future studies to elucidate the structure of potential actives in SL root extract and to test their potential as possible antibacterials and antifungals. Additionally, formulation researchers can also explore the possibility of developing novel cost-effective herbal formulations of SL for the treatment of common bacterial and fungal infections.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank the University of Botswana for providing the necessary laboratory facilities for carrying out this work.

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Received on 03.11.2022 Modified on 13.03.2023

Research J. Pharm. and Tech 2023; 16(9):4016-4020.

DOI: 10.52711/0974-360X.2023.00658