nach oben

BMC Complementary Medicine and Therapies

Erschienen in:

Open Access 01.12.2023 | Research

Evaluation of the antibacterial activity of selected Kenyan medicinal plant extract combinations against clinically important bacteria

verfasst von: Elizabeth A Odongo, Peggoty C Mutai, Beatrice K Amugune, Nelly N Mungai, Mary O Akinyi, Julia Kimondo

Erschienen in: BMC Complementary Medicine and Therapies | Ausgabe 1/2023

Abstract

Background

Infectious diseases are a major global public health concern as antimicrobial resistance (AMR) currently accounts for more than 700,000 deaths per year worldwide. The emergence and spread of resistant bacterial pathogens remain a key challenge in antibacterial chemotherapy. This study aims to investigate the antibacterial activity of combined extracts of various Kenyan medicinal plants against selected microorganisms of medical significance.

Methods

The antibacterial activity of various extract combinations of Aloe secundiflora, Toddalia asiatica, Senna didymobotrya and Camelia sinensis against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Methicillin Resistant Staphylococcus aureus was assessed using the agar well diffusion and the minimum inhibitory concentration in-vitro assays. The checkerboard method was used to evaluate the interactions between the various extract combinations. ANOVA test followed by Tukey’s post hoc multiple comparison test was used to determine statistically significant differences in activity (P < 0.05).

Results

At concentrations of 100 mg/ml (10,000 µg/well), the different combinations of the aqueous, methanol, dichloromethane and petroleum ether extracts of the selected Kenyan medicinal plants revealed diverse activity against all the test bacteria. The combination of methanolic C. sinensis and A. secundiflora was the most active against E. coli (14.17 ± 0.22 mm, diameter of zones of inhibition (DZI); MIC 2500 µg/well). The combination of methanolic C. sinensis and S. didymobotrya was the most active against S. aureus (16.43 ± 0.10 mm; MIC 1250 µg/well), K. pneumonia (14.93 ± 0.35 mm, DZI; MIC 1250 µg/well), P. aeruginosa (17.22 ± 0.41 mm, DZI; MIC 156.25 µg/well) and MRSA (19.91 ± 0.31 mm, DZI; MIC 1250 µg/well). The Minimum Inhibitory Concentration of the different plant extract combinations ranged from 10,000 µg/ well to 156.25 µg/well. The ANOVA test indicated statistically significant differences (P < 0.05) between single extracts and their combinations. The fractional inhibitory concentration indices (FICI) showed that the interactions were either synergistic (10.5%), additive (31.6%), indifferent (52.6%), or antagonistic (5.3%) for the selected combinations.

Conclusion

This study findings validate the ethnopractice of selectively combining medicinal plants in the management of some bacterial infections in traditional medicine.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Background

Antibiotics have made a considerable contribution to the control of infectious diseases that have over time contributed to human morbidity and mortality for most of human existence [1]. In spite of the existing range of conventional antimicrobial agents in clinical use, antimicrobial resistance (AMR) remains a constant threat with regular antibiotic use [2, 3]. Among bacterial infections, the so-called “ESKAPE” pathogens have caused the most concerns based on their prevalence and overall mortality [4]. Failure to take the appropriate measures to combat the progress of antimicrobial resistance may result in the loss of approximately 10 million lives and cost about US$100 trillion per year by 2050 [5]. The significant gaps in the surveillance of antimicrobial resistance coupled with a lack of quality data on the impact of antimicrobial resistance is a common observation in most African countries [5].

There is a continuous need to develop new medicines that are capable of overcoming microbial resistance. Approximately, 30 to 40% of the commercially available antimicrobial drugs are from natural products and primarily from microbial origins [6]. Plants are a promising alternative in the search for new antimicrobials based on their utilization in traditional medicine for the management of bacterial diseases and potential to provide an unlimited range of chemical compounds for exploration [6, 7]. Over 1340 plants possess defined antimicrobial activity and about 30,000 antimicrobial compounds have been isolated from plants [8].

Drug combination is a recognized approach in both traditional and conventional medicine systems. It is based on synergistic interactions to improve the therapeutic efficacy and lifespan of drugs [9‐11]. For example, locals around the Lake Victoria basin in Tanzania reportedly utilize multi-plant extracts in the management of secondary opportunistic infections [12]. Polyherbalism is also famous in Ayurveda [13]. While the combination of conventional drugs is a common practice and a successful tactic in the management of drug resistant microorganisms, the outcome of combinations between herbal drugs remains obscure due to limited scientific appraisal [14].

Based on a previous systematic review on the antibacterial activity of Kenyan medicinal plants, Camelia sinensis, Aloe secundiflora, Toddalia asiatica and Senna didymobotrya were selected for pharmacological assay as they exhibited high mean inhibition zone values and/ or low minimum inhibitory concentration (MIC) values [15, 16].

Camelia sinensis L. (Theaceae) is a common evergreen shrub widely grown in many parts of the world. It is used as an astringent, stimulant, diuretic and de-flatulent in traditional medicine [17]. It has antioxidant, antimicrobial, cholesterol lowering and cardio protective effects [18]. The bioactive constituents include caffeine, L-theanine and polyphenols/flavonoids, proteins, minerals, vitamins, and amino acids [17, 19].

Aloe secundiflora Engl. (Asphodelaceae) widely famed for its medicinal and cosmetic properties is the most commonly used Aloe species in Kenya [20]. It remedies constipation, sore throat and promotes wound healing [15]. The chemical constituents comprise tannins, terpenoids and flavonoids [21].

Toddalia asiatica L. (Rutaceae) is a traditional remedy for coughs, dysentery and malaria [22]. It has anti-inflammatory, analgesic, hemostatic coagulation anti-tumor effects. The main chemical constituents are coumarins and alkaloids [23].

Senna didymobotrya (Fres.) Irwin & Barneby (Fabaceae) is abundant across East Africa and the traditional preparations relieve diarrhea, malaria and ringworm [24]. Its pharmacological effects include antibacterial, antifungal and antioxidant [25]. The chemical constituents consist of steroids, terpenoids, anthraquinones, tannins, saponins, glycosides, flavonoids, alkaloids and phenols [24].

This study presents the first report on the antibacterial activities of various plant extract combinations of four Kenyan medicinal plants.

Methods

Collection of plant material

C. sinensis (leaves) was collected from Shinyalu (Kakamega County). The leaves of S. didymobotrya were collected from Kangundo road (Machakos County). The stem bark of T. asiatica was collected from Tala (Machakos County) while the leaves of A. secundiflora were collected from Matuu (Machakos County). The plants were authenticated at the Department of Botany, University of Nairobi and voucher specimen deposited at the University of Nairobi herbarium. Voucher numbers were allocated as follows: C. sinensis (EAO UON 2021/001), A. secundiflora (EAO UON 2021/002), S. didymobotrya (EAO UON 2021/003) and T. asiatica (EAO UON 2021/004). The plant materials were individually air-dried under shade and ground into powder using a laboratory mill [14].

Extraction procedures

Extraction was separately done using four solvents of different polarities (petroleum ether (PET), dichloromethane (DCM), methanol (MeOH) and water (H₂O).

Hot aqueous extraction

About 200 gm of each dried plant powder was separately extracted using 800 ml of distilled water by heating at 60 °C for 30 min. After cooling, the mixture was then filtered through Whatman No.1 filter paper. Reduction was done using a rotary evaporator. The dry aqueous extracts were obtained via lyophilization [26].

Organic solvent extraction

Approximately 100 gm of each dried powder were separately extracted using 500 ml of petroleum ether, dichloromethane and methanol. Maceration with stirring was done for 72 h at room temperature. The mixture was filtered through Whatman No.1 filter paper. The extracts were then concentrated at 40 °C (for petroleum ether and dichloromethane extracts) and at 65 °C (for methanol extracts) using a rotary evaporator (Heidolph WB2000, Germany) [27]. The organic solvent extracts were further evaporated to dryness at 40 °C in an oven before storing at 4 °C for future use [28].

Collection of bacterial cultures

Pure bacteria cultures of; Staphylococcus aureus ATCC 25,923, Escherichia coli ATCC 25,922, Klebsiella pneumoniae ATCC 70,063 and Pseudomonas aeruginosa 15,422 (from Department of Pharmacy, University of Nairobi) and MRSA ATCC 1385 (from Department of Biology, University of Nairobi) were maintained on nutrient broth slants at 4 °C [29]. The standard inoculum suspensions were adjusted to turbidity equivalent to 0.5 McFarland standards and modified to give a density of 1 × 10⁶ cells or spores/ml [29, 30].

Sterilization and equipment

All the glassware, nutrient media and distilled water used in the antibacterial activity studies were sterilized in an autoclave Memmert Universal oven (Memmet GmbH and Co, KG, Schwabach, Germany) at 121 °C for 15 min before use [28]. The bacteriological wire loop and cork borer were sterilized by flaming using a Bunsen burner flame. All bench work involving use of microorganisms was carried out in a Bioflow laminar flow cabinet (Vermeulen, L.J. BVBA, Westmalle, Belgium) while a Freezer-1 incubator (Analis, Suarlee, Belgium) was used for incubation of the microorganisms [28, 31].

Preparation of stock solutions

The extracts stock solutions were prepared by dissolving 500 mg in 5 ml of 10% dimethyl sulfoxide (DMSO). Stock solutions of the extract- extract combinations were prepared by combining the two extracts (ratio 1:1) [30]. Gentamicin (30 mg/ 100 ml sterile distilled water) was used as positive control for Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae [31]. Mupirocin (10 mg/ 100 ml sterile distilled water) was used as positive control for Staphylococcus aureus and Methicillin Resistant Staphylococcus aureus. A 10% DMSO solution served as the negative control [32, 33].

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was done by agar well diffusion method according to the Clinical and Laboratory Standards Institute [34]. The bacterial test organisms were cultured on tryptone soya agar. The nutrient agar was inoculated uniformly with the standardized test organisms. Reservoir wells were formed by cutting out cylindrical plugs from the solidified nutrient agar at equidistant points (30 mm), using a sterile cork borer, to produce wells (diameter 10 mm) [31, 33, 35]. On each petri-dish, the respective wells were separately filled with 100 µl of the stock solutions (10,000 µg/well) single plant extracts, 100 µl plant extract combination, gentamicin 0.3 mg/ml (30 µg/well), mupirocin 0.1 mg/ml (10 µg/well) or 10% DMSO [32]. The inoculated petri-dishes with test solutions in wells were allowed to diffuse for 30 min before overnight (18 h) incubation at 37 °C. All determinations were done in triplicate. The antimicrobial activity was recorded as the diameter (mm) of the of inhibition after incubation [31, 36].

Minimum inhibitory concentration (MIC) determination

The MICs of the extracts against the test microorganisms were determined by the agar well diffusion method [37‐40]. MIC for single extracts and extract combinations that failed to meet minimum activity threshold in the susceptibility studies were not determined [36]. For the single extracts, double serial dilution of the stock solution was carried out resulting in concentration range of 10,000 µg/ml to 78.1 µg/ml [41].

Separate petri-dishes were used for each of the test solutions (single extracts, extract combinations and antibiotic standards). On each petri-dish, the respective wells were separately filled with 100 µl of the respective dilutions [32, 36], The inoculated petri-dishes with test solutions in wells were allowed to diffuse for 30 min before overnight (18 h) incubation at 37 °C. All determinations were done in triplicate. The MIC was determined as the lowest concentration that inhibited visible bacterial growth on the agar subculture [30].

Determination of fractional inhibitory concentration index

The antibacterial effects of combining selected plant extracts were assessed using the checkerboard method [30]. The fractional inhibitory concentration (FIC) was derived from the lowest concentrations of the extract and the extract in combination permitting no visible growth of the test organisms after incubation [42]. FIC indices were calculated using the formula described [43]: FIC index = (MIC of extract 1 in combination/MIC of extract 1 alone) + (MIC of extract 2 in combination /MIC of extract 2 alone). The following criterion was used in the interpretation of the FIC Index in relation to the mode of plant extract interactions: FICI ≤ 0.5 = synergistic effect; FICI > 0.5 but ≤ 1 = additive effect; FICI > 1, but ≤ 4 = indifferent effect and FICI > 4 = antagonistic effect [43]. The data were analyzed by using MS Excel 2016 and presented as mean ± SD of three replicates. The significance was evaluated by analysis of variance (ANOVA) test and by Tukey’s post hoc multiple comparison test using Statistical Package for the Social Sciences (SPSS) 21.0. Significant differences in the data were established at the 5% level of significance [44].

Results

Both the single and the combined plant extracts in this study displayed activity against the test bacteria. The patterns of antibacterial activity varied with the plant, test microorganism and the solvent used for extraction. Generally, C. sinensis displayed activity against the widest range of microorganisms and the polar extracts from all the four plants demonstrated higher antibacterial activity than the non-polar extracts. For example, the methanol extract of S. didymobotrya and that of C. sinensis (Table 1) individually displayed low activity against E. coli but exhibited an increase in the zone of inhibition in combination (Table 2). This scenario is replicated with the combination of the methanol extract of A. secundiflora and methanol C. sinensis (Table 2). The combination of dichloromethane extracts of S. didyobotrya and T. asiatica were not effective in inhibiting MRSA. A few (5.26%) of the extract combinations resulted in lower zones of inhibition than the single plant extracts (Tables 1 and 2).

Table 1

Table showing mean diameter of zones of inhibition and Minimum inhibitory concentration (MIC) of single plant extracts against P. aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus and Methicillin Resistant Staphylococcus aureus

	Pseudomonas aeruginosa		Klebsiella pneumoniae		Escherichia coli		Staphylococcus aureus		Methicillin Resistant Staphylococcus aureus
EXTRACT (100 mg/ ml)	DZI (mm) ± SD	MIC (µg/ well)	DZI (mm) ± SD	MIC (µg/ well)	DZI (mm) ± SD	MIC (µg/ well)	DZI (mm) ± SD	MIC (µg/ well)	DZI (mm) ± SD	MIC (µg/ well)
Aloe MeOH	17.88 ± 0.22	5000	14.60 ± 0.18	5000	13.41 ± 0.18	10,000	14.39 ± 0.21	10,000	11.43 ± 0.12	10,000
Toddalia MeOH	16.11 ± 0.14	5000	13.79 ± 0.02	5000	14.96 ± 0.36	10,000	13.82 ± 0.16	5000	14.07 ± 0.05	2500
Senna MeOH	17.91 ± 0.02	312.5	14.40 ± 0.05	10,000	11.25 ± 0.13	10,000	15.75 ± 0.40	5000	12.03 ± 0.18	10,000
Camelia MeOH	21.67 ± 0.08	312.5	14.01 ± 0.21	5000	13.03 ± 0.04	5000	16.74 ± 0.31	1250	16.57 ± 0.26	1250
Aloe DCM	10.24 ± 0.01	ND	10.15 ± 0.03	ND	10.08 ± 0.02	ND	10.83 ± 0.14	ND	10.13 ± 0.04	ND
Toddalia DCM	16.87 ± 0.23	1250	10.23 ± 0.07	ND	12.55 ± 0.11	ND	11.46 ± 0.15	ND	13.48 ± 0.12	5000
Senna DCM	10.17 ± 0.06	ND	10.38 ± 0.12	ND	10.12 ± 0.03	ND	12.29 ± 0.18	ND	10.04 ± 0.01	ND
Camelia DCM	11.15 ± 0.08	ND	10.00 ± 0.01	ND	10.36 ± 0.04	ND	10.03 ± 0.01	ND	10.08 ± 0.01	ND
Aloe PET	10.30 ± 0.04	ND	10.21 ± 0.04	ND	10.10 ± 0.02	ND	10.65 ± 0.13	ND	10.06 ± 0.02	ND
Toddalia PET	10.22 ± 0.02	ND	10.28 ± 0.02	ND	10.16 ± 0.05	ND	11.61 ± 0.04	ND	11.13 ± 0.03	ND
Senna PET	10.12 ± 0.06	ND	10.32 ± 0.09	ND	10.17 ± 0.09	ND	12.55 ± 0.12	ND	10.10 ± 0.02	ND
Camelia PET	11.04 ± 0.01	ND	10.24 ± 0.03	ND	11.05 ± 0.14	ND	10.11 ± 0.01	ND	10.04 ± 0.01	ND
Aloe H₂O	11.33 ± 0.25	ND	10.49 ± 0.11	ND	10.04 ± 0.01	ND	10.35 ± 0.19	ND	10.24 ± 0.02	ND
Toddalia H₂O	10.31 ± 0.16	ND	10.33 ± 0.08	ND	10.10 ± 0.02	ND	11.06 ± 0.02	ND	10.26 ± 0.01	ND
Senna H₂O	19.72 ± 0.29	312.5	11.39 ± 0.13	10,000	10.42 ± 0.16	10,000	13.12 ± 0.04	10,000	10.50 ± 0.06	10,000
Camelia H₂O	15.66 ± 0.23	2500	13.48 ± 0.03	2500	17.04 ± 0.24	2500	14.75 ± 0.27	2500	15.05 ± 0.13	2500
DMSO	0.00		0.00		0.00		0.00		0.00
Gentamycin sulphate (0.3 mg/ ml)	23.24 ± 0.20	4.375	14.00 ± 0.02	8.75	16.02 ± 0.08	8.75
Mupirocin (0.1 mg/ml)							25.92 ± 0.23	2.315	25.9 ± 0.10	8.75

Table 2

Table showing mean diameter of zones of inhibition, Minimum inhibitory concentration (MIC) and Fractional Inhibitory Concentration Index (FICI) of plant extract combinations against Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus and Methicillin Resistant Staphylococcus aureus

	Pseudomonas aeruginosa			Klebsiella pneumoniae			Escherichia coli			Staphylococcus aureus			Methicillin Resistant Staphylococcus aureus
EXTRACT (100 mg/ ml)	DZI* (mm) ± SD	MIC (µg/well)	Σ FICI	DZI* (mm) ± SD	MIC (µg/well)	Σ FICI	DZI* (mm) ± SD	MIC (µg/well)	Σ FICI	DZI* (mm) ± SD	MIC (µg/well)	Σ FICI	DZI* (mm) ± SD	MIC (µg/well)	Σ FICI
Camelia MeOH/Senna MeOH	27.22 ± 0.41	156.25	1.0 ^**	14.93 ± 0.35	1250	0.375 ^*	14.02 ± 0.03	625	0.188 ^*	16.43 ± 0.10	1250	1.25^***	19.91 ± 0.31	1250	0.5625 ^*
Camelia MeOH/Aloe MeOH	22.07 ± 0.38	312.5	1.1^***	14.19	2500	1.0 ^**	14.17 ± 0.22	2500	0.75 ^**	14.72 ± 0.34	2500	2.25^***	16.24 ± 0.22	1250	1.125^***
Camelia MeOH/Toddalia MeOH	18.35 ± 0.22	625	2.1^***	13.72 ± 0.16	2500	1.0 ^**	10.48 ± 0.17	ND	ND	14.43 ± 0.12	2500	2.5^***	17.06 ± 0.41	1250	1.5^***
Camelia MeOH/Toddalia DCM	19.03 ± 0.46	625	2.5^***	13.51 ± 0.10	ND	ND	10.05 ± 0.01	ND	ND	13.49 ± 0.23	ND	ND	16.28 ± 0.32	1250	1.25^***
Senna DCM/Toddalia DCM	ND	ND	ND	12.77 ± 0.01	ND	ND	ND	ND	ND	14.33 ± 0.16	ND	ND	10.70 ± 0.06	ND	ND
Camelia H2O/Senna H2O	22.22 ± 0.15	156.25	0.5625 ^*	10.36 ± 0.10	ND	ND	10.15 ± 0.04	ND	ND	14.1 ± 0.02	10,000	5^****	13.96 ± 0.24	1250	0.625 ^**
DMSO	0.00			0.00			0.00			0.00			0.00
Gentamycin sulphate (0.3 mg/ml)	23.24 ± 0.20	4.375		14.00 ± 0.02	8.75		16.02 ± 0.08	8.75
Mupirocin (0.1 mg/ml)										25.92 ± 0.23	2.315		25.9 ± 0.10	2.315	ND

^*Synergy ^** Additive ^***Indifferent ^****Antagonism

The absolute values of the diameter of zones of inhibition (DZI) varied from 10.04 to 27.22 mm (Tables 1 and 2). The minimum inhibitory concentration range for the extract combinations was 10,000 µg/well – 156.25 µg/well and 10,000 µg/well – 1250 µg/well for single extracts (Tables 1 and 2). The ANOVA test indicated significant difference (P < 0.05) in bioactivity between these combinations. The fractional inhibitory concentration indices (FICI) showed that the interactions were synergistic (10.5%), additive (31.6%), indifferent (52.6%), and antagonistic (5.3%). The fractional inhibitory concentration indices (FICI) ranged from 0.5 to 2.5 for P. aeruginosa, 0.375 to 1.0 for K. pneumoniae, 0.188 to 0.75 for E. coli, 1.25 to 5 for S. aureus and 0.5625 to 0.625 for MRSA strains. The best synergistic interaction (FICI 0.188) appeared with Camelia methanol and Senna methanol combination against E. coli strain.

Discussion

The polar single extracts had higher activity than the non-polar single extracts. This is in agreement with the findings of a previous studies [36, 45‐49]. The methanol crude extracts showed more inhibition than the aqueous extracts (Table 1). This observation is similarly reported in previous studies [50‐52]. It is possible that the aqueous crude extracts may contain a lower concentration of antibacterial constituents and this may explain why large quantities of decoctions are taken over a relatively long period to achieve therapeutic success [53, 54].

It is evident that combining some plant extracts improved bioactivities over individual plant extracts. In this study, the polar compounds interacted more synergistically than the non-polar compounds. These findings are comparable to previous studies [7, 10, 45, 55, 56]. Combination drug therapies target multiple pathologic processes thus are capable of suppressing bacterial resistance mechanisms to remedy bacteria [8, 57].

The observed synergistic activity may be explained by the ability of compounds within the plants extracts to interact with one another to improve their solubility, enhance their bioavailability and subsequent antibacterial activity. Possible differences in modes of action of different compounds present in the combined extracts may also result in synergism [58‐60]. Pharmacodynamic synergy may have also occurred resulting in different agents regulating either the same or different target in various pathways [61]. The combinations that displayed these positive interactions can be considered as a potential strategy to combat bacterial resistance.

As previously reported elsewhere, non-polar extracts seem less potent than the polar extracts (Tables 1 and 2) [62, 55]. For combinations with non- polar constituents, higher doses but within safety levels can be explored in future [36, 55].

The lower activity in some combinations may be attributed to the respective compounds either neutralizing each other’s activity or forming inactive complexes when in combination [63, 64]. Combination of compounds with minor structural differences that may compete for the same molecular target could also result in antagonism [65]. For the combinations that displayed antagonistic activity, different combination ratios could be further explored [66, 67].

The observed variation in the antibacterial activities for specific plant extract combinations could be due to the differences in chemical composition and concentrations [44, 68]. Some constituents from the plants have reported antibacterial activity through various mechanisms. Ulopterol, a coumarin compound from T. asiatica has been shown to inhibit the growth of K. pneumoniae and E. coli [69]. An alkaloid (chelerythrine) isolated from T. asiatica exerts its antibacterial activity via destroying the cell wall and membrane [70].

Tannins present in C. sinensis are shown to react with proteins of the bacterial cell wall to form stable water-insoluble components [71]. Flavonoids bind with intracellular proteins as well as soluble proteins present in the bacterial cell walls. Steroids are shown to form complexes with membrane lipids thus resulting in leakage [21, 72, 73]. These compounds present in In C. sinensis, may have contributed to the observed antibacterial effect. The exhibited antibacterial activity of S. didymobotrya may be due to the presence of alkaloids that are known to interchelate with DNA of both Gram positive and negative bacteria and interfere with cell division [74].

Essential oils have been shown to disrupt the cell wall and lipid bilayer of gram-positive bacteria, resulting in the disarray of metabolic processes and cell lysis [75]. This may account for the antibacterial activity observed in non-polar extracts of T. asiatica and S. didymobotrya against S. aureus and MRSA.

In this study, the combination of extracts with a similar phytochemical profile displayed increased bioactivity as in the case of S. didymobotrya and C. sinensis. This may be due to increased concentrations of the similar antibacterial compounds thus resulting in higher potency.

Conclusion and recommendations

Plants remain a valuable resource while bioprospecting for novel antimicrobial drugs. The findings of this study support the use of multiple herbs to manage bacterial infections as an appreciable proportion of tested combinations exhibited synergistic and additive properties. To optimize the use of such combinations, there may be need to first standardize the herbal preparations in a bid to ensure efficacy and for safe delivery. Further studies on combination of isolated compounds responsible for the observed antibacterial activity may guide into realization of novel antibacterial agents.

Acknowledgements

The authors acknowledge Raphael Ingwela for technical assistance with the extraction. Hannington Mugo for technical assistance with the antibacterial assays and Patrick Mutiso Kyalo for identification of plant species.

Declarations

Ethical approval was obtained from the University of Nairobi-Kenyatta National Hospital Ethical Review Committee (P387/07/2020). It does not report on or involve the use of any animal or human data or tissue. Permissions to collect the respective medicinal plants were obtained. All methods were performed in accordance with the relevant guidelines and regulations.

Not applicable.

Competing interests

The authors declare no competing interests.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Aminov RI. A brief history of the antibiotic era: lessons learned and challenges for the future. Front Microbiol. 2010;8:1:134. https://doi.org/10.3389/fmicb.2010.00134.CrossRef

Silveira GP, Nome F, Gesser JC, Sá MM. Estratégias Utilizadas no Combate a Resistência Bacteriana. Química Nova. 2006;29(4):844–55.CrossRef

Guimarães DO, Momesso LS, Pupo MT. Antibióticos: importância terapêutica e perspectivas para a descoberta e desenvolvimento de novos agentes. Química Nova. 2010;33(3):667–79.CrossRef

Spengler G, Gajdács M, Donadu MG, Usai M, Marchetti M, Ferrari M, Mazzarello V, Zanetti S, Nagy F, Kovács R. Evaluation of the Antimicrobial and Antivirulent Potential of Essential Oils Isolated from Juniperus oxycedrus L. ssp. macrocarpa Aerial Parts. Microorganisms. 2022;10:758.

O’Neill J. Tackling Drug-Resistant Infections Globally: Final Report and Recommendations. 2016. (http://amrreview.org/sites/default/files/160525_Final%20paperwith%20cover.pdf). Accessed 23/11/2022.

Chattopadhyay D, Sarkar MC, Chatterjee T, et al. Recent advancements for the evaluation of anti-viral activities of natural products. N Biotechnol. 2009;25(5):347–68.PubMedPubMedCentralCrossRef

Ríos JL, Recio MC. Medicinal plants and antimicrobial activity. J Ethnopharmacol. 2005;100:1–2.

Tajkarimi M, Ibrahim S, Cliver D. Antimicrobial Herb and spice compounds in Food. Food Control. 2010;21:1199–218.CrossRef

Terrie YC, Monitoring Combination Drug T. 2010. Monitoring Combination Drug Therapy (https://www.pharmacytimes.com/view/rxfocuscombination-0110). Accessed on 10/01/2023

10.

Sun W, Sanderson PE, Zheng W. Drug combination therapy increases successful drug repositioning. Drug Discov Today. 2016;21(7):1189–95.PubMedPubMedCentralCrossRef

11.

Yarnell E. Synergy in herbal medicines. J Restor Med. 2015;4(1):60?73.

12.

Otieno JN, Hosea KM, Lyaruu HV, Mahunnah RL. Multi-plant or single-plant extracts, which is the most effective for local healing in Tanzania? Afr J traditional Complement Altern Med. 2008;5(2):165–72.

13.

Karole S, Shrivastava S, Thomas S, Soni B, Khan S, Dubey J, Dubey SP, Khan N, Jain DK. Polyherbal Formulation Concept for Synergic Action: a review. J Drug Discovery Ther. 2019;9(1–s):453–66.

14.

Muregi FW, Chhabra SC, Njagi EN, et al. In vitro antiplasmodial activity of some plants used in Kisii, Kenya against malaria and their chloroquine potentiation effects. J Ethnopharmacol. 2003;84(2–3):235–9.PubMedCrossRef

15.

Odongo EA, Mutai PC, Amugune BK, Mungai NN. A Systematic Review of Medicinal Plants of Kenya used in the Management of Bacterial Infections. Evid Based Complement Alternat Med. 2022:9089360.

16.

Zaidan MR, Noor Rain A, Badrul AR, Adlin A, Norazah A, Zakiah I. In vitro screening of five local medicinal plants for antibacterial activity using disc diffusion method. Trop Biomed. 2005;22(2):165–70.PubMed

17.

Aboulwafa MM, Youssef FS, Gad HA, Altyar AE, Al-Azizi MM, Ashour ML. A comprehensive insight on the health benefits and phytoconstituents of Camellia sinensis and recent approaches for its Quality Control. Antioxidants (Basel). 2019;8(10):455.CrossRef

18.

Naveed M, BiBi J, Kamboh AA, Suheryani I, Kakar I, Fazlani SA, FangFang X, Kalhoro SA, Yunjuan L, Kakar MU, El-Hack A, Noreldin ME, Zhixiang AE, LiXia S, C., XiaoHui Z. Pharmacological values and therapeutic properties of black tea (Camellia sinensis): a comprehensive overview. Biomed Pharmacother. 2018;100:521–31.CrossRef

19.

Saeed M, Naveed M, Arif M, Kakar MU, Manzoor R, Abd El-Hack ME, Alagawany M, Tiwari R, Khandia R, Munjal A, Karthik K, Dhama K, Iqbal H, Dadar M, Sun C. Green tea (Camellia sinensis) and l-theanine: Medicinal values and beneficial applications in humans-A comprehensive review. Biomed Pharmacother. 2017;95:1260–75.

20.

Bjora CS, Wabuyele E, Grace OM, Nordal I, Newton LE. The uses of kenyan aloes: an analysis of implications for names, distribution and conservation. J Ethnobiol Ethnomed. 2015;11:82.PubMedPubMedCentralCrossRef

21.

Mariita RM, Orodho JA, Okemo PO, Kirimuhuzya C, Otieno JN, Magadula JJ. Methanolic extracts of Aloe secundiflora Engl. Inhibits in vitro growth of tuberculosis and diarrhea-causing bacteria. Pharmacognosy Res. 2011;3(2):95–9.PubMedPubMedCentralCrossRef

22.

Alagaraj P, Muthukrishnan S. Toddalia asiatica L. - A Rich source of phytoconstituents with potential pharmacological actions, an appropriate plant for recent global Arena. Cardiovasc Hematol Agents Med Chem. 2020;18(2):104–10.

23.

Zeng Z, Tian R, Feng J, Yang NA, Yuan L. A systematic review on traditional medicine Toddalia asiatica (L.) Lam.: Chemistry and medicinal potential. Saudi Pharm J. 2021;29(8):781–98.PubMedPubMedCentralCrossRef

24.

Nyamwamu LB, Ngeiywa M, Mulaa M, Lelo EA, Ingonga J, Kimutai A. Phytochemical constituents of Senna didymobotrya Fresen irwin roots used as a traditional Medicinal plant in Kenya. Int J Educ Res. 2015;2:435–6.

25.

Ngule CM, Anthoney ST, Obey JK. Phytochemical and bioactivity evaluation of Senna didymobotrya Fresen Irwin used by the Nandi community in Kenya. Int J Bioassays. 2013;207:1037–43.

26.

Woranan N, Munjit R, Kannika S, Niramol S, Somchit D. Effects of drying and extraction methods on phenolic compounds and in vitro assays of Eclipta prostrata Linn leaf extracts. Sci Asia. 2019;45:127–37.CrossRef

27.

Sultana B, Anwar F, Ashraf M. Effect of extraction Solvent/Technique on the antioxidant activity of selected Medicinal Plant extracts. Molecules. 2009;14(6):2167–80.PubMedPubMedCentralCrossRef

28.

Atwaa ESH, Shahein MR, Radwan HA, Mohammed NS, Aloraini MA, Albezrah NKA, Alharbi MA, Sayed HH, Daoud MA, Elmahallawy EK. Antimicrobial activity of some plant extracts and their applications in Homemade Tomato paste and pasteurized cow milk as natural preservatives. Fermentation. 2022;8(9):428.CrossRef

29.

Owk AK, Lagudu MN. Litsea glutinosa (Lauraceae): evaluation of its Foliar Phytochemical constituents for antimicrobial activity. Not Sci Biol. 2018;10(1):21–5.CrossRef

30.

Basri DF, Sandra V. Synergistic Interaction of Methanol Extract from Canarium odontophyllum Miq. Leaf in Combination with Oxacillin against Methicillin-Resistant Staphylococcus aureus (MRSA) ATCC 33591. Int J Microbiol. 2016;2016:5249534.

31.

Omosa LK, Amugune B, Ndunda B, Milugo TK, Heydenreich M, Yenesew A, Midiwo JO. Antimicrobial flavonoids and diterpenoids from Dodonaea angustifolia. South Afr J Bot. 2014;91:58–62.CrossRef

32.

Sato M, Tanaka H, Yamaguchi R, Kato K, Etoh H. Synergistic effects of mupirocin and an isoflavanone isolated from Erythrina variegata on growth and recovery of methicillin-resistant Staphylococcus aureus. Int J Antimicrob Agents. 2004;24(3):241–6.PubMedCrossRef

33.

Chaturvedi P, Singh AK, Singh AK, Shukla S, Agarwal L. Prevalence of Mupirocin Resistant Staphylococcus aureus isolates among patients admitted to a Tertiary Care Hospital. North Am J Med Sci. 2014;6(8):403–7.CrossRef

34.

Clinical and Laboratory Standards Institute; CLSI. CLSI document M100-S23. Vol. 33. Wayne PA. Performance Standards for Antimicrobial Susceptibility Testing;Twenty-Third Informational Supplement; 2013.pp. 74–85.

35.

Munir MT, Pailhories H, Eveillard M, Irle M, Aviat F, Dubreil L, Federighi M, Belloncle C. Testing the antimicrobial characteristics of Wood materials: a review of methods. Antibiotics. 2020;9(5):225.PubMedPubMedCentralCrossRef

36.

Perera MMN, Dighe SN, Katavic PL, Collet TA. Antibacterial Potential of Extracts and Phytoconstituents Isolated from Syncarpia hillii Leaves In Vitro. Plants. 2022;11:283.

37.

Matu EN, Kirira PG, Kigondu EVM, Moindi E, Amugune B. Antimicrobial activity of organic total extracts of three Kenyan medicinal plants. Afr J Pharmacol Ther. 2012;1(1).

38.

Gonelimali FD, Lin J, Miao W, Xuan J, Charles F, Chen M, Hatab SR. Antimicrobial Properties and Mechanism of Action of Some Plant Extracts Against Food Pathogens and Spoilage Microorganisms. Front Microbiol. 2018;9:1639. https://doi.org/10.3389/fmicb.2018.01639. PMID: 30087662; PMCID: PMC6066648

39.

Singh BR. Evaluation of antibacterial activity of Salvia officinalis [L.] sage oil on veterinary clinical isolates of bacteria. Noto-are: Medicine. 2013. https://www.notoare.com/index.php/index/explorer/getPDF/15341289

40.

Boyan Bonev J, Hooper J, Parisot. Principles of assessing bacterial susceptibility to antibiotics using the agar diffusion method. J Antimicrob Chemother. 2008;61:1295–301. https://doi.org/10.1093/jac/dkn090.

41.

Afrasiabi S, Bahador A, Partoazar A. Combinatorial therapy of chitosan hydrogel-based zinc oxide nanocomposite attenuates the virulence of Streptococcus mutans. BMC Microbiol. 2021;21:62.PubMedPubMedCentralCrossRef

42.

Olajuyigbe OO, Coopoosamy RM. Influence of first-line antibiotics on the Antibacterial Activities of Acetone Stem Bark Extract of Acacia mearnsii De Wild. Against Drug Resistant Bacterial isolates. Evid Based Complement Alternat Med. 2014;2014:423751. https://doi.org/10.1155/2014/423751. Epub 2014 Jul 1. PMID: 25101132; PMCID: PMC4102002.CrossRefPubMedPubMedCentral

43.

Meletiadis J, Pournaras S, Roilides E, Walsh TJ. Defining fractional inhibitory concentration index cutoffs for additive interactions based on self-drug additive combinations, Monte Carlo simulation analysis, and in vitro-in vivo correlation data for antifungal drug combinations against aspergillus fumigatus. Antimicrob Agents Chemother. 2010;54(2):602–9.PubMedCrossRef

44.

Hemeg, et al. Antimicrobial effect of different herbal plant extracts against different microbial population. Saudi J Biol Sci. 2020;3221?7.

45.

Saraiva AM, Castro RHA, Cordeiro RP, et al. In vitro evaluation of antioxidant, antimicrobial and toxicity properties of extracts of Schinopsis brasiliensi engl. (Anacardiaceae. Afr J Pharm Pharmacol. 2011;5(14):1724–31.CrossRef

46.

Narod FB, Gurib-Fakim A, Subratty AH. Biological investigations into Antidesma madagascariense Lam. (Euphorbiaceae), Faujasiopsis flexuosa (Lam.) C. Jeffrey (Asteraceae), Toddalia asiatica (L.) Lam. and Vepris lanceolata (Lam.) G. Don (Rutaceae). J Cell Mol Biol. 2004;3:15?21.

47.

Asan-Ozusaglam M, Erzengin M, Darilmaz DO, Erkul SK, Teksen M, Karakoca K. Antimicrobial and antioxidant activity of various solvent extracts of Salsola stenoptera Wagenitz and Petrosimonia nigdeensis Aellen (Chenopodiaceae) plants. Chiang Mai J Sci. 2015;42(1):156–72.

48.

Kahlout E, Borsh K, Aksoy WA, Kichaoi A, Hindi EM, Ashgar EN. Evaluation of Antibacterial and Synergistic/Antagonistic effect of some Medicinal plants extracted by microwave and conventional methods. J Biosci Med. 2020;8:69–79. https://doi.org/10.4236/jbm.2020.89006.CrossRef

49.

Khanal, et al. Green Synthesis of Silver Nanoparticles from Root Extracts of Rubus ellipticus Sm. and Comparison of Antioxidant and Antibacterial Activity. J Nanomater. 2022. Article ID 1832587.

50.

Komape NPM, Bagla VP, Kabongo-Kayoka P, et al. Anti-mycobacteria potential and synergistic effects of combined crude extracts of selected medicinal plants used by Bapedi traditional healers to treat tuberculosis related symptoms in Limpopo Province, South Africa. BMC Complement Altern Med. 2017;17:128. https://doi.org/10.1186/s12906-016-1521-2.CrossRefPubMedPubMedCentral

51.

Jigna P, Chanda SV. In vitro Antimicrobial Activity and Phytochemical Analysis of some indian Medicinal plants. Turk J Biol. 2007;3:53–8.

52.

Yineger H, Kelbessa E, Bekele, Lulekal T. Plants used in traditional management of human ailments at Bale Mountains National Park, Southeastern Ethiopia. J Med plant Res. 2008;2(6):132–53.

53.

Kitonde C. Antimicrobial activity and phytochemical screening of Senna didymobotry used to treat bacterial and fungal infections in Kenya. Int J Educ Res. 2014;2(1).

54.

Burman S, Bhattacharya K, Mukherjee D, Chandra G. Antibacterial efficacy of leaf extracts of Combretum album pers. Against some pathogenic bacteria. BMC Complement Altern Med. 2018;18(1):213.PubMedPubMedCentralCrossRef

55.

Obuekwe IS, Okoyomo EP, Anka US. Effect of Plant Extract Combinations on some bacterial pathogens. J Appl Sci Environ Manag. 2020;24:627–32.

56.

Vaou N, Stavropoulou E, Voidarou C, Tsakris Z, Rozos G, Tsigalou C, Bezirtzoglou E. Interactions between Medical Plant-Derived Bioactive Compounds: Focus on Antimicrobial Combination Effects. Antibiotics. 2022;11:1014. https://doi.org/10.3390/antibiotics11081014

57.

Worthington RJ, Melander C. Combination approaches to combat multidrug-resistant bacteria. Trends Biotechnol. 2013;31(3):177–84.PubMedPubMedCentralCrossRef

58.

Parekh J, Jadeja D, Hanlin RL. Efficacy of aqueous and methanol extracts of some medicinal plants for potential antibacterial activity. Turk J Biol. 2005;29:203–10.

59.

Wagner H, Ulrich-Merzenich G. Synergy Research: approaching a New Generation of Phytopharmaceuticals. Phytomedicine. 2009;16:97–110.PubMedCrossRef

60.

Chouhan S, Sharma K, Guleria S. Antimicrobial activity of some essential Oils-Present Status and Future Perspectives. Medicines (Basel). 2017;4(3):58. https://doi.org/10.3390/medicines4030058. Published 2017 Aug 8.CrossRef

61.

Yang Y, Zhang Z, Li S, Ye X, Li X, He K. Synergy effects of herb extracts: pharmacokinetics and pharmacodynamic basis. Fitoterapia. 2014;92:133 – 47. https://doi.org/10.1016/j.fitote.2013.10.010. Epub 2013 Oct 28. PMID: 24177191

62.

Nazemi M, Moradi Y, Rezvani GF, Ahmaditaba MA, Gozari M, Salari Z. Antimicrobial activities of semi polar-nonpolar and polar secondary metabolites of sponge Dysidea pallescens from Hengam Island, Persian Gulf. Iran J Fisheries Sci. 2017;15(5):200–9.

63.

Idowu OA, Babalola AS, Olukunle J. Antagonistic effects of some commonly used herbs on the efficacy of artemisinin derivatives in the treatment of malaria in experimental mice. Bull Natl Res Cent. 2020;44:176. https://doi.org/10.1186/s42269-020-00429-2.CrossRef

64.

Masoumian M, Zandi M. Antimicrobial activity of some Medicinal Plant extracts against Multidrug resistant Bacteria. Zahedan J Res Med Sci. 2017;19(11):e10080.CrossRef

65.

Caesar LK, Cech NB. Synergy and antagonism in natural product extracts: when 1 + 1 does not equal 2. Nat Prod Rep. 2019;36(6):869–88. https://doi.org/10.1039/c9np00011a.CrossRefPubMedPubMedCentral

66.

Kharsany K, Viljoen A, Leonard C, van Vuuren SV. The New Buzz: investigating the antimicrobial interactions between Bioactive Compounds found in south african Propolis. J Ethnopharmacol. 2019;24:1732.

67.

Wang FY, Xu JK. Liu New geranyloxycoumarins from. J Asian Nat Prod Res. 2009;11:752–6.PubMedCrossRef

68.

Sukieum S, Sang-Aroon W, Yenjai C. Coumarins and alkaloids from the roots of Toddalia asiatica. Nat Prod Res. 2018;32:944–52.PubMedCrossRef

69.

Karunai RM, Balachandran C, Duraipandiyan V, Agastian P, Ignacimuthu S. Antimicrobial activity of Ulopterol isolated from Toddalia asiatica (L.) Lam.: a traditional medicinal plant. J Ethnopharmacol. 2012;140(1):161–5.CrossRef

70.

He N, Wang P, Wang P, et al. Antibacterial mechanism of chelerythrine isolated from root of Toddalia asiatica (Linn) Lam. BMC Complement Altern Med. 2018;18:261.PubMedPubMedCentralCrossRef

71.

Khan, et al., antimicrobial potential of aqueous extract of Camellia sinensis against representative microbes. Pak J Pharm Sci. March 2019;32(2):631–6.

72.

Dangoggo SM, Hassan LG, Sadig IS, Manga SB. Phytochemical analysis and antibacterial screening of leaves of diospyrosespiliformis and ziziphusspina-christi. J Chem Eng. 2012;1(1):31–7.

73.

Wang C, Han J, Pu Y, Wang X. Tea (Camellia sinensis): a review of nutritional composition, potential applications, and Omics Research. Appl Sci. 2022;12(12):5874.CrossRef

74.

Bukar AM, Kyari MZ, Gwaski PA, Gudusu M, Kuburi FS, Abadam YI. Evaluation of phytochemical and potential antibacterial activity of ziziphusspina-christi against some medically important pathogenic bacteria. J Pharmacogen Phytochem. 2015;3(5):98–101.

75.

Spengler G, Gajdács M, Donadu MG, Usai M, Marchetti M, Ferrari M, Mazzarello V, Zanetti S, Nagy F, Kovács R. Evaluation of the antimicrobial and antivirulent potential of essential oils isolated from Juniperus oxycedrus L. ssp. macrocarpa Aer Parts. Microorganisms. 2022;10:758. https://doi.org/10.3390/microorganisms10040758.CrossRef

Titel: Evaluation of the antibacterial activity of selected Kenyan medicinal plant extract combinations against clinically important bacteria
verfasst von: Elizabeth A Odongo
Peggoty C Mutai
Beatrice K Amugune
Nelly N Mungai
Mary O Akinyi
Julia Kimondo
Publikationsdatum: 01.12.2023
Verlag: BioMed Central
Erschienen in: BMC Complementary Medicine and Therapies / Ausgabe 1/2023
Elektronische ISSN: 2662-7671
DOI: https://doi.org/10.1186/s12906-023-03939-4

Springer Medizin

Abstract

Background

Methods

Results

Conclusion

Publisher’s Note

Background

Methods

Collection of plant material

Extraction procedures

Hot aqueous extraction

Organic solvent extraction

Collection of bacterial cultures

Sterilization and equipment

Preparation of stock solutions

Antimicrobial susceptibility testing

Minimum inhibitory concentration (MIC) determination

Determination of fractional inhibitory concentration index

Results

Discussion

Conclusion and recommendations

Acknowledgements

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Publisher’s Note

Weitere Artikel der Ausgabe 1/2023

A real-world study and network pharmacology analysis of EGFR-TKIs combined with ZLJT to delay drug resistance in advanced lung adenocarcinoma

Tai Chi for the prophylaxis of episodic migraine: protocol of a non-inferiority randomized controlled trial with mechanism exploration

Isha yoga practices, vegan diet, and participation in Samyama meditation retreat: impact on the gut microbiome & metabolome – a non-randomized trial

The effect of bovine milk lactoferrin-loaded exosomes (exoLF) on human MDA-MB-231 breast cancer cell line

Indigenous medicinal plants used in folk medicine for malaria treatment in Kwara State, Nigeria: an ethnobotanical study

What are the ways in which social media is used in the context of complementary and alternative medicine in the health and medical scholarly literature? a scoping review