전통 의학에 사용되는 케냐 토착 식물 5종의 항산화 및 항균 특성 탐구 2025 네이처

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Exploring the antioxidant and antimicrobial properties of five indigenous Kenyan plants used in traditional medicine

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Exploring the antioxidant and antimicrobial properties of five indigenous Kenyan plants used in traditional medicine

• Virginia Gichuru, 
• Irene Sbrocca, 
• Michela Molinari, 
• Teodora Chiara Tonto, 
• Vittoria Locato, 
• Sara Cimini & 
• Laura De Gara 

Scientific Reports volume 15, Article number: 1459 (2025) Cite this article

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Abstract

Defined by the World Health Organization (WHO) as indigenous knowledge and practices used for maintaining health and treating illnesses, traditional medicine (TM) represents a rich reservoir of ancient healing practices rooted in cultural traditions and accumulated wisdom over centuries. Five indigenous Kenyan plant species traditionally used in African TM, named Afzelia quanzensis, Azadirachta indica, Gigasiphon macrosiphon, Grewia bicolor, and Lannea schweinfurthii, represent a valuable resource in healing practices, yet their chemical composition and bioactivity remain understudied. To depict a primary bio-chemical characterization of these plants, their antioxidant and antimicrobial features have been eval‎uated by the use of methods validated in this context. G. bicolor, and G. macrosiphon were found to have great potential as sources of bioactive metabolites, such as chlorophyll a (1456.29 µg/ g DW; 1104.33 µg/ g DW), chlorophyll b (712.48 µg/ g DW; 443.31 µg/ g DW), and carotenoids (369.71 µg/ g DW; 300 µg/ g DW) as well as phenols (31.78 mg GAE/g DW; 27.54 GAE/g DW), and exhibiting high antioxidant activity, according to TEAC, DPPH and FRAP assays. Additionally, L. schweinfurthii and G. macrosiphon demonstrated antimicrobial activity against the Gram-negative bacteria E. coli, as well as against Gram-positive ones, S. aureus and B. subtilis.

초록

세계보건기구(WHO)가 건강 유지 및 질병 치료를 위해 사용되는 토착 지식과 관행으로 정의한 전통의학(TM)은 수세기에 걸쳐 축적된 문화적 전통과 지혜에 뿌리를 둔 고대 치유 관행의 풍부한 보고입니다.

아프리카 전통의학에서 전통적으로 사용되는

케냐 토착 식물 5종인

Afzelia quanzensis, Azadirachta indica, Gigasiphon macrosiphon, Grewia bicolor, Lannea schweinfurthii는

치유 관행에서 귀중한 자원을 이루지만,

이들의 화학적 구성과 생물학적 활성은 아직 충분히 연구되지 않았다.

이 식물들의 주요 생화학적 특성을 파악하기 위해, 이 분야에서 검증된 방법을 사용하여 항산화 및 항균 특성을 평가하였다. G. bicolor 및 G. macrosiphon은 엽록소 a(1456.29 µg/g 건조중량; 1104.33 µg/g 건조중량), 엽록소 b(712.48 µg/g 건조중량; 443.31 µg/g DW), 카로티노이드(369.71 µg/g DW; 300 µg/g DW) 및 페놀(31.78 mg GAE/g DW; 27.54 GAE/g DW)과 같은 생리활성 대사산물 공급원으로 큰 잠재력을 지니며, TEAC, DPPH 및 FRAP 분석법에 따른 높은 항산화 활성을 나타냄이 확인되었다. 또한, L. schweinfurthii 및 G. macrosiphon은 그람 음성균인 E. coli뿐만 아니라 그람 양성균인 S. aureus 및 B. subtilis에 대해서도 항균 활성을 나타냈다.

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Introduction

Traditional medicine (TM), also referred to as complementary and alternative medicine, stands as one of the oldest healthcare systems1. Defined by the World Health Organization (WHO) as “the sum total of the knowledge, skills, and practices based on the theories, beliefs, and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health, as well as in the prevention, diagnosis, improvement, or treatment of physical and mental illnesses”, TM has been, since ancient times, fundamental to human survival and well-being2. According to the WHO, up to 80% of the population in some African countries relies on TM for primary health care coexisting alongside other forms of modern healthcare2,3,4. This high utilization rate is due to several factors such as ready accessibility, lower costs compared to modern drugs and therapies and high affordability. On the other hand, modern medicine has capitalized on the resources provided by nature and from the knowledge gained from TM regarding the utilization of medicinal plants, herbs, roots, and bark to treat diseases. The interest in alternative medicine is growing among Western populations, driven by factors such as concerns about the side effects of pharmaceutical drugs, and a desire for more natural and personalized approaches to healthcare5. Therefore, an effective integration of TM with modern treatments has been one of the strategic objectives of the WHO Traditional Medicine Strategy 2014–20236. In this context, the lack of a comprehensive understanding of the chemical composition, biochemical and biological properties of the plants traditionally used for medical purposes may pose a challenge in their integration into modern healthcare systems normally based on scientific evidences.

In this context, Grewia bicolor, Afzelia quanzensis, Gigasiphon macrosiphon, Azadirachta indica, and Lannea schweinfurthii are African species currently used in many rural communities mainly for medical purposes, even if an in-depth characterization of their phytochemical and biological properties is still lacking. In particular, G. bicolor, commonly known as white raisin or false brandy bush, belongs to the Malvaceae family and is currently widespread across the African continent. G. bicolor is a small tree, typically reaching up to 5 m in height, though it may grow to 10 m under favorable conditions. The leaves are elliptical with serrated margins, the flowers are small, and the fruits are rounded edible drupes. G. bicolor is traditionally employed in the ethnomedicine as an abortifacient. Infusions of roots and leaves are also used as vermifuge, diuretic, and laxative, intestinal inflammation, and to treat syphilis. Additionally, in East Africa, the bark of G. bicolor is used for its therapeutic properties in treating intestinal inflammation, worms, fever, syphilis and as antidote for common poisons, while in Kenya, the leaves are used for the management of epilepsy and for the maintenance of dental hygiene7,8,9,10,11. A. quanzensis, commonly known as pod mahogany, belongs to the Fabaceae family. It originates from sub-Saharan Africa and is nowadays found in several countries such as Angola, Zambia, Tanzania, Mozambique, Malawi, and Zimbabwe. A. quanzensis is a deciduous tree that can grow to heights of 20–30 m. Its leaves are compound, with numerous elliptical leaflets. The tree produces large white or pink flowers, and its fruits are pods containing large, hard seeds. A. quanzensis is traditionally used for the treatment of a wide range of conditions including pneumonia, malaria, gonorrhoea, chest pains, kidney problems, bilharzia, eye problems and snakebites9,11,12. Another member of the Fabaceae family, G. macrosiphon, commonly known as holy grail tree, is native to regions from Kenya to Tanzania, where it is also currently distributed. G. macrosiphon is a large, rare tree distinguished by its large, light-colored, tubular flowers. The leaves are pinnate, and the branches have smooth, light-colored bark. This is the most poorly studied species taken into account. L. schweinfurthii, commonly known as False Marula, from the Anacardiaceae family, is native to areas spanning from Tropical Africa to Namibia and is currently widespread across these regions. L. schweinfurthii is a deciduous tree that can reach about 15–20 m in height. The leaves are compound with elliptical leaflets, and it produces small flowers followed by small, edible red fruits. This plant is utilized in TM in case of respiratory disorders, stomachache, headache, blood system disorders, infections, and gastro-intestinal disorders9,13. Moving to the Meliaceae family, A. indica, also known as neem, is a spontaneous plant native to the tropical regions of West and Central Africa as well as South Asia, with its current distribution including Nigeria, Cameroon, Gabon, the Republic of Congo, and Madagascar. This plant is an evergreen tree that can reach heights of 20–30 m. Its leaves are compound, composed of small, bright green ovate segments. The flowers are white and clustered, and the fruits are yellow-green oval drupes. A. indica is the most studied species taken into account in this study, with significantly more information available regarding its biochemical and antimicrobial properties. In particular, it has been reported to exhibit various therapeutic proprieties, including antidiabetic, immunomodulatory, diuretic, antiseptic, anti-inflammatory, antipyretic, antiulcer, antiarthritic, antimalarial, spermicidal, antifungal, antibacterial, hypoglycemic, antioxidant and anticarcinogenic ones14,15,16,17,18,19,20,21.

In this setting, the aim of this study is to fill the gap between the traditional use of the selected African plants and the knowledge of their chemical composition and biological activity. To reach this goal, bioactive metabolite accumulation, antioxidant properties and antimicrobial activity against specific bacteria commonly associated with human infections were selected as the more promising attributes describing their biological activities.

Materials and methods

Plant material

Afzelia quanzensis, Gigasiphon macrosiphon, Grewia bicolor, Lannea schweinfurthii, and Azadirachta indica plants were grown at the Pwani University Botanical Garden (Kilifi, Kenya). The botanical specimens were identified at the Pwani University Botanical Garden (Kilifi, Kenya). The voucher number is not available. Twenty completely developed leaves were collected from at least four plants. The leaves were washed using distilled water, and subsequently dried at ambient temperature conditions for about fourteen days. The dried leaves were weighed and subsequently pulverized. The plant material was stored at room temperature, shielded from light, and analyzed within one month of collection. In these experiments, at least four biological replicates were used, each with three technical replicates. The Pwani University Botanical Garden granted the author permission to collect the plant material required for this study.

Determination of pigment levels

The pigment levels were determined as previously described by Tonto et al.22. Briefly 0.2 g of leaf was powdered using liquid nitrogen and 80% acetone (Merck KGaA, Darmstadt, Germany) was added in a 1:5 weight to volume (w: v) ratio. The mixture was homogenized and then centrifuged at maximum speed, 12,000 xg, for 10 min at 4° C. The supernatant containing the liposoluble pigments was recovered. The absorbance was measured by spectrophotometer UV-1800 Shimidazu, at the peak wavelengths: 663 (chlorophyll a), 646 (chlorophyll b), 470 (carotenoids) nm. The results were expressed as µg of pigments per g of dry weight (µg / g DW).

Ascorbate and glutathione quantification

Total ascorbate, given by the sum of ascorbate (ASC) and dehydroascorbate (DHA), and total glutathione given by the sum of oxidized glutathione (GSSG) and reduced glutathione (GSH) levels were measured as described by Cimini et al.23. Briefly, 0.3 g of leaf was powdered using liquid nitrogen and homogenized with 6 volumes of cold 5% metaphosphoric acid at 4 °C. The homogenate was centrifuged at maximum speed, 12,000 xg, for 15 min at 4 °C and the supernatant was collected.

Total ascorbate was determined after reduction of DHA to ASC with DTT (Merck KGaA, Darmstadt, Germany). The reaction mixture for total ascorbate pool contained a 0.1 ml aliquot of the supernatant, 0.25 ml of 150 mM phosphate buffer (pH 7.4) containing 5 mM EDTA (Merck KGaA, Darmstadt, Germany), and 0.05 ml of 10 mM DTT. After incubation for 10 min at room temperature, 0.05 ml of 0.5% N-ethylmaleimide (Merck KGaA, Darmstadt, Germany) was added to remove excess DTT. Color was developed in the reaction mixture after addition of the following reagents: 0.2 ml of 10% trichloroacetic acid (Merck KGaA, Darmstadt, Germany), 0.2 ml of 44% ortho-phosphoric acid (Merck KGaA, Darmstadt, Germany), 0.2 ml of 4% α,α’-dipyridyl (Merck KGaA, Darmstadt, Germany) in 70% ethanol (EtOH) (Merck KGaA, Darmstadt, Germany) and 0.3% (w/v) FeCI3 (Merck KGaA, Darmstadt, Germany). After vortexing, the mixture was incubated at 40 °C for 40 min and the absorbance (A525) was read. A standard curve was developed based on ASC (Merck KGaA, Darmstadt, Germany) in the range of 0–10 mg/ml (y = 0.0051x + 0.0433, R2 = 0.9992). Results were converted to nmol of ascorbic acid per g of dry weight (nmol / g DW).

The glutathione pool was assayed according to Zhang et al.24 utilizing 0.4 ml aliquots of supernatant neutralized with 0.6 ml of 0.5 M phosphate buffer (pH 7.5). For total glutathione pool (GSH plus GSSG) assay 20 µl of H20 were added to the aliquots. Tubes were mixed until an emulsion was formed. Glutathione content was measured in 1 mI of reaction mixture containing 0.2 mM NADPH, 100 mM phosphate buffer (pH 7.5), 5 mM EDTA, 0.6 mM 5,5’- dithiobis(2-nitrobenzoic acid), and 0.1 ml of sample obtained as described above. The reaction was started by adding 3 units of glutathione reductase and was monitored by measuring the change in absorbance at 412 nm for 1 min. Results were expressed as nmol glutathione / g DW.

Total phenolic content determination

Total phenolic content was eval‎uated using the Folin–Ciocalteau method22,25. Briefly 0.2 g of leaf was powdered using liquid nitrogen and methanole (Merck KGaA, Darmstadt, Germany) was added in a 1:5 weight to volume (w: v) ratio. The mixture was homogenized and then centrifuged at maximum speed, 12,000 xg, for 10 min at 4° C. The obtained supernatant, consisting in the methanolic fraction of the leaf sample, was recovered and 20 µL were mixed with 1.58 mL of 50% EtOH. Then, 100 µL of Folin–Ciocalteau reagent (Merck KGaA, Darmstadt, Germany) was added to both samples and standards. After 8 min, 300 µL of Na2CO3 1.886 M were added. Samples were incubated for 2 h in the dark and then centrifuged at room temperature for 5 min. The absorbance was measured at 765 nm. A standard curve was developed based on gallic acid in the range of 0-1.5 mg/ml (y = 0.0007x − 0.0135, R2 = 0.9983). Concentration of total phenolic compounds was expressed as mg of gallic acid equivalents (GAE) / g DW.

Antioxidant capacity assays

For all antioxidant assays, the same sample preparation was used: 0.3 g of fresh leaf tissue was powdered in liquid nitrogen and homogenized in 100% EtOH with a w: v ratio of 1:10. The mixture was then centrifuged at maximum speed for 10 min at 4 °C, and the resulting supernatant was collected and diluted when needed.

DPPH (1,1-Diphenyl-2-Picrylhydrazyl) radical scavenging assay was analyzed as reported by Padmanabhan and Jangle26 with some modifications. 20 µL of samples and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) standards were incubated with 180 µL of 0.1 mM DPPH (Merck KGaA, Darmstadt, Germany) for 20 min in the dark. Absorbance was measured at time 0 and after the incubation using a microplate reader (TECAN Infinite M200PRO) at 518 nm. A standard curve was developed based on Trolox in the range of 25–250 µM (y = 0.001x − 0.0141, R2 = 0.9969). Results were expressed as µmol Trolox equivalent (TE) / g DW.

FRAP (ferric-reducing antioxidant power) assay was performed based on Fu et al.27. Briefly, 10 µL of the supernatant were incubated with 190 µL of FRAP solution (acetate buffer: TPTZ: Fe 10:1:1, Merck KGaA, Darmstadt, Germany). The absorbance was measured at 593 nm after 6 min of incubation. A standard curve was developed based on Trolox in the range of 25–400 µM (y = 0.001x − 0.0031, R2 = 0.9956). Results were expressed as µmol TE / g DW.

TEAC (Trolox equivalent antioxidant capacity) assay was eval‎uated as previously described by Kupe et al.28. Briefly, 20 µL of samples and Trolox standards were incubated with 180 µL of ABTS+ solution (7 mM ABTS, 2.5 mM potassium persulfate, Merck KGaA, Darmstadt, Germany) for 20 min in the dark. Absorbance was measured at time 0 and after the incubation at 734 nm. A standard curve was developed based on Trolox in the range of 50–400 µM (y = 0.001x − 0.0052, R2 = 0.9955). Results were expressed as µmol TE / g DW.

Agar well diffusion assay

The agar well diffusion assay was performed as described in29,30. The Muller-Hinton (MH) agar plate surface, poured to a uniform depth of approximately 4 mm, was inoculated by spreading 100 µl of Bacillus subtilis, Escherichia coli and Staphylococcus aureus cell suspension (cell density 1.5 × 10^8 CFU/ml). The surface was then dried under the sterile flow bench for 15 min. The wells in the agar plate were made using a sterile cork borer (4–8 mm in diameter). 100 mg of the plant material was homogenated with 1 ml of Dimethyl sulfoxide (DMSO) and centrifuged at 12,000 xg. The supernatant was collected, and 50 µl was added to each well and incubated for 24 h at 37 °C. The diameters of growth inhibition zones around the wells were measured. In these assay, 5% DMSO was used as negative control. Different grades of growth inhibition were indicated as follow:

Diameter of the inhibition zone (mm)Grade of growth inhibition (–/+)

0–5	–
6–10	+
11–15	++
16–20	+++
21–25	++++

No or poor inhibition was indicated as – when a null or small diameter of inhibition zone (0–5 mm) was registered; whereas with the increase of inhibition zone diameter (from 6 to 25 mm), the grade of inhibition progressively increases, and it is indicated with a growing number of +. Figure S1 represented the results obtained from the agar well diffusion (Fig. S1).

Statistical analysis

All statistical analyses were performed with GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA). One-way ANOVA followed by Tukey test correction was performed. p < 0.05 was set as the significance cut-off. All values were presented as means ± SD. Each experiment was performed with n > 4 biological replicates, with each biological replicate representing independent extractions from separate plants. Technical replicates > 3 were also included to ensure measurement consistency.

Results

Leaf phytochemical composition of Kenya plant species

While the therapeutic efficacy of medicinal plants is generally associated with the accumulation of secondary metabolites, such as polyphenols, and vitamins, the relevance of photosynthetic pigments, particularly chlorophyll a and b, is also emerging for their biological implications31,32.

In the leaves of higher plants, chlorophylls a and b are the most abundant pigments in the light-harvesting antenna system, where they play a central role in absorbing light and transmitting energy to specific molecules of chlorophyll a able to transform solar energy in chemical energy33.The concentration of chlorophyll a was found to significantly vary between the species under investigation. Specifically, G. bicolor exhibited the highest value of chlorophyll a and chlorophyll b among the five analyzed species (1456.29 µg/g DW and 712.48 µg/g DW, respectively). A. quanzensis had the lowest chlorophyll a and chlorophyll b contents with average values of 646.23 µg/g DW and 403.83 µg/g DW, respectively (Fig. 1A and B). G. macrosiphon and A. indica displayed intermediate concentrations of chlorophyll a, with statistically comparable values standing at 1104.33 and 1055.88 µg/g DW, respectively (Fig. 1A). Intermediate level of chlorophyll b was also observed for A. indica, which has a chlorophyll b content of 573.5 µg/g DW, which is 22% lower than that of G. bicolor and by L. schweinfurthii, whose chlorophyll b content was slightly but significantly higher than those observed in A. quanzensis and G. macrosiphon (Fig. 1B).

Besides chlorophylls, carotenoids represent another group of pigments abundant in the leaves of higher plants, known for their well-established biological activity and notable implications for human health. Among the plant species under investigation, G. bicolor stood out for exhibiting the highest content of carotenoids in its leaves, with an average value of 369.71 µg/g DW. G. macrosiphon and A. indica demonstrated a comparable concentration of carotenoids that stand out at 300 and 290 µg/g DW, which are between 15% and 16% lower than that of G. bicolor. On the other hand, A. quanzensis and L. schweinfurthii showed notably inferior levels of these compounds in their leaves, with an average value of 203.76 µg/g DW and 245.82 µg/g DW, respectively (Fig. 1C).

Fig. 1

Photosynthetic pigments in the leaves of A. quanzensis, A. indica, G. macrosiphon, G. bicolor and L. schweinfurthii. Content of chlorophyll a (A), chlorophyll b (B), and carotenoids (C). Values are means ± SD. Different lowercase letters indicate a statistical difference (p < 0.05) based on one-way ANOVA followed by Tukey test correction.

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Ascorbate and glutathione are the main soluble antioxidants present in plant leaves, being mainly involved in the protection of photosynthetic machinery34,35,36. A. indica and G. bicolor exhibited the highest levels of total ascorbate with an average content of 390 and 380 nmol/g DW, respectively (Fig. 2A). Total ascorbate content of G. macrosiphon (220 nmol/g DW) was approximately 40% lower than that of A. indica. On the other hand, A. quanzensis and L. schweinfurthii showed significantly lower total ascorbate concentration compared to the other three species analyzed. In particular, A. quanzensis had a total ascorbate content of 29.9 nmol/g DW, while L. schweinfurthii contained 109.9 nmol/g DW (Fig. 2A).

Another antioxidant molecule generally accumulated at high concentrations in leaves and with significant implications for human health, is glutathione. A. indica had a significantly higher glutathione content than the other analyzed species, with a concentration of 258 nmol/g DW. G. bicolor and L. schweinfurthii showed a total glutathione amount that was slightly lower, but statistically comparable, to that of A. indica. G. macrosiphon displayed the lowest amount of glutathione, with a concentration of 107 nmol/g DW (Fig. 2B). Ascorbate and glutathione, were both mainly present in their oxidized form as consequence of the leaf essication process, normally used to store these plants. Indeed, the redox state of both metabolites, measured as ratio between the reduced form and the total pool, ranged from 0.1 to 0.3.

Fig. 2

Ascorbate Pool (ASC + DHA) (A) and Glutathione Pool (GSH + GSSG) (B) content in A. quanzensis, A. indica, G. macrosiphon, G. bicolor and L. schweinfurthii. Values are means ± SD. Different lowercase letters indicate a statistical difference (p < 0.05) based on one-way ANOVA followed by Tukey test correction.

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Phenols are a group of antioxidant molecules known for their beneficial properties. Indeed, the therapeutic effects and overall health benefits of many plants in TM are frequently attributed to their high levels of phenolic compounds37. In the analyzed plants, G. bicolor and G. macrosiphon showed the highest concentration of total phenolic compounds, with an amount standing at 31.78 and 27.54 mg GAE/g DW, respectively (Fig. 3). The other three analyzed species were characterized by a significantly lower total phenolic content. Specifically, the total phenolic content in A. indica and L. schweinfurthii was more than four times lower than the total phenolic content found in G. bicolor (7.38 and 7.48 mg GAE/g DW, respectively). Finally, A. quanzensis exhibited the lowest total phenolic content at a concentration of 1.46 mg GAE/g DW, which was more than twenty times lower than that of G. bicolor (Fig. 3).

Fig. 3

Total phenolic content, in A. quanzensis, A. indica, G. macrosiphon, G. bicolor and L. schweinfurthii. Values are means ± SD. Different lowercase letters indicate a statistical difference (p < 0.05) based on one-way ANOVA followed by Tukey test correction.

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Antioxidant and antimicrobial activities of Kenya plant species

The presence of photosynthetic pigments, ascorbate, glutathione, and phenolic compounds plays a crucial role in shaping the antioxidant capacity of a specific plant and, consequently, of its extract, thereby contributing to delineating its beneficial properties. To quantify the antioxidant power of the extracts obtained from the considered plants, determined by molecules that can neutralize free radicals, three different methods were employed, TEAC, FRAP and DPPH. The use of multiple methods is a gold standard experimental approach to include the contributions of different kinds of antioxidants in the total antioxidant power of a complex matrix38,39.

The different methods used to quantify the antioxidant power showed a consistent trend among the considered plants. Specifically, G. macrosiphon and G. bicolor exhibited the highest antioxidant capacity in all the different assays (Fig. 4A-C). A. indica and L. schweinfurthii showed statistically comparable results across all three assays, with intermediate levels of antioxidant activity (Fig. 4A-C). Finally, the plant species displaying the lowest antioxidant activity in all the assays was A. quanzensis (Fig. 4A-C).

Fig. 4

Antioxidant activity in A. quanzensis, A. indica, G. macrosiphon, G. bicolor and L. schweinfurthii, measured by DPPH (A), FRAP (B) and TEAC (C) assays. Values are means ± SD. Different lowercase letters indicate a statistical difference (p < 0.05) based on one-way ANOVA followed by Tukey test correction.

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Many plants used in TM have beneficial effects against various types of infections40. To eval‎uate the potential benefits of the selected plants against possible bacterial infections, their antimicrobial activity was measured. In detail, the agar well diffusion assay was performed against a Gram-negative bacterium, Escherichia coli, and two Gram-positive bacteria, Staphylococcus aureus, and Bacillus subtilis.

The agar well diffusion assay against E. coli indicated that only two out of five species, G. macrosiphon and L. schweinfurthii, were effective against this bacterium (Table 1). Conversely, the agar well diffusion assay conducted on S. aureus revealed significant efficacy of A. indica, G. macrosiphon and L. schweinfurthii extracts against S. aureus growth. Specifically, A. indica and G. macrosiphon showed the highest level of inhibition (Table 2). On the contrary, S. aureus demonstrated no sensitivity to extracts obtained from (A) quanzensis and G. bicolor (Table 2). Finally, the agar well diffusion assay against (B) subtilis revealed that (A) indica exhibited the largest zone of inhibition, indicating high sensitivity of (B) subtilis to the plant extract obtained from this species (Table 3). B. subtilis also showed an intermediate sensitivity to G. macrosiphon and to L. schweinfurthii extracts, showing an inhibition zone of 19 and 13 mm, respectively (Table 3). Again, no growth inhibition was observed with G. bicolor and A. quanzensis extracts (Table 3).

Table 1 Inhibitory effect of plant extracts onE. coli growth. The inhibitory effect of plant extracts on E. Coli growth was performed using a sample concentration of 100 mg/ml.

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Table 2 Inhibitory effect of plant extracts onS. aureus growth. The inhibitory effect of plant extracts on S. Aureus growth was performed using a sample concentration of 100 mg/ml.

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Table 3 Inhibitory effect of plant extracts on B. subtilis growth. The inhibitory effect of plant extracts on B. subtilis growth was performed using a sample concentration of 100 mg/ml.

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Discussion

This study aimed to investigate the biochemical and antimicrobial properties of five indigenous Kenyan plant species commonly utilized in African TM. Despite their longstanding use in traditional healing practices, the chemical composition of these plants and their bioactivity have remained largely understudied. Only the chemical composition of A. indica is largely documented as well as various therapeutic properties attribuited to this species14,15,16,17,18,19,20,21. Therefore, the inclusion of A. indica in this study has been considered in order to have a standard reference for the other understudied species. Indeed, the levels of the analysed phytochemicals in A. indica leaf extract was comparable to those reported in previous studies, also considering the sample variability in terms of geographical origin, developmental stage and harvesting, storing and extraction procedures41,42. Through the determination of bioactive molecule levels in the leaves of the selected African plants, A. indica, G. macrosiphon, and G. bicolor have emerged as highly interesting species, capable of accumulating high levels of ascorbate, chlorophylls a and b, carotenoids, and phenolic compounds within their leaves (Fig. 1A-C). All these molecules have been gathering attention for their potential beneficial effects on human health. In recent years, chlorophylls have been described for their antioxidant, anti-inflammatory, and potentially anticancer properties43,44. Recent studies have shown that native chlorophylls, ingested through the consumption of chlorophyll-rich foods, such as green leafy vegetables, undergo significant transformations during digestion, and a considerable amount of these chlorophyll derivatives is taken up by human intestinal cells under in vitro culture conditions45,46. Notably, both native chlorophylls and chlorophylls derivatives have been reported to form molecular complexes with aromatic carcinogens (aflatoxins, polycyclic aromatic hydrocarbons, heterocyclic amines), thus reducing the bioavailability and uptake of carcinogens, and inhibiting carcinogenesis43,47. In rat treated with aflatoxins B1, chlorophylls and chlorophyllin demonstrated protective effects against the occurrence of carcinogenesis in the liver and colon, possibly through the formation of complexes, thereby reducing aflatoxins B1 bioavailability43,48. Moreover, chlorophylls in the gastrointestinal tract are known to provide anti-inflammatory actions43,49. Indeed, chlorophyll a and its degradation product pheophytin a are reported to inhibit TNF-α gene expression‎, thereby exhibiting anti-inflammatory activity50. The anti-inflammatory effect of chlorophylls is supported by their capability to scavenge free radicals. Indeed, chlorophylls’ antioxidant properties are based on their scavenging effects towards harmful free radicals known to contribute to oxidative stress-induced cellular damage associated with several chronic diseases involving inflammation51,52,53,54. In vitro studies demonstrated that derivatives of chlorophylls a and b have a high antioxidant capacity55,56. Moreover, in vivo experiments revealed that pretreatment with chlorophyll b reduced the oxidative stress and lipid peroxidation induced by cisplatin in in mice57. Similar to chlorophylls, numerous beneficial effects of carotenoids on human health have been documented, mainly attributed to their antioxidant and anti-inflammatory properties58,59. They are major precursors of vitamin A, essential for various physiological processes, such as vision and growth. Moreover, specific carotenoids, such as β-carotene, lutein, and zeaxanthin, exhibit additional mechanisms of action, including potential cancer mitigation and prevention of cardiovascular diseases60,61,62,63,64. Here, the high levels of chlorophylls and carotenoids present in the leaves of G. bicolor are coherent with the beneficial and therapeutic effects traditionally attributed to this plant in African TM. The chlorophyll and carotenoid contents in G. bicolor are even higher than that in A. indica, a plant known for its numerous beneficial properties65, whose photosynthetic pigments’ content was reported as higher than that observed in other medicinal plants leaves extracts66. To the best of our knowledge, this work reports for the first time the chlorophyll and carotenoid content in G. macrosiphon leaves. The levels of these bioactive molecules in this species are comparable to those found in A. indica, suggesting the potential use of G. macrosiphon as medicinal plant.

Regarding the total ascorbate pool (Fig. 2A), G. bicolor exhibits the highest content among the analyzed plants, followed by A. indica and G. macrosiphon. To the best of our knowledge, this study provides the first data related to the concentration of ascorbate in the leaves of these plant species. It is important to underline that the traditional use of these plants is often based on the use of dried and stored tissues, causing ascorbate oxidation. However, although a consistent part of the ascorbate pool is present in its oxidized form in dried leaves, its biological active form can be recycled in human body by the endogenous reducing enzymes that are ubiquitous in all the organisms using ascorbate as vitamin. In addition to its role as Vitamin C, ascorbate works in synergy with other bioactive molecules such as tocopherols, acting in several redox reactions as both reducing and oxidizing agents67.

The accumulation of glutathione, another redox molecule highly abundant in plant tissues showed no significant differences among the selected plants (Fig. 2B). It is interesting to notice that A. indica and G. bicolor showed the highest levels of both ascorbate and glutathione, consistently with the metabolic interconnection of these metabolites in plant cells68,69.

In this study, the simultaneous use of different assays to test global antioxidant activity is employed as a robust experimental approach in order to achieve trustable results, since each method may exhibit different sensitivity in measuring the scavenging capacity of the molecules composing the extract towards different radical species38,39. Specifically, G. bicolor showed the highest antioxidant power, followed by G. macrosiphon, which has a slightly but significantly lower values. This observation is consistent with the fact that some Grewia species are used traditionally for their capacity to protect against the oxidative damages that underlies several diseases70. Indeed, G. bicolor was found to exhibit cytoprotection by reducing ROS level and preventing oxidative stress triggered cell death71. Several Grewia species have also been reported to have very high levels of total phenolic content, flavonoids, tannins, and anthocyanins71,72,73, even if the antioxidant capacity of Grewia bicolor was poorly documented.

By comparing the levels of different molecules with the results obtained with all antioxidant assays, it is possible to suggest that total phenols (Fig. 3), accumulated at high concentration in G. macrosiphon and G. bicolor, are those that better mirror the total antioxidant capacity (Fig. 4A-C) even if both carotenoids and chlorophylls could also contribute to this capacity. On the other hand, ascorbate and glutathione pool, are probably less relevant due to the predominance of their oxidized forms.

Antimicrobial effects have also been documented for many plants commonly used in traditional ethnobotanical medicine74. Here, the antimicrobial activity of the investigated plant species was eval‎uated by analyzing the effect of plant extracts on the ability to inhibit the growth of Gram-positive and Gram-negative bacteria, which are notoriously associated with widely spread diseases, such as B. subtilis, S. aureus and E. coli75,76,77. It is worth noting that the antibacterial activities of the different species show independent trend compared their antioxidant activity. Indeed, the extracts of A. quanzensis and G. bicolor, which are characterized by the lowest and the highest global antioxidant capacity respectively, were both ineffective in reducing the growth of all three bacterial species. This aligns with previous findings reporting no antimicrobial activity against Salmonella enterica and S. aureus for methanol and water extracts obtained from the bark of A. quanzensis grown in South Africa9,78. Previous studies on G. bicolor cultivated in Nigeria demonstrated that root and bark extracts exhibited antimicrobial activity against various bacterial strains including S. aureus, E. coli, and Klebsiella pneumoniae. The ethanolic extract of the leaves of this species showed no activity against S. aureus and Streptococcus pyogenes79. While, previous studies reported variable antimicrobial activity against E. coli, K. pneumoniae, and S. enterica9,79, the different antibacterial activity of G. bicolor against E. coli may be due to a different concentration of antimicrobial metabolites as a consequence of the environment and the developmental stage in which leaves are collected. The ethanolic leaf extracts of the other three analyzed species, A. indica, G. macrosiphon, and L. schweinfurthii, showed a variable growth inhibitory effect depending on the bacterial species, thus suggesting that different mechanisms could be involved in the interaction between the metabolites present in the plant extracts and specific bacteria. Indeed, (A) indica exhibited the highest antimicrobial properties against S. aureus and (B) subtilis, while it had no inhibitory effect against E. coli (Tables 1, 2 and 3). Previous studies indicate that extracts from leaves, barks and seeds of (A) indica possessed antimicrobial activity against the Gram-negative Pseudomonas aeruginosa in addition to the Gram-positive S. aureus80,81. G. macrosiphon, and L. schweinfurthii showed antimicrobial properties against all the three bacterial strains considered; in this case a difference in the antimicrobial effects was evident only in term of efficacy, since G. macrosiphon was more effective against the Gram-positive S. aureus and (B) subtilis, while it was less effective against the Gram-negative E. coli (Tables 1, 2 and 3). To our knowledge, this is the first evidence of antimicrobial activity for G. macrosiphon, while our results confirm‎ the remarkable antimicrobial activity against S. aureus and B. subtilis by leaf extract of L. schweinfurthii previously reported in the literature82.

Fig. 5

Antioxidant and antimicrobial activities of Kenya plant species.

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Conclusion

The continued exploration and sustainable use of natural resources hold promise for enhancing global health outcomes and expanding the scope of medical treatments available today ensuring that their benefits are maximized while preserving biodiversity. The interdisciplinary approach employed in this study allows to reveal the potential bioactivity of under-investigated indigenous Kenyan plant species mainly utilized in TM. The obtained results indicate that the leaves of G. bicolor, G. macrosiphon, and A. indica, are excellent sources of carotenoids, chlorophylls, phenols, ascorbate and glutathione, compounds known for their great antioxidant potential, which could play a critical role in mitigating oxidative stress linked to numerous chronic diseases. Furthermore, the specificity of the different plant extracts in inhibiting the growth of different bacterial species could be useful for identifying new antimicrobial metabolites of particular interest in the context of antibiotic resistance. Merging the antioxidant and anti-microbial properties of the analysed leaf extracts, G. macrosiphon resulted as a new species of interest. On the other hand, Afzelia quanzensis did not show relevant antioxidant and antimicrobial activities (Fig. 5). Taken together, these findings contribute to the valorization of plants belonging to ethnomedicinal traditions but still very poorly investigated, promoting a greater understanding of their biochemical characteristics and biological properties, to bridge the existing gap between TM and contemporary medical practices. Based on the results obtained, future studies will be undertaken to elucidate the mechanisms underlying the observed biological activities. This will involve the eval‎uation of specific phytochemicals and classes of compounds, such as phenols, that will be identified in the extracts, in relation to their demonstrated antioxidant and antimicrobial effects.

Data availability

Data Availability: The data presented in this study are available on request from the corresponding author.

References

1. Sofowora, A. Research on medicinal plants and traditional medicine in Africa. J. Altern. Complement. Med. 2, 365–372 (1996).

   Article CAS PubMed Google Scholar 

2. Traditional medicine. definitions. Geneva: World Health Organization (WHO). https://www.who.int/health-topics/traditional-complementary-and-integrative-medicine#tab=tab_1.

3. Kigen, G. K., Ronoh, H. K., Kipkore, W. K. & Rotich, J. K. Current trends oftraditional herbal medicine practice in Kenya: a review. Afr. J. Pharm. 2, 32–37 (2013).

   Google Scholar 

4. Kiringe, J. W. Ecological and anthropological threats to Ethno-Medicinal Plant Resources and their utilization in Maasai Communal ranches in the Amboseli Region of Kenya. Ethnobot Res. App. 3, 231 (2005).

   Article Google Scholar 

5. Frass, M. et al. Use and acceptance of complementary and alternative medicine among the general population and medical personnel: a systematic review. Ochsner J. 12, 45–56 (2012).

   PubMed PubMed Central Google Scholar 

6. Traditional Complementary and Integrative Medicine. https://www.who.int/health-topics/traditional-complementary-and-integrative-medicine#tab=tab_2

7. Mohamed, A. E. et al. Pharmacological activities of Grewia bicolor roots. J. Ethnopharmacol. 28, 285–292 (1990).

   Article CAS PubMed MATH Google Scholar 

8. Kingo, R. M. & Maregesi, S. M. Ethnopharmacological study on some medicinal plants used in ujiji, kigoma, tanzania. J. Phytopharmacol. 9, 102–109 (2020).

   Article Google Scholar 

9. Innocent, E., Marealle, A. I., Imming, P. & Moeller, L. An annotated inventory of tanzanian medicinal plants traditionally used for the treatment of respiratory bacterial infections. Plants 11, (2022).

10. Bussmann, R. W., Paniagua-Zambrana, N. Y. & Njoroge, G. N. Grewia bicolor Juss. GTembensisbensis Fresen. Grewia tenax (Forssk.) Fiori Grewia villosa Willd. Malvaceae. In Ethnobotany of the Mountain Regions of Africa Ethnobotany of Mountain Regions. (ed Bussmann, R. W.) (Cham: Springer International Publishing), 567–575. (2021).

    Chapter Google Scholar 

11. Palgrave, K. C., Palgrave, M. C. & Duggan, T. Trees of Southern Africa (B H B Distribution), 1983).

12. Hutchings, A., Scott, A. H., Lewis, G. & Cunningham, A. Zulu Medicinal Plants: An Inventory (University Of Kwazulu-natal), 1996).

13. Maroyi, A. Review of Ethnomedicinal, Phytochemical and Pharmacological Properties of Lannea schweinfurthii (Engl.) Engl. Molecules. 24, (2019).

14. Subapriya, R. & Nagini, S. Medicinal properties of neem leaves: a review. Curr. Med. Chem. Anticancer Agents. 5, 149–146 (2005).

    Article CAS PubMed Google Scholar 

15. Dastan, D., Pezhmanmehr, M., Askari, N., Ebrahimi, S. N. & Hadian, J. Essential oil compositions of the leaves of Azadirachta indica A. Juss from Iran. J. Essent. Oil Bearing Plants. 13, 357–361 (2010).

    Article CAS Google Scholar 

16. Mahmoud, D. A., Hassanein, N. M., Youssef, K. A. & Abou Zeid, M. A. Antifungal activity of different neem leaf extracts and the nimonol against some important human pathogens. Braz J. Microbiol. 42, 1007–1016 (2011).

    Article CAS PubMed PubMed Central Google Scholar 

17. Eid, A., Jaradat, N. & Elmarzugi, N. A Review of chemical constituents and traditional usage of Neem plant (Azadirachta Indica). Palestinian Medical and Pharmaceutical Journal. 2. (2017).

18. Ojewumi Modupe, E. et al. Phytochemical and antimicrobial activities of the Leaf Oil Extract of Mentha Spicata and its efficacy in repelling Mosquito. Int. J. Pharm. Res. Allied Sci. 6, 17–27 (2017).

    Google Scholar 

19. Ojewumi, M. E. et al. Analytical investigation of the extract of lemon grass leaves in repelling mosquito. IJPSR. 8, 1000–1009 (2017).

    MATH Google Scholar 

20. Ojewumi, M. E., Adeyemi, A. O. & Ojewumi, E. O. Oil extract from local leaves - an alternative to synthetic mosquito repellant. Pharmacophore 9, 1–6 (2018).

    Google Scholar 

21. Babatunde, D. E., Otusemade, G. O., Ojewumi, M. E., Agboola, O. & Oyeniyi, E. Antimicrobial activity and phytochemical screening of neem leaves and lemon grass essential oil extracts. IJMET. 10, 882–889. (2019).

22. Tonto, T. C. et al. Methodological pipeline for monitoring post-harvest quality of leafy vegetables. Sci. Rep. 13, 20568 (2023).

    Article ADS CAS PubMed PubMed Central MATH Google Scholar 

23. Cimini, S., Locato, V., Giacinti, V., Molinari, M. & De Gara, L. A Multifactorial Regulation of Glutathione Metabolism behind Salt Tolerance in Rice. Antioxidants (Basel). 11. (2022).

24. Zhang, J. & Kirkham, M. B. Antioxidant responses to drought in sunflower and sorghum seedlings. New. Phytol. 132, 361–373 (1996).

    Article CAS PubMed MATH Google Scholar 

25. Della Posta, S., Gallo, V., Dugo, L., De Gara, L. & Fanali, C. Development and Box-Behnken design optimization of a green extraction method natural deep eutectic solvent-based for phenolic compounds from barley malt rootlets. Electrophoresis. 43, 1832–1840 (2022).