Milkwood (Mimusops zeyheri Sond.) in South Africa: diversity, traditional knowledge, and sustainable utilisation.

Abstract

Mimusops zeyheri is an indigenous wild fruit tree of ecological, nutritional, cultural, and economic importance in southern Africa, yet comprehensive scientific documentation of its traditional uses, population ecology, genetic and morphological variation, nutritional value, and phytochemical properties remains limited. This thesis provides an integrated multi disciplinary assessment of M. zeyheri across two regions, Vhembe in the Limpopo province and Ehlanzeni in the Mpumalanga province using ethnobotanical, ecological, morphological, sensory, nutritional, and chemical approaches. An ethnobotanical survey was conducted to collect data from 116 participants over the age of 18 through semi-structured random face-to face interviews. Ethnobotanical indices, such as Relative Frequency of Citation (RFC) and Fidelity Level (FL), were used for data analysis. The results demonstrate the multifaceted use of M. zeyheri by different ethnic groups to treat various ailments and conditions. A total of 16 other uses of M. zeyheri were reported in Limpopo, and 27 reported in Mpumalanga. The RFC values ranged from 0.29 to 1 in Limpopo and from 0.03 to 1 in Mpumalanga. Fruits eaten whole as a snack received an FL value of 100% in both provinces, as recorded from all participants. In Limpopo, the ethnomedicinal use of M. zeyheri to treat sexually transmitted infections received an FRC value of 1, recorded from all participants (FL = 100%), with efficacy reported when the species is mixed with other plant species, such as Aloe vera, and boiled to form a decoction that is taken orally. Similar use of M. zeyheri for treating sexually transmitted infections was also reported (RFC=0.50) in Mpumalanga, highlighting similar use of the same species by different ethnic groups. Other commonly reported uses include the use of M. zeyheri for treating skin ailments and conditions, such as ringworm (RFC = 0.57) in Mpumalanga and (RFC = 1) in Limpopo. In Mpumalanga, the use of M. zeyheri extends to spiritual and cultural practices, such as using the leaves to remove bad omens (RFC = 0.100) and to collect the spirit of the deceased from where they last took their breath (RFC = 0.05). Different plant parts are utilized in both provinces, with Limpopo having a slightly higher preference for roots (17%), while Mpumalanga has a slightly higher preference for nuts (17%). Both study sites share a similar preference for the use of leaves (22%) and a strong preference for decoction in preparing ethnomedicine (36% and 34%) for Limpopo and Mpumalanga, respectively. Internal and external applications were cited by participants in the study, with oral administration being the most preferred mode of delivery in both Limpopo (75%) and Mpumalanga (55%). Participants in Mpumalanga (40%) identified land expansion and infrastructure development as perceived major threats to the M. zeyheri population, while overharvesting (30%) was identified as a significant concern in Limpopo. These findings are based on community perceptions and are interpreted alongside ecological observations, including size-class distribution patterns, which indicate both active recruitment and potential limitations in the progression of individuals into mature stages. The population dynamics, spatial distribution, tree size variation, and ecological condition of M. zeyheri in two distinct regions, the Ehlanzeni District (Mpumalanga) and the Vhembe District (Limpopo), were assessed to understand the species ecology and use patterns. Data collection focused on size class distribution, with statistical analyses applied to assess population structure and variation using ecological modelling and scoring indices. Significant differences were found in stem circumference (F = 31.98, p = 0.000031), tree height (p = 0.017813), and inter-tree distances (p = 0.01005) between the two sites. Spatial analysis revealed a dispersed pattern in Vhembe (mean distance = 5.54 m), likely driven by wind dispersal (anemochory), and a clustered pattern in Ehlanzeni (mean distance = 2.68 m), suggesting animal-mediated dispersal (zoochory). Ecological health assessments revealed greater anthropogenic disturbance in Vhembe, where over half the population fell within the 50–100% disturbance index range, whereas Ehlanzeni showed minimal disturbance (0–5% index). Despite high regeneration potential, a dominance of smaller size classes suggests possible regeneration bottlenecks linked to harvesting pressures. Mimusops zeyheri morphological characterization was based on sampling 40 trees (20 per region), with fruit, nut, and leaf traits measured using standard morphometric procedures. Sensory evaluation was conducted with 100 adult participants (50 per site) using an eight-attribute, 9-point hedonic scale. Genetic diversity analysis employed chloroplast markers matK and trnH-psbA, with a 75% sequencing success rate. Mpumalanga fruits were larger, with mean fruit lengths of 29.41 ± 0.61 mm and widths of 24.14 ± 0.55 mm, compared to Limpopo fruits measuring 27.41 ± 0.47 mm in length and 23.22 ± 0.36 mm in width. Nut size followed the same pattern, averaging 2.11 ± 0.36 cm in Mpumalanga and 1.81 ± 0.31 cm in Limpopo. By contrast, leaf morphology remained stable across regions, with overlapping mean leaf lengths (7.23–7.52 cm), widths (3.93–4.03 cm), and leaf areas (21.69–22.80 cm²). The results of the sensory evaluation show that Limpopo fruits received markedly higher ratings across all eight sensory attributes, with mean scores for taste, aroma, and mouthfeel ranging from 7.7 to 8.0. In contrast, Mpumalanga fruits scored between 5.0 and 5.4 on the 9 point hedonic scale. Genetic analyses using the matK and trnH-psbA chloroplast markers (with a 75% sequencing success rate) identified two major genetic lineages, distinguished by regional clustering, along with rare haplotypes such as LP3, MP9, and MP10, which exhibited longer branch lengths and unique nucleotide substitutions. Together, the morphological, sensory, and genetic evidence demonstrates strong regional structuring in M. zeyheri populations, shaped by both environmental gradients and historical lineage divergence. The nutritional composition of M. zeyheri plant parts was determined using standard AOAC methods and elemental analysis by microwave-assisted digestion followed by ICP-OES. Data were analyzed using one-way ANOVA and Pearson correlation in R. Nuts contained the highest dry matter (~94 %), crude fat (~54 %), protein (~19 %), and energy value (~595 kcal/100 g⁻¹), together with the greatest concentrations of calcium (≈210 mg/100 g⁻¹), magnesium (≈143 mg 100 g⁻¹), zinc (≈95 mg/kg⁻¹), and potassium (≈495 mg/100 g⁻¹). Leaves were rich in protein (≈24 %) and fibre (≈22 %), while pulp and fruit fibre provided high moisture, modest vitamin C (5–7 mg/100 g⁻¹), and β-carotene (~1.5–2.1 mg/100 g⁻¹). Correlation analysis revealed strong positive associations (r > 0.95, p < 0.01) between dry matter, crude fat, protein, and divalent cations (Ca, Mg, Zn), indicating that nutrient concentration increases as moisture decreases. Provincial differences were minor, indicating ecological consistency. Mimusops zeyheri offers a unique combination of energy-dense nuts, protein, mineral-rich leaves, and vitamin-containing pulp, making it a valuable food resource for improving dietary diversity and combating micronutrient deficiencies in rural South Africa. Its climate resilience is suggested by its ability to persist across contrasting agro-ecological zones, including semi-arid and low-input environments, as observed in this study. Lastly, the total phenolics, flavonoids, and alkaloids were quantified using spectrophotometry techniques, and the antioxidant activities using the 2, 2-diphenyl-1 picrylhydrazyl (DPPH) radical scavenging method and Ferric Reducing Antioxidant Power assay. Untargeted secondary metabolites from the different plant parts of M. zeyheri, collected in various Agro-ecological zones, were identified using Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-QT-MS/MS). Leaves recorded significantly higher phenolic content (19.13 mg GAE g⁻¹ in Limpopo; 18.20 mg GAE g⁻¹ in Mpumalanga) than nuts (≈14 mg GAE g⁻¹), pulp (≈8 mg GAE g⁻¹), and fibre (≈6 mg GAE g⁻¹). Similar to phenolic content, the total flavonoid content (TFC) of Mimusops zeyheri followed a clear pattern across the different plant parts. Leaves exhibit the highest concentration of TFC. .83 ± 0.25 µg QE g⁻¹ in Limpopo and 9.30 ± 0.20 µg QE g⁻¹ in Mpumalanga. Nuts followed (6.23 ± 0.25 and 5.80 ± 0.15 µg QE g⁻¹), while pulp (around 4 µg QE g⁻¹), with the lowest TFC observed in fruit fibre ( around 3 µg QE g⁻¹ in both Limpopo and Mpumalanga). Differences in TFC between the WTO provinces are small, emphasizing plant tissue as the main driver of concentration rather than the location where the plant grows. Alkaloid content was again highest in leaves: 4.67 ± 0.15 mg AE g⁻¹ (LLP) and 4.37 ± 0.15 mg GAE g⁻¹ (LMP). Nuts followed (3.33–3.10 mg AE g⁻¹), then pulp (~2 mg AE g⁻¹) and fibre (~1.5 mg AE g⁻¹). The pattern mirrors that of phenolics and flavonoids, reflecting the defensive role of alkaloids against herbivores and pathogens, which is strongest in leaves and reproductive tissues. Antioxidant activity differed between plant parts and provinces. Mpumalanga nuts showed the strongest radical-scavenging capacity in the DPPH assay (EC₅₀ 0.0581 µg/mL) compared with Limpopo nuts (EC₅₀ 0.1767 µg/mL) and much lower than the positive control. Fibre and pulp extracts were more effective in the β-carotene–linoleic acid system, with Mpumalanga fibre showing the lowest EC₅₀ (18.24 µg/mL) and pulp extracts ranging between 64.10–77.43 µg/mL. Leaf extracts showed moderate activity in both assays. The heatmap indicated clear tissue- and site-specific patterns, with Mpumalanga samples generally outperforming those from Limpopo. Fruit fibre from Mpumalanga is rich in flavonoids with dominant compounds such as quercetin-3-O-glucoside (Rt 7.82 min, 463.12 m/z) with congeners identified as isoquercitrin (Rt 5.81 min, 463.09 m/z) and rutin (Rt 4.62 min, 609.14 m/z) known for their distinct structural and biological properties while M. zeyheri fruit fibre from Limpopo has notable phenolic glycosides such as β-glucogallin (Rt 1.08 min, 367.05 m/z). β-glucogallin has been identified as having potent antioxidant and antimicrobial properties, which also support good gut health function in humans and aid in the preservation of food. M. zeyheri fruit pulp from Mpumalanga has a notable array of various flavanol glycosides and nutrient compounds that together create a flavorful and health-promoting profile of tentatively identified compounds. By contrast, the Limpopo pulp is dominated by hydrolysable tannins and phenolic glycosides, reflecting a more therapeutic, preservative-leaning profile. The Mpumalanga nut profile is flavonoid rich with notable quercetin–catechin profile and compounds such as Free quercetin (Rt 5.75 min, 301.03 m/z) alongside its glycosides-isoquercitrin (quercetin-3-O glucoside; Rt 5.81 min, 463.09 m/z) and a rutin isomer (quercetin-3-O-rutinoside; Rt 5.90 min, 609.14 m/z) known for antioxidant and anti-inflammatory activity relevant to vascular protection, insulin sensitivity and neuroprotection. By contrast, the Limpopo nuts present a tannin-dense fingerprint with higher oligomerization and a striking acylated anthocyanin. A hallmark proanthocyanidin trimer, specifically gallocatechin-(4α→8)-gallocatechin-(4α→8) gallocatechin (Rt 3.02 min, 913.19 m/z), together with a dimeric feature at 577.14 m/z (identified as endotelon), indicates an extensive condensed-tannin spectrum. In contrast, leaves from Limpopo are characterized by high levels of galloyl phenolics and oligomeric proanthocyanidins (OPCs), associated with vascular and antioxidant benefits, with compounds such as Glucosyringic acid (Rt 3.08 min, 359.10 m/z ) previously reported for its antioxidant and anti-inflammatory effects. This study presents the most comprehensive multidimensional assessment of Mimusops zeyheri to date, integrating ethnobotanical knowledge, ecological analysis, morphological and genetic characterization, nutritional profiling, and phytochemical screening. Together, the findings reveal M. zeyheri as a culturally valued, nutritionally important, and chemically rich indigenous fruit tree with significant potential for conservation, domestication, and rural economic development. The clear regional structuring observed across ecological, genetic, nutritional, and phytochemical dimensions underscores the importance of context-specific management and utilization strategies. Accordingly, the study recommends the integration of M. zeyheri into local conservation and agroforestry programmes, the protection of genetically distinct populations and rare haplotypes, and the promotion of sustainable harvesting practices to reduce pressure from overexploitation and land-use change. In line with the United Nations Sustainable Development Goals, the study contributes directly to SDG 1 (No Poverty) by identifying pathways for livelihood diversification and income generation through the sustainable use and value addition of M. zeyheri, SDG 2 (Zero Hunger) by demonstrating its potential to enhance dietary diversity and nutrition security through climate-resilient indigenous food resources, and SDG 13 (Climate Action) by highlighting the role of resilient indigenous tree species in supporting adaptation strategies under increasing climate variability. Future research should incorporate high-resolution spatial mapping using GIS to identify priority conservation areas and harvesting hotspots, alongside biological assays and targeted compound isolation to validate the bioactivity, safety, and therapeutic relevance of key phytochemicals. In addition, the development of community-based, value-added products from M. zeyheri has strong potential to strengthen local value chains and rural livelihoods, while long-term ecological monitoring is essential to track population dynamics, regeneration success, and anthropogenic disturbance. Collectively, these measures will support the sustainable conservation, responsible commercialization, and policy integration of M. zeyheri as a climate-resilient indigenous food and medicinal resource.