Phytochemicals, bioactivity, and ethnopharmacological potential of selected indigenous plants

acids, and terpenoids, which demonstrate significant antioxidant, antimicrobial and anti-inflammatory activities. The plants studied possess anti-epileptic, antiplasmodial, and snake antivenom qualities. Here we highlight the views of different reports on ethnopharmacological relevance, phytochemistry, and bioactivity of the selected South African indigenous medicinal plants. We found a research gap in the phytochemical composition and phytopharmacological activity evaluation of Carissa macrocarpa and Englerophytum magalismontanum.


Introduction
Africa is endowed with indigenous fruit plants rich in beneficial phytonutrients. 1 The fruits are the most popular part of the fruit-bearing plants and they appeal to the young, the old, and the ill as they are a useful source of essential metals and bioactive phytochemicals such as polyphenolics, vitamin C, and beta-carotene (provitamin A). 1 The minerals are building blocks for teeth, blood, muscle, and bone, and are essential in brain cell development. 2 Daily consumption of a reasonable quantity of fruits substantially reduces the risk of contracting severe respiratory infections and long-lasting diarrhoea, while inadequacy of these in children's diets results in stunted growth and poor academic ability. 2 The rural populace of South Africa utilises traditional medicine and medicinal plants to augment their nutrition and in treating ailments such as skin cancer, tuberculosis, diarrhoea, malaria, fever, diabetes, gastritis, smallpox, chickenpox, snakebites, and erectile dysfunction. [3][4][5][6][7][8][9][10] However, wasteful harvesting practices threaten the existence of these indigenous plants. This threat is exacerbated by periodic droughts and wildfires. These medicinal plants include milk plum (Englerophytum magalismontanum), Natal plum (Carissa macrocarpa), wild custard apple (Annona senegalensis), and wild medlar (Vangueria infausta). They are ubiquitous in the Eastern Cape, Free State, Gauteng, KwaZulu-Natal, Limpopo, Northern Cape, and North West Provinces. 11,12 Reports reveal that approximately 80% of the South African population uses indigenous plants to meet their primary healthcare needs. 2,6,13 This finding is congruent with a worldwide growing acceptance of traditional medicines, and hence the safe use of herbal concoctions has also become a medical issue. 13 Information on indigenous medicinal plants is therefore necessary to enhance their phytopharmacological value and utilisation.
This review summarises different opinions and research findings on the ethnopharmacological uses, phytochemical constituents, and bioactivities of the four selected South African medicinal plants, thus highlighting their importance in addressing the nutritional and medicinal needs of rural communities. We recommend their sustainable harvesting/preservation as well as further exploratory studies of their potential utilisation as food and cosmetics enrichment sources.

Data sources
The major databases explored for this review were Web of Science, PubMed, Google Scholar, and Scopus. The key search words for each plant were ethnopharmacological uses, phytochemicals, and biological activities of the plant. Articles included in the study constitute those that were published in English with a focus on the four plants of interest. Primary consideration was on peer-reviewed articles, although some relevant non-peer-reviewed quality publications from government bodies were considered. The search covered the period from 2000 to 2021. The record identification process followed is presented in the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) flow diagram ( Figure 1).

Indigenous medicinal plants
Communities worldwide commonly use therapeutic herbs and plants to treat various health conditions. The widespread usage of multipurpose South African indigenous plants could therefore be attributed to their being ubiquitous and affordable.
As a multipurpose plant, A. senegalensis is used traditionally as food and as a concoction to treat many diseases. In folk medicine, reports reveal that the leaves, stem bark, and root bark are used in treating various health disorders 1,14-21 (Table 1). One report notes that the leaves of the plant are locally used to protect cereals against weevil attacks. 14 A plethora of studies have reported the phytochemical profiles and bioactivities of A. senegalensis, highlighting their significant health benefits 4,7,8,15,[21][22][23] , as presented in Table 2. The investigations of the leaves, twigs, and stem-bark extracts revealed a substantial constituent of bioactive phytochemicals. The studies established that extracts of the leaves and the stem bark were laden with polyphenols and phenolic acids 24 , hence, their exhibition of significant antioxidant and hepatoprotective activities 25 .
The cytotoxicity of A. senegalensis parts was also investigated. The highest cytotoxicity (93%) was demonstrated by the crude oil extract of the leaf, administered at a dose of 50 ppm in the brine shrimp lethality bioassay. 34 Conversely, the phytochemical fractions obtained on further purification of the crude oil fraction caused a lower lethality (49%) in the brine shrimp larvae at the same concentration of 50 ppm. Caryophyllene oxide was identified as the major compound in the lower cytotoxic fraction. However, conducting a similar test with a pure caryophyllene oxide compound showed no toxicity on the target substrate. It was concluded that caryophyllene oxide works most effectively in synergy with other phytochemicals. 34 An in vitro test reported that the leaf, stem bark, and root bark of A. senegalensis water/methanol extracts administered at 10 mg/mL demonstrated significant activity on Trypanosoma evansi by inhibiting its motility. 35 Endophytes are endosymbionts that enhance the growth of the host by improving the medicinal plant's nutrient acquisition, resistance to pathogens, repugnance to insects and herbivores, and tolerance ability over abiotic stresses, for example, drought. [35][36][37] The methanol extract of A. senegalensis leaves showed dose-dependent activity against the malarial parasite Plasmodium berghei. 15 However, the hexane and chloroform fractions from the leaf methanolic extract caused more mortality in the immature mosquito vectors. 38 The aqueous extract of A. senegalensis leaves exhibited drug detoxification possibilities. 39 A study recorded that the diethyl ether fraction of the stem bark exhibited significant antimicrobial activity against Escherichia coli, Salmonella enteritidis, and Shigella dysenteriae. 22 Another investigation established that the crude methanolic extract from the stem bark of the plant demonstrated a minimum inhibitory concentration (MIC) at a dose of 100 mg/mL against Shigella species and E. coli strain and repressed Salmonella typhi growth at 50 mg/mL. 40,41 The root-bark methanolic extract was reported to detoxify snake venom and stimulate a reduction in hyperthermia. 42 A study evaluated the leaf ethanolic extract efficacy as an antimalarial remedy and found the extract to show moderate inhibitory effect on Plasmodium falciparum and Plasmodium yoelii malaria parasite strains, as well as cytotoxicity towards leukaemia cell lines. 21 The methanol, hexane, chloroform, and ethyl acetate extracts of the A. senegalensis leaves demonstrated significant (p<0.001) toxicity against Aedes aegypti larvae, which transmits yellow fever, dengue fever, Chikungunya, and Zika fever. 43 However, the efficacy of the solvent extract fractions was dose-dependent, and the most effective was the hexane extract at a LC 50 of 379.3 mg/L, that is, at a lethal concentration causing 50% death. Recent evaluations of oral toxicity of A. senegalensis extracts indicated that a lethal oral dose (LD 50 ) of about 5 g/kg body weight (b.w.) of the plant parts extract administered to mice did not cause acute toxicity or motility in the mice. 44 Another study confirmed that a mixture of aqueous root extracts of A. senegalensis and Asparagus africanus was given orally at a dose of about 12 800 mg/kg b.w. of mice and did not cause mortality in the experimental mice. However, the mice displayed mild signs of drowsiness, inactivity, and loss of appetite. 45

Natal plum (Carissa macrocarpa)
The Natal plum belongs to the family Apocynaceae. The plant is indigenous to the province of KwaZulu-Natal and widely distributed along the coastal areas of the Eastern Cape, as well as inland in the Free State and Limpopo Provinces. 1 The plant's common name is Natal plum, and the indigenous names are amatungula, noemnoem, umbethankunzi, and ditokolo. 40 White star-shaped flowers and protective Y-shaped thorns characterise this twig-like evergreen medicinal shrub. The fruit of C. macrocarpa is small, ovoid, and oozes delicious flavour. The sweetish and slightly sour cherry-red ripe fruit contains numerous small, flat, brown seeds. The fruits are used in the preparation of salads, cakes, puddings, ice-cream, pies, tarts, and sweet jelly 46 , with the leaves used to treat diarrhoea in livestock. According to the South African National Research Council, all of the parts of the plant are used in treating infections such as sexually transmitted ailments and coughs. 1 The phytochemical composition of C. macrocarpa has not been thoroughly studied. However, some investigations have recorded that Natal plum parts are richly endowed with bioactive polyphenolics, vitamin C, and cardiac healthy fatty acids. 2,[47][48][49][50][51] The studies also noted that the fruit is rich in essential metals such as Mg, Ca, P, Fe, Zn, Mn, Cu, Se, Cr, and Ni 47 , and thus contributes significantly to the consumer's dietary needs for micronutrients and bioactive phytochemicals. 33,47 Zn, in particular, featured prominently as one of the key essential metals in combating the COVID-19 global pandemic. 52 Mg is an essential element for cellular metabolism, protein digestion, Ca absorption, muscle growth, parathyroid hormone release, and proper functioning of the nervous system. 53 Conversely, Mg deficiency in the body results in low vitamin activity and poor Ca absorption, which contributes to immune compromise, and accelerates ageing of the skin because of free radicals and uncontrollable movement of muscle twitching/tremor/ spasm/cramps. 53,54 Ca improves muscle contraction and blood clotting, as well as controls the passage of nutrients through cell walls, and reduces insomnia. A deficiency in dietary Ca causes absorption of Ca from the bones, resulting in the bones becoming weak and brittle. 54 This condition is known as osteoporosis. K acts as a buffer that prevents acidity change in body fluids. 55 Fe assists in the formation of blood and in the transportation of oxygen and carbon dioxide in the body tissues. Fe deficiency causes anaemia and impaired learning and behavioural changes in children. 54 Zn boosts hair growth, and promotes functioning of taste, touch and smell sense organs while contributing to the proper metabolism of carbohydrates and protein. Mn assists in carrying oxygen from the lungs to other organs in the body. In this way, Mn acts as an enzyme metabolic reaction activator necessary for food metabolism. Deficiency in Mn results in growth retardation and abnormal loss of weight, while Co facilitates vitamin B12 metabolism. 54 Dietary intake of fibre reduces serum cholesterol and contributes to several other health benefits. 54 A recent study reported that incorporation of C. macrocarpa fruit into mango fruit pulp affords functional food possessing betterquality dietary phytochemical content and nutritional value. 56 In consideration of the utilisation of C. macrocarpa as an agricultural resource, crude phenolic and terpenoid extracts of the leaves, administered at a concentration of 10 mg/mL, demonstrated significant in vitro inhibition of Aphis fabae (a devastating bean pest). Accordingly, the leaf extracts can act as a natural pesticide alternative to petroleumbased synthetic pesticides. 57 The phytochemical constituents of essential oils from different parts of C. macrocarpa and their antimicrobial activities were investigated. The results showed pentadecanal and tetradecan-1-ol as the main compounds in the essential oils from the fresh leaves. 58 The fruit essential oils contained nerolidol (a naturally occurring sesquiterpene alcohol extensively used in cosmetics and detergents) and caryophyllene oxide. The essential oils from the stem possessed linalool and hexahydrofarnesyl acetone, and the flower essential oils showed the presence of benzyl benzoate, which is used in treating lice and in repelling insects. The essential oils exhibited significant antimicrobial activities, which presented the plant oil as a potential source of antimicrobial compounds. 58 An earlier study of the C. macrocarpa phytoconstituent profile reported pentacyclic oleanane triterpenes isolated from the fruits. The oleanane triterpenes included amyrin, methyl oleanolate, oleanolic acid, and hydroxyolean-11-en-28,13β-olide, whereas ursolic acid was obtained from the leaves. The oleanane triterpenes and the ursolic acid demonstrated good antibacterial activity against bacterial strains (Klebsiella pneumoniae, Pseudomonas aeruginosa, E. coli, Staphylococcus saprophyticu, Staphylococcus aureus, and Enterococcus faecium) at MICs ranging from 0.06 mg/mL to 1.0 mg/mL. 5

Wild medlar (Vangueria infausta)
The Vangueria infausta from the Rubiaceae family is known by the common name wild medlar. Its local names are wildemispel, mmilo, muzwilu, mavelo, umviyo, umtulwa, mothwanye, umvile, and amantulwane. The plant is indigenous to southern Africa. In South Africa, it is widely distributed in the KwaZulu-Natal, Eastern Cape, Free State, Gauteng, Limpopo, and North West Provinces. 1 The fruit of V. infausta is round and dark green when unripe and light brown when ripe. The ripe fruit is about 20-50 mm in diameter with soft and fleshy pulp. The orange pulp that contains roughly five seeds is slightly astringent and sweet-sour-bitter in taste. The flesh is usually eaten fresh and occasionally cooked. The fresh pulp, as a tradition, is processed into juice, jam, marmalade, puddings, and porridge, or sundried for storage. 56 Diverse parts of V. infausta are used in the traditional treatment of various kinds of diseases and health disorders, as summarised in Table 1.
Reports on the phytochemical investigations of V. infausta plant parts available to us during this review were limited. However, the accessed papers reported the identification of the presence of flavonoids, tannins, coumarins, lignans, phenolic acids, saponins, fatty acids, terpenoids, and anthraquinones in different parts of the V. infausta plant extracts. 58,59 The phytochemicals isolated from the extracts are listed in Table 2.
The biological activities of V. infausta have been extensively investigated, relative to its phytochemistry. The reviewed literature shows that all parts of the plant have exhibited both antimalarial 60 and antifungal activities [60][61][62] . Inhibitory activities of leaf extracts against seven pathogenic fungi were observed with acetone and dichloromethane (DCM) extracts at a MIC of 0.32 mg/mL. 36 An indole alkaloid, morindolide, isolated from the leaf displayed good antimalarial activity at IC 50 = 107.1±0.6 µM. 60 The hexane, DCM, acetone, and methanol extracts of V. infausta leaves demonstrated inhibitory activities against Enterococcus faecalis at a low MIC value of 0.02 mg/mL. 60 The DCM extract of V. infausta root exhibited high inhibitory effects on the Leishmania protozoan with an IC 50 value of 4.51 µg/mL 63 , a significant antiplasmodial activity at IC 50 of 1.84 µg/mL, and a selectivity index of 25 against Plasmodium falciparum 64 . The report resonated with earlier findings, which identified chloroform extracts from the V. infausta root bark that markedly inhibited the activities of two Plasmodium falciparum strains at IC 50

Annona senegalensis
The ripe fruit is eaten raw.

Carissa macrocarpa
Processed into a salad, cake, pudding, ice-cream, pies, tarts, and sweet jelly. Treats hepatitis and HIV infection.
Treats diarrhoea in animals. Treats cough and venereal infections.

Treats epilepsy
Remedy for diabetes mellitus, pain, and rheumatoid arthritis.
Treats Alzheimer's disease, inflammation, rheumatism, abdominal pain, and wounds. 6,13,67 The cause of health conditions such as inflammatory and arthritic ailments (spondylitis, osteoarthritis, and rheumatoid arthritis) is related to protein denaturation. Therefore, any substance that could prevent or limit protein denaturation will consequently contribute to the prevention of inflammation and arthritic conditions of the body and skin. An investigation of V. infausta proved that this wild plant showed significant antiinflammatory activity from different phytochemical extracts of the plant parts on a substrate 66 , thus proving its health benefit.

Milk plum (Englerophytum magalismontanum)
The Englerophytum magalismontanum plant family is Sapotaceae. The common name is milk plum, and local names are stamvrug, motlhatswa, munombelo, amanumbela, and umnumbela. 1 It grows in the riverine forest suburbs of Gauteng, North West, Mpumalanga, Limpopo, and the northern part of KwaZulu-Natal. 11 The flesh of E. magalismontanum berry fruit is ellipsoid to round. The fruit ripens to a bright red colour and contains sticky, milky latex. The edible fruit serves as a thirst quencher. During the harvest season, the fruit is processed into syrup, jelly, jam, wine, vinegar, and a strong alcoholic drink, known as mampoer. 13 68 Interestingly, Mg, Mn, Zn, and Se, which are known to be very beneficial minerals for human nutrition, are present in the fruit in significant amounts. These essential elements (Mg, Mn, Zn, and Se) are popular in nourishing and rejuvenating skin, particularly when incorporated into a product for topical application. Furthermore, the root is traditionally pulverised into powder and rubbed into incisions, to assist in healing. 13 Research has revealed that E. magalismontanum leaf extracts exhibit significant activity against acetylcholinesterase at a dose of 160 µg/mL. Extracts of different parts of the plant demonstrated strong activity in managing oxidative stress related diseases and ailments, such as Alzheimer's disease, inflammation, and pain. 6 Furthermore, extracts from E. magalismontanum parts exhibited significant antimicrobial activities against oral microorganisms such as Actinomyces naeslundii and Actinomyces israelii. The ethanol extract exhibited MIC values from 12.5 mg/mL 6.3 mg/mL, while the cytotoxicity test showed an IC 50 value of about 98.8 mg/mL. 69

Conclusion
Our review and summary of the literature on four indigenous medicinal plants in South Africa indicate that different parts of the plants have long been used in folk medicines and other remedies, including as dietary supplements, based on traditional beliefs. The phytochemical composition and biological activity profile of three of the reviewed plants were sparsely studied; hence, not much was reported. However, this review indicates the presence of the bioactive phytochemicals flavonoids, tannins, coumarins, lignans, phenolic acids, saponins, fatty acids, terpenoids, and anthraquinones in these plants, which relate to the demonstration of strong antioxidant, anti-inflammatory, antiparasitic, anthelmintic, anticonvulsant, antimalarial, antitrypanosoma, antinociceptive and antivenom activities.
The study reveals that there is sufficient evidence to promote the consumption of V. infausta and A. senegalensis to manage various ailments, especially in the era of the COVID-19 pandemic that undermines the immune system.
We therefore recommend intense research on the phytochemical composition and bioactivity profiles of C. macrocarpa and E. magalismontanum. Such studies will improve their worth and facilitate their incorporation into communal medicaments such as cosmetics and other functional products.