Medicinal Uses, Phytochemistry and Pharmacological Properties of Elaeodendron transvaalense

Elaeodendron transvaalense is a plant species, which is in high demand as a herbal medicine in southern Africa. This study critically reviewed the medicinal applications, phytochemistry and pharmacological activities of E. transvaalense. The literature on medicinal applications, phytochemical, and pharmacological activities of E. transvaalense, was collected from multiple internet sources including Elsevier, Google Scholar, SciFinder, Web of Science, Pubmed, BMC, Science Direct, and Scopus. Complementary information was gathered from pre-electronic sources, such as books, book chapters, theses, scientific reports, and journal articles obtained from the University library. This study revealed that the species is used as herbal medicine in 62.5% of the countries where E. transvaalense is native in southern Africa. It is mainly used as herbal medicine for diarrhoea, menorrhagia, stomach aches, skin infections, inflammations, and rashes. Phytochemical compounds identified from the species, include flavonoids, peltogynoid, phenols, proanthocyanidins, tannin, and triterpenes. Ethnopharmacological research revealed that extracts and phytochemical constituents isolated from E. transvaalense have antibacterial, antifungal, anti-HIV, anti-inflammatory, antioxidant, antiplasmodial, anti-protozoan, anti-pyretic, hypoglycaemic, larvicidal, cytotoxicity, and mutagenic activities. Elaeodendron transvalense should to be subjected to detailed phytochemical, pharmacological, and toxicological evaluations aimed at correlating the medicinal uses of the species with the ethnopharmacological properties of the species.


Phytochemical Constituents of Elaeodendron transvaalense
A wide range of minerals and phytochemicals ( Table 2) have been isolated from the stem bark, leaves and the bark of E. transvaalense. Phytochemical screening of ethanol, hexane, and hexane: Ethyl acetate (80: 20) extracts of root and stem bark yielded carbohydrate, flavonoid, peltogynoid and triterpenes (Table 3; Figure 1).

Antibacterial Activities
McGaw et al. [58] evaluated the antibacterial activities of aqueous, ethanol, and hexane bark extracts of E. transvaalense against Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, and

Anti-Fungal Activities
Steenkamp et al. [61] evaluated the anti-fungal activities of methanol and the water bark extracts of E. transvaalense against Candida albicans standard strain (ATCC 10231), and five clinical isolates using the plate-hole diffusion and broth microdilution methods, with amphotericin B as the positive control (Table 4). Only the methanol extract was active against the standard strain (ATCC 10231) exhibiting an MIC value of 20.2 mg/mL, while the positive control amphotericin B inhibited growth of all strains tested with an MIC value of <10 µg/mL [61]. Samie et al. [22] evaluated the anti-fungal activities of acetone and hexane bark extracts of E. transvaalense against Candida albicans, Candida krusei, and Cryptococcus neoformans using the agar diffusion and the microdilution methods, with nystatin and flucytosine as positive controls. Only hexane extract exhibited activities with the zone of inhibition ranging from 8 mm to 16 mm in comparison to 22 mm exhibited by both nystatin and flucytosine, the two positive controls. The MIC values against tested pathogens ranged from 0.5 mg/mL to 1.9 mg/mL, while the positive controls, nystatin and flucytosine, exhibited MIC values of 0.2 µg/mL, and 1.9 µg/mL, respectively. The minimum fungicidal concentration (MFC) values ranged from 1.9 mg/mL to 7.5 mg/mL ( Table 4). The time-to-kill experiments indicated an intense time-dependent fungicidal effect of the hexane extract against Candida albicans, able to kill >90% of all the cells at a concentration of 1.9 mg/mL after a 10 hour incubation [22]. Mamba et al. [46] evaluated the antifungal activities of ethanol bark extracts of E. transvaalense and the compounds lup-20(30)-ene-3α,29-diol, lup-20(29)-ene-30-hydroxy-3-one and 4'-O-methyl-epigallocatechin, isolated from the species against Candida albicans, using the serial broth microdilution assay. The extracts and compounds exhibited activities with MIC values ranging from 3.1 mg/mL to <12.5 mg/mL [46]. These documented antifungal activities corroborate the use of the species as herbal medicine against candidiasis in South Africa [40], skin infections, and rashes [19,20,[30][31][32].

Anti-HIV Activities
Morobe et al. [62] evaluated the anti-HIV activities of methanolic bark extracts of E. transvaalense, using the anti-HIV-1 iiiB assay ( Table 4). The extract exhibited the ability to inhibit HIV-1 iiiB with half maximal effective concentration (EC 50 ) values of 0.1 µg/mL and 0.2 µg/mL [62]. Bessong et al. [44] evaluated the anti-HIV activities of aqueous and methanol root extracts of E. transvaalense by assessing their inhibitory properties against HIV-1 reverse transcriptase (RT). The strongest inhibition was against the ribonuclease H (RNase H) activity of RT with methanol and aqueous extracts exhibiting half maximal inhibitory concentration (IC 50 ) values of 30.0 µg/mL, and 31.2 µg/mL, respectively, while the inhibitory on RNA-dependent-DNA polymerase (RDDP) activity of RT for aqueous and methanol extracts exhibited IC 50 values of 80.0 µg/mL, and 131.0 µg/mL, respectively [44]. Tshikalange et al. [50] evaluated the anti-HIV activities of 70% acetone, chloroform and ethyl acetate stem bark extracts of E. transvaalense by assessing their inhibition against α-glycohydrolase, reverse transcriptase, and viral proteins (NF-kB and Tat), which play a significant role in the HIV life cycle with mesuol as a positive control. In the in vitro assay of α-glycohydrolase, the extracts showed no inhibition against α-glycohydrolase, but the chloroform and ethyl acetate extracts showed good inhibitory activities of 64%, and 76%, respectively at the lowest concentration tested (1 µg/mL) in the NF-kB assay (Table 4). At the highest concentration 1 µg/mL, 70% acetone extract exhibited an inhibition of 54%, chloroform (73%) and ethyl acetate (75%), which was comparable to 84% exhibited by mesuol, the positive control. Chloroform and ethyl acetate extracts showed a high Tat inhibitory activity of 73%, and 75%, respectively at 15 µg/mL, while 70% acetone extract demonstrated a lower activity of 43%. The extracts showed lower cell death percentages, ranging from 17.1% to 27.6% after 36 h at the highest concentration tested (15 µg/mL) [50]. Mthethwa et al. [23] evaluated anti-HIV activities of E. transvaalense bark extracts using the anti-HIV-1 iiiB assay. The extract exhibited the ability to inhibit HIV-1 iiiB with half the maximal effective concentration (EC 50 ) value of 3.5 µg/mL [23]. Mamba et al. [46] evaluated anti-HIV activities of ethanol bark extracts of E. transvaalense and the compounds lup-20(30)-ene-3α,29-diol, lup-20(29)-ene-30-hydroxy-3-one, and 4'-O-methyl-epigallocatechin isolated from the species against recombinant HIV-1 enzyme, using non-radioactive HIV-RT colorimetric assay with doxorubicin as a positive control. The ethanol extract exhibited low inhibitory activity of 20%, 4'-O-methyl-epigallocatechin showed moderate activity of 63.7%, while the positive control doxorubicin showed 96.5% inhibitory activity [46]. Sigidi et al. [63] evaluated the anti-HIV activities of aqueous bark extract of E. transvaalense using the reverse transcriptase (RT) assay. The extract showed inhibition ranging from 25% to 40% [63]. These documented anti-HIV activities corroborate the use of the species as herbal medicine against HIV opportunistic infections in South Africa [16,34,35,44].

Antioxidant Activities
Motlhanka et al. [65] evaluated the antioxidant activities of water and ethanol root extracts of E. transvaalensis and a compound 4'-O-methyl-epigallocatechin, isolated from the species using the 2,2-dipheny-l-picrylhydrazyl (DPPH) free radical scavenging assay with quercetin, rutin, and ascorbic acid as positive controls. Above 100 µg/mL, the ethanolic extract showed an 80% scavenging activity, which was similar to the activities exhibited by the control antioxidant compounds quercetin, rutin, and ascorbic acid, and the water extract reached a similar of activity (80%) at 200 µg/mL (Table 4). Between 25.0 µg/mL to 50 µg/mL, the compound 4'-O-methyl-epigallocatechin exhibited a 65% scavenging activity, which was greater than the activities exhibited by both water and ethanol extracts. But at concentrations above 50 µg/mL, the scavenging activity of the ethanol extract exceeded that of the compound 4'-O-methyl-epigallocatechin [65]. Motlhanka et al. [39] evaluated the antioxidant activities of water and ethanol root extracts of E. transvaalensis and a compound 4'-O-methyl-epigallocatechin, isolated from the species, using the DPPH free radical scavenging assay with quercetin, rutin, and ascorbic acid as positive controls. Both the crude extract and the compound 4'-O-methyl-epigallocatechin showed activities, and at 100 µg/mL, the ethanolic extract showed 80% scavenging activity, which was similar to the activities exhibited by the control antioxidant compounds quercetin, rutin, and ascorbic acid; while the water extract reached a similar level at 100 µg/mL [39]. Nethengwe et al. [48] evaluated the antioxidant activities of methanolic bark extracts of E. transvaalense, using the DPPH free radical scavenging, 2,2'-azinobis-3-ethylbenzothiazoline-6-sulphonate (ABTS), hydroxyl ( • OH) radical scavenging, super oxide (SO), nitric oxide (NO) radical scavenging, iron chelating property assays, total antioxidant capacity, and the sulphur hydryl (SH) content ( Table 4). The IC 50 values for the DPPH assay was 0.7 µg/mL, ABTS (4.1 µg/mL), iron chelating (3.9 µg/mL), • OH (3.6 µg/mL), NO (3.6 µg/mL) and SO (1.6 µg/mL) [48]. Makhafola et al. [57] evaluated the antioxidant activities of methanolic leaf extracts of E. transvaalense, using the DPPH free radical scavenging assay with ascorbic acid as the positive control. The extract exhibited activities with EC 50 value of 2.8 µg/mL, which was comparable to EC 50 value of 2.3 µg/mL exhibited by ascorbic acid, the positive control [57]. The antioxidant activities exhibited by the crude extracts of E. transvaalense are probably due to flavonoids and phenolics, which have been isolated from the species [48,53,55,57].

Antiplasmodial Activities
Nethengwe et al. [48] evaluated the anti-plasmodial activities of aqueous, dichloromethane, and methanolic bark extracts of E. transvaalense against the chloroquine sensitive strain of Plasmodium falciparum (D10), using the parasite lactate dehydrogenase assay ( Table 4). The other extracts were not active with the exception of dichloromethane, which exhibited IC 50 value of 5.1 µg/mL [48]. These findings support the general view that E. transvaalense is a potential source of antimalarial agents and to some extent corroborate the traditional use of the species as herbal medicine against fever [10,20,21,23,26,29] and malaria [48].

Anti-pyretic Activities
Nethengwe et al. [48] evaluated the anti-pyretic activities of dichloromethane and methanolic bark extracts of E. transvaalense, using both female and male Sprague-Dawley rats with paracetamol as the reference drug ( Table 4). The extracts exhibited the potential to reduce pyrexia in the induced rats and the activities were time-and concentration-dependent, with the extracts showing activity as early as 30 minutes, even at the lowest concentration of 100 mg/kg. The methanol extract showed significant activity that was comparable to paracetamol, the reference drug [48]. These findings corroborate the use of E. transvaalense as herbal medicine against fever [10,20,21,23,26,29].

Hypoglycaemic Activities
Deutschländer et al. [67] evaluated the hypoglycaemic activities of acetone stem bark extracts of E. transvaalense, by assessing their inhibiting effects on carbohydrate-hydrolising enzymes α-glucosidase and α-amylase. The acetone extracts were screened against C2C12 myocytes, 3T3-L1 preadipocytes and Chang liver cells by measuring their glucose uptake ( Table 4). The in vitro assay in 3T3-L1 preadipocytes indicated that the extracts had potential of 138.6% to lower blood glucose levels at a concentration of 50 µg/mL. The α-glucosidase and α-amylase 50% inhibitory concentrations (IC 50 ) of the extracts was found to be 50.6 µg/mL, and 1.1 µg/mL, respectively [67]. These results somehow support the usage of E. transvaalense as a herbal medicine against diabetes [66].

Larvicidal Activities
Nethengwe et al. [48] evaluated larvicidal activities of aqueous, dichloromethane, and methanolic bark extracts of E. transvaalense, using the mosquito larvicidal assay by the use of Culex quinquefascitus larvae. The results of the percentage mortality of the fourth instar larvae of Culex quinquefascitus showed that the aqueous extracts had least larvicidal activity of 35%, methanol (47%) and dichloromethane (60%) ( Table 4). The IC 50 values of methanol and dichloromethane extracts were 9.8 µg/mL and 18.2 µg/mL, respectively [48]. These findings corroborate the use of E. transvaalense as herbal medicine against malaria [48].

Cytotoxicity and Mutagenic Activities
Deutschländer et al. [67] evaluated the cytotoxic activities of stem bark extracts of E. transvaalense, by assessing its effects on preadipocytes and hepatocytes cell lines ( Table 4). The extract exhibited cytotoxicity at 12.5 µg/mL to 3T3-L1 preadipocytes, and Chang liver cells [67]. Tshikalange and Hussein [53] [62] evaluated the cytotoxic activities of methanolic and aqueous extracts of E. transvaalense against MAGI CCR5+ cells, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The extracts exhibited activities with half maximal cytotoxic concentration (CC 50 ) value of 3.7 mg/mL [62]. Nethengwe et al. [48] evaluated the cytotoxic activities of aqueous, dichloromethane, and methanolic bark extracts of E. transvaalense, using the MTT cell proliferation assay against human embryonic kidney (HEK293) and human hepatocellular carcinoma (HepG2) cells. The other extracts were not active with the exception of dichloromethane, which exhibited the median lethal concentration (LC 50 ) value of 512.0 µg/mL and 394.0 µg/mL against HEK293, and HepG2, respectively [48]. Mthethwa et al. [23] evaluated the cytotoxic activities of E. transvaalense bark extracts, using the MTT assay with berberine as a positive control. The CC 50 value of the extract was 200.0 µg/mL, which was higher than 27 µg/mL exhibited by berberine, the control and a selective index (SI) value of 57.1 [23]. Sigidi et al. [63] evaluated the cytotoxicity activities of aqueous bark extract of E. transvaalense on U937, MeWo, and Vero cell lines, using the MTT cell proliferation assay. The extract exhibited activities in all the three human tumour cancer cell lines [63].
Makhafola et al. [57] evaluated mutagenicity activities of methanolic leaf extracts of E. transvaalense, using the Ames test on Salmonella typhimurium strains TA98 and TA100. The authors also evaluated the antimutagenicity of the plant extracts against 4-nitroquinoline 1-oxide (4-NQO) using the Ames test. The extract did not exhibit any mutagenic activities, but showed weak antimutagenic activities ( Table 4). The percentage inhibition of 4-NQO was 23.2% in Salmonella typhimurium TA98 and 21.3% in strain TA100 at the assayed concentration of 5 mg/mL [57].

Conclusion
The present review summarizes the medicinal uses, phytochemistry, and pharmacological properties E. transvaalense. The diverse pharmacological activities of E. transvaalense are somehow directly or indirectly involved in a range of physiological processes, which offer protection against both free radicals and harmful pathogens. In the past 30 years, E. transvaalense has been the subject of phytochemical and pharmacological research, but there is not yet enough data correlating the medicinal uses of the species with its phytochemical and pharmacological properties. Detailed studies on the pharmacokinetics, in vivo, and clinical research involving compounds isolated from E. transvaalense and extracts of the species are required. Therefore, future research should focus on the molecular modes or mechanisms of action, pharmacokinetics, and physiological pathways for specific extracts of the species, including the identification of the bioactive compounds of the species and their associated pharmacological activities. These studies need to be complemented with experimental animal studies, randomized clinical trials, and target-organ toxicity studies. The bark of E. transvaalense is known to be poisonous and there is need to do detailed toxicological evaluations that strike a balance between the medicinal potential, and adverse and toxic effects on the species. There is very little information on the toxicological properties of E. transvaalense, whether it causes superficial discomfort when ingested as herbal medicine or serious poisoning. In the absence of such detailed toxicological evaluations, the intake of E. transvaalense as a herbal medicine should, therefore, be done with caution as the species has potential to cause long-term damage in patients. The wide usage of E. transvaalense as a herbal medicine in southern Africa has resulted in an increased collection of its bark from the wild. The species population is declining due to harvesting for the medicinal plant trade, and this calls for conservation strategies and mechanisms to ensure sustainable utilization of the species.