Review of Ethnomedicinal Uses, Phytochemistry and Pharmacological Properties of Euclea natalensis A.DC.

Euclea natalensis is traditionally used as herbal medicine for several human diseases and ailments in tropical Africa. This study reviews information on ethnomedicinal uses, botany, phytochemical constituents, pharmacology and toxicity of E. natalensis. Results of this study are based on literature search from several sources including electronic databases, books, book chapters, websites, theses and conference proceedings. This study showed that E. natalensis is used as traditional medicine in 57.1% of the countries where it is indigenous. Euclea natalensis has a high degree of consensus on abdominal pains, antidote for snake bites, diabetes, diarrhoea, malaria, roundworms, stomach problems, toothache, venereal diseases and wounds. Several ethnopharmacological studies have shown that crude extracts and chemical compounds from E. natalensis demonstrated many biological activities both in vitro and in vivo, which included antibacterial, antidiabetic, antifungal, antimycobacterial, antiviral, antioxidant, antiplasmodial, larvicidal, antischistosomal, molluscicidal, dentin permeability and hepatoprotective activities. Future studies should focus on the mechanism of biological activities of both crude extracts and chemical compounds from the species, as well as structure–function relationships of bioactive constituents of the species.


Introduction
Euclea natalensis A.DC. (family Ebenaceae) is traditionally used as herbal medicine to treat several human diseases and ailments in tropical Africa. The wide usage of the species as herbal medicine in tropical Africa has resulted in a major resurgence in interest in the ethnopharmacological properties of E. natalensis, a plant species characterized by several uses which are recognized culturally, medicinally and commercially. According to Van Wyk [1,2], the roots of E. natalensis have commercial potential as remedies for chest ailments, toothache, bronchitis, pleurisy, asthma, headache, and urinary tract infections, as well as mouth rinses or toothbrush sticks that may be developed into pharmaceutical drugs and health promoting products. The twigs and roots of E. natalensis are traditionally used as chewing sticks, toothbrushes and mouthwash for oral hygiene, for cleaning teeth and the gums in the belief that these plant parts benefit the health of the mouth and teeth [3]. In southern Africa, the root or twig of E. natalensis is peeled from the end of a small root or twig and the end is chewed to a fibrous brush, with the root changing in colour from white to yellow as it is chewed, imparting a pungent and refreshing taste to the mouth [4]. In East Africa, the twigs are used as toothbrushes and roots of E. natalensis are also chewed by women to impart a red colour to their mouths [5]. Research by Van Wyk and Gericke [4] showed that, in southern Africa, the root bark of E. natalensis is moistened and applied to the lips as a yellow-brown cosmetic by women. Research by Cunningham [6] revealed that roots of E. natalensis are sold as herbal medicines in herbal medicine informal markets in Mozambique and South Africa. Similarly, leaves of E. natalensis are sold as herbal medicines in herbal medicine informal markets in Tanzania [7].

Ethnomedicinal Uses of E. natalensis
The different uses of E. natalensis are summarized in Table 1, including data about herbal preparation and countries where such practices are applied. Information on phytochemicals is summarized in Table 2 and associated pharmacological properties are discussed separately. Ethnomedicinal uses of E. natalensis in Table 1 are validated by bibliography shown in Table 2 and pharmacological properties of the species discussed in Section 5 of the manuscript.
Euclea natalensis has been recorded in Angola, Botswana, the Democratic Republic of Congo, Ethiopia, Kenya, Malawi, Mozambique, Namibia, Somalia, South Africa, Swaziland, Tanzania, Zambia and Zimbabwe. The species is found in arid and rocky habitats, termite mounds, dune bush, open grassveld, thickets, forests, forest margins, river banks and swamps, with altitude ranging from sea level to about 1600 m above sea level [5,22,23]. It is a shrub or small to medium-sized dioecious tree. The roots, bark, twigs and leaves of E. natalensis exhibit several medicinal applications and used to treat or manage various human diseases and ailments throughout the distributional range of the species ( Table 1). The roots were the most used plant parts (83.3%), followed by bark and leaves with 6.7% each, leaf sap (3.3%) and twigs (1.7%). A total of 51 ethnomedicinal uses of E. natalensis are documented in the literature (Table 1)

Antimicrobial Activities
Euclea natalensis is widely used as herbal medicine for a wide range of infectious diseases caused by microorganisms. Such diseases or ailments include amoebic dysentery, bronchitis, chest complaints, diarrhoea, sexually transmitted infections, sores, syphilis, toothache, tuberculosis, urinary tract infections, venereal diseases and wounds [7,8,13,16,21,24,25,29,30,38,39,41]. Such wide use of E. natalensis against bacterial, fungal and viral infections in traditional medicine prompted several researchers to assess antibacterial, antifungal, antimycobacterial and antiviral activities of crude extracts and compounds isolated from the species. The antibacterial, antifungal, antimycobacterial and antiviral investigations reported mixed results as highlighted in the relevant sub-sections below.

Antibacterial Activity
Khan and Nkunya [40] evaluated antibacterial activities of E. natalensis root bark extract against Escherichia coli and Staphylococcus aureus. The extract was active against Staphylococcus aureus exhibiting 15-20 mm inhibition zone. Khan and Nkunya [40] evaluated antibacterial activities of the compounds mamegakinone 4, diospyrin 11 and 7-methyljuglone 12 isolated from E. natalensis roots against Bacillus anthracis, Bacillus cereus, Clostridium perfringens, Corynebacterium diphtheriae, Escherichia coli, Haemophilus influenzae, Klebsiella aerogenes, Neisseria gonorrhoeae, Pseudomonas aureginosa, Salmonella Heidelberg, Shigella dysenteriae, Shigella flexnerii and Staphylococcus aureus. The compounds were active against most of the bacteria except Escherichia coli and Pseudomonas aureginosa, with inhibition zone demonstrated by the pathogens ranging from 8 mm to 24 mm [40]. A preliminary antibacterial assay showed that the petroleum ether and chloroform extracts of E. natalensis root bark gave an inhibitory zone of 15 mm, at an extract concentration of 0.3 mg/mL against Staphylococcus aureus [50]. Lall and Meyer [61] evaluated antibacterial activities of water and acetone root extracts of E. natalensis against Bacillus cereus, Bacillus pumilus, Bacillus subtilis, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Micrococcus kristinae, Pseudomonas aeruginosa, Serratia marcescens and Staphylococcus aureus. The water and acetone extracts inhibited the growth of Bacillus cereus, Bacillus pumilus, Bacillus subtilis, Micrococcus kristinae and Staphylococcus aureus at concentrations ranging between 0.1 mg/mL and 6.0 mg/mL. The water extract did not exert any inhibitory action on Gram-negative bacteria, while the acetone extract showed inhibitory activity at a concentration of 5.0 mg/mL against all the Gram-negative bacteria investigated [61]. Weigenand et al. [52] evaluated antibacterial activities of betulin 1, lupeol 2, shinanolone 13, 20(29)-lupene-3β-isoferulate 14 and octahydroeuclein 15 compounds isolated from root bark of E. natalensis against Bacillus cereus, Bacillus pumilus, Bacillus subtilis, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pantoea agglomerans, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus and Streptococcus faecalis using the agar plate method with streptomycin sulphate as control. The compound shinanolone 13 showed inhibitory activity against Gram-positive bacterial strains at a concentration of 0.1 mg/mL and 20(29)-lupene-3β-isoferulate 14 showed inhibitory activity against Bacillus pumilus at a concentration of 0.1 mg/mL [52]. More et al. [35] evaluated antimicrobial activities of ethanol leaf extracts of E. natalensis against oral pathogens, namely Actinobacillus actinomycetemcomitans, Actinomyces naeslundii, Actinomyces israelii, Porphyromonas gingivalis, Prevotella intermedia and Streptococcus mutans, using the disk diffusion method. The extracts were active against the tested bacteria with both minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values ranging from 1.6 mg/mL to 25 mg/mL [35]. Van Vuuren and Naidoo [62] evaluated antibacterial activities of aqueous and a mixture of methanol and dichloromethane (1:1) leaf extracts of E. natalensis against bacterial pathogens associated with urogenital or sexually transmitted infections which included Gardnerella vaginalis, Neisseria gonorrhoeae, Oligella ureolytica and Ureaplasma urealyticum. All methanol and dichloromethane extracts exhibited noteworthy activities with MIC values ranging from 1.5 mg/mL to 2.0 mg/mL against all tested pathogens. The exhibited antibacterial activities validate the ethnobotanical use of E. natalensis as herbal medicine against vaginal discharge in Kenya [30], sexually transmitted infections and syphilis in South Africa [8,13,24,38] and venereal diseases in South Africa and Tanzania [8,13,25]. Sharma and Lall [63] evaluated antimicrobial activities of leaf and root ethanol extracts of E. natalensis against pathogenic bacteria, Propionibacterium acnes using the broth dilution method with tetracycline as positive control. The extracts showed weak activities with MIC value of 250 µg/mL in comparison to MIC value of 3.1 µg/mL demonstrated by tetracycline, the positive control.
Otieno et al. [25] evaluated antimicrobial activities of root ethanol extracts of E. natalensis, and extracts of E. natalensis mixed with root extracts of Carissa spinarum, Ximenia caffra and Harrisonia abyssinica against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus and Streptococcus faecalis using the disc diffusion assay with ampicillin and dimethylsulphoxide (DMSO) as positive and negative controls, respectively. The multi-plant extracts of E. natalensis mixed with Carissa spinarum, Ximenia caffra and Harrisonia abyssinica were more active against all tested microbes with inhibition zones ranging from 18 to 22 mm, MIC values ranging 8.3 ± 0.6 µg/mL to 55 ± 2.6 µg/mL and MBC values ranging from 0.02 mg/mL to 0.335 mg/mL [25]. The single extract of E. natalensis showed some activity against Staphylococcus aureus only with inhibition zone of 14 mm and MIC value of 55 ± 4.4 µg/mL. These results support use and preference of E. natelensis mixed with other plant species as remedy for stomach complaints in South Africa [14,24], amoebic dysentery, opportunistic infections and venereal diseases in Tanzania [25].

Antimycobacterial Activity
Lall and Meyer [39] evaluated antimycobacterial activities of acetone and water root extracts of E. natalensis against drug-resistant and drug-sensitive strains of Mycobacterium tuberculosis H37Rv using the agar plate method. Acetone and water extracts inhibited the growth of Mycobacterium tuberculosis at a concentration of 0.5 mg/mL. Lall and Meyer [39] evaluated the acetone and water extracts using a rapid radiometric method against Mycobacterium tuberculosis and obtained a MIC value of 0.1 mg/mL against the strains. In 2001, Lall and Meyer [64] evaluated the antimycobacterial activities of diospyrin 11 isolated from E. natalensis against the drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis using the radiometric respiratory BACTEC assay. The compound diospyrin 11 was active against Mycobacterium tuberculosis with MIC value of 100 µg/mL for all strains. Weigenand et al. [52] evaluated antimycobacterial activities of betulin 1, lupeol 2, shinanolone 13, 20(29)-lupene-3β-isoferulate 14 and octahydroeuclein 15 compounds isolated from root bark of E. natalensis against a drug-sensitive strain of Mycobacterium tuberculosis H37Rv using a rapid radiometric method with TB drugs, streptomycin and ethambutol as controls. The compound shinanolone 13 showed inhibitory activity against a drug sensitive strain of Mycobacterium tuberculosis at a concentration of 0.1 mg/mL [52]. Lall et al. [17] evaluated antimycobacterial activities of crude extracts, the compounds betulin 1, lupeol 2, diospyrin 11 and 7-methyljuglone 12 isolated from the roots of E. natalensis against Mycobacterium tuberculosis both in axenic medium and in a macrophage cell line. Crude extract, diospyrin 11 and 7-methyljuglone 12 isolated from the plant exhibited MIC values of 8.0, 8.0 and 0.5 µg/mL, respectively, against Mycobacterium tuberculosis while betulin 1 and lupeol 2 were inactive. The MIC value of 7-methyljuglone 12 against a panel of clinical pan-sensitive and drug-resistant strains of Mycobacterium tuberculosis ranged from 0.32 to 1.25 µg/mL [17]. Similarly, Van der Kooy et al. [52] evaluated antimycobacterial activities of isodiospyrin 3, mamegakinone 4, neodiospyrin 10, diospyrin 11, 7-methyljuglone 12 and shinanolone 13 isolated from root extracts of E. natalensis against Mycobacterium tuberculosis using the radiometric respiratory BACTEC assay. The MIC values of isodiospyrin 3 (10.0 µg/mL), neodiospyrin 10 (10.0 µg/mL), diospyrin 11 (8.0 µg/mL) and 7-methyljuglone 12 (0.5 µg/mL) compared well to those of the known antimycobacterial drugs ethambutol, isoniazid and rifampicin [55]. McGaw et al. [65] evaluated antimycobacterial activities of root extracts of E. natalensis and the compound diospyrin 11 isolated from the species against Mycobacterium bovis, Mycobacterium fortuitum and Mycobacterium smegmatis using a twofold serial dilution assay with anti-TB drug isoniazid as positive control. The root extracts showed some activities with MIC values ranging from 5.7 µg/mL to 16.3 µg/mL against the tested organisms, and the compound diospyrin 11 also showed some activities with MIC values ranging from 15.6 µg/mL to 62.5 µg/mL against the tested organisms [65]. McGaw et al. [66] evaluated antimycobacterial activities of acetone, chloroform and methanol root extracts of E. natalensis and the compounds lupeol 2, neodiospyrin 10, diospyrin 11, 7-methyljuglone 12 and shinanolone 13 isolated from the species against Mycobacterium bovis, Mycobacterium fortuitum and Mycobacterium smegmatis using a twofold serial dilution assay with anti-TB drug isoniazid as positive control. The plant extracts demonstrated activity with MIC values ranging from 5.7 to 664.1 µg/mL against Mycobacterium bovis, Mycobacterium fortuitum and Mycobacterium smegmatis [66]. The MBC values were relatively high, ranging from 625 to > 2 500 µg/mL against Mycobacterium bovis, Mycobacterium fortuitum and Mycobacterium smegmatis [66]. Compound lupeol 2 was inactive against all tested pathogens while neodiospyrin 10, diospyrin 11, 7-methyljuglone 12 and shinanolone 13 demonstrated activities with MIC values ranging from 1.6 to 166.7 µg/mL and MBC values ranging from 15.6 to 250.0 µg/mL against Mycobacterium bovis, Mycobacterium fortuitum and Mycobacterium smegmatis [66]. Lall et al. [67] evaluated in vitro antimycobacterial activities of ethanolic shoot extracts of E. natalensis against Mycobacterium tuberculosis H37Rv using a 96-well microtitre to determine the minimum inhibitory concentration of the extract. The E. natalensis extracts were also evaluated for in vivo antimycobacterial activities in Mycobacterium tuberculosis H37Rv infected mice. The MIC value of the extract was found to be 125 µg/mL against Mycobacterium tuberculosis compared to MIC value of 0.25 µg/mL exhibited by the positive control isoniazid. The antimycobacterial activities of E. natalensis extracts evaluated on Mycobacterium tuberculosis (H37Rv) infected Balb/c mice showed substantial decrease in bacterial loads when comparing the infected control group to the treatment groups, showing a decrease in lung homogenate colony forming units from 1.5 × 10 6 (control) to 7.1 × 10 3 (drug control) [67]. Therefore, the traditional use of E. natalensis extract against sores, purulent lesions and skin infections, cough could possibly be attributed to the activities of compounds such as of isodiospyrin 3, neodiospyrin 10, diospyrin 11, 7-methyljuglone 12 and shinanolone 14 against Mycobacterium tuberculosis. Evaluation of antimycobacterial activities of naphthoquinone compounds isolated from E. natalensis [54,[68][69][70] seem to suggest that 7-methyljuglone 12 and diospyrin 11 are the most active constituents.

Antifungal Activity
Lall et al. [53] evaluated antifungal activities of the compounds betulin 1, lupeol 2, shinanolone 13, 20(29)-lupene-3β-isoferulate 14, octahydroeuclein 15 and β-sitosterol 16 isolated from the root bark of E. natalensis against Aspergillus flavus, Aspergillus niger, Cladosporium cladosporioides and Phytophthora spp. Aspergillus niger was significantly inhibited by shinanolone 13, 20(29)-lupene-3β-isoferulate 14 and β-sitosterol 16 at 0.01 mg/mL. Of all the compounds tested, only octahydroeuclein 15 was found to be significantly effective against Phytophthora spp. at 0.1 mg/mL. The compounds octahydroeuclein 15 and β-sitosterol 16 significantly inhibited the growth of Cladosporium cladosporioides at 0.01 mg/mL [53]. None of the isolated compounds exhibited significant activities against Aspergillus flavus at 0.01 mg/mL [53]. Van Vuuren and Naidoo [62] evaluated antifungal activities of aqueous and a mixture of methanol and dichloromethane (1:1) leaf extracts of E. natalensis against Candida albicans, a pathogen associated with genital candidiasis or thrush. The aqueous and a mixture of methanol and dichloromethane extracts exhibited noteworthy activities with MIC values of 0.5 mg/mL and 3.0 mg/mL against Candida albicans, respectively. The exhibited anticandidal activities validates the ethnobotanical use of E. natalensis as herbal medicine against vaginal discharge in Kenya [30], sexually transmitted infections in South Africa [24,38] and venereal diseases in South Africa and Tanzania [8,13,25].

Antiviral Activities
Lall et al. [18] evaluated antiviral activities of acetone and water extracts of E. natalensis and the compound diospyrin 11 isolated from this species against herpes simplex virus Type 1 (HSV-1). The acetone extract of E. natalensis showed moderate antiviral activity against HSV-1, at concentrations of 0.1 to 0.02 mg/mL as shown by the reduction of virus-induced cytopathogenic effects and the protection of cells in a cell viability assay. The compound diospyrin 11 exhibited no inhibitory effects while water extracts exhibited weak activity at a concentration of 0.2 mg/mL which corresponded to a 42% cytopathic effect [18]. Mahapatra et al. [71] evaluated the HIV-1 reverse transcriptase inhibition activities of the compound 7-methyljuglone 12 isolated from the roots of E. natalensis and its synthetic derivatives against recombinant HIV-1 enzyme using non-radioactive HIV-RT colorimetric assay. The compound 7-methyljuglone 12 and synthesized compounds exhibited potent inhibitory activities ranging from 70% to 100% at 100 µg/mL [71]. These findings will provide baseline data to future research focusing on correlating the traditional use of E. natalensis as herbal medicine for viral infections and the antiviral properties of the species.

Antioxidant Activities
Nkobole et al. [72] evaluated antioxidant activities of acetone root extracts of E. natalensis using 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) free radical assay. The DPPH scavenging activity of the plant extract was 94.4 ± 0.01% which was comparable to 95.8 ± 0.01% demonstrated by the control, Vitamin C. Nkobole et al. [72] also evaluated antioxidant activities of the compounds lupeol 2 and β-sitosterol 16 isolated from the stem bark of Terminalia sericea using DPPH free radical assay. The compound lupeol 2 demonstrated high radical activity, exhibiting half maximal effective concentration (EC 50 ) values of 3.66 µM, which was comparable to the EC 50 values of 2.52 µM demonstrated by the control, Vitamin C [72]. Lall et al. [67] evaluated antioxidant activities of E. natalensis ethanolic shoot extracts using theDPPH free radical assay. The IC 50 value of the extracts against DPPH free radical was found to be 22.55 ± 2.93 µg/mL against 4.34 ± 0.48 µg/mL exhibited by the control, ascorbic acid [67]. These results obtained by Lall et al. [67] and Nkobole et al. [72] are important as intake of antioxidant rich herbal medicines scavenge free radicals and modulate oxidative stress-related degenerative effects.

Antiplasmodial and Larvicidal Activities
Clarkson et al. [73] evaluated antiplasmodial activities of aqueous, dichloromethane, dichloromethane and methanol (1:1) root and stem extracts of E. natalensis against Plasmodium falciparum using the parasite lactate dehydrogenase assay. Euclea natalensis dichloromethane and methanol (1:1) root and leaf extracts showed promising activities with IC 50 values of 5.1 µg/mL and 5.3 µg/mL, respectively [73]. The antiplasmodial properties demonstrated by E. natalensis imply that the species could be a promising candidate for further investigation as plant-based antimalarial agent. Historically, some of the antimalarial drugs have been derived from herbal medicines or from structures modelled on medicinal plant lead compounds and these include the quinoline-based antimalarials as well as artemisinin and its derivatives. Maharaj et al. [74] evaluated larvicidal activities of roots and stem dichloromethane extracts of E. natalensis by exposing the third instar Anopheles arabiensis larvae to the extracts with acetone and distilled water as controls. The root and stem extracts exhibited 100% mortality after 48 and 96 h of exposure, respectively [74]. These results provide a scientific basis to the traditional uses of E. natalensis as herbal medicine for malaria in East Africa [16], Mozambique [15], Tanzania [7,19] and Zimbabwe [20].

Antischistosomal and Molluscicidal Activities
Sparg et al. [75] evaluated antischistosomal properties of crude extracts of E. natalensis against the schistosomula of Schistosoma haematobium with praziquantel and a culture medium blank as controls. The schistosomula were placed into a culture medium to which the plant extracts were added. Euclea natalensis was active, killing 66.7% of the schistosomula worms at a concentration of 3.13 mg/mL. The schistosomula worms that were placed in the culture medium blank survived between 12 and 24 h of exposure while the praziquantel MIC value was 1 µg/mL [75]. Ojewole [76] evaluated molluscicidal activities of E. natalensis by exposing adult Bulinus africanus and Biomphalaria pfeifferi to sublethal and lethal doses of crude and aqueous bark, leaf and twig extracts of the species for a period of 24 h using niclosamide (Bayluscide ® ) (Coating Place Inc., Washington DC, WA, US) as reference molluscicide for comparison. The extracts demonstrated moderate to strong molluscicidal activity with lethal dose 90% (LD 90 ) value of 50-100 ppm compared to the positive control, niclosamide (Bayluscide ® ) which killed all the snails at a dose of 1 ppm [76]. These pharmacological evaluations are of importance in the traditional use of E. natalensis as remedy for schistosomiasis in South Africa [8,13] and remedy for intestinal worms in East Africa [5,16] and Malawi [28] and South Africa [13,14].

Dental Health
Sales-Peres et al. [77] evaluated the effect of an experimental gel containing E. natalensis extract on dentin permeability. The study assessed the in vitro variations in hydraulic conductance of dentin after treatment with E. natalensis gel and acidified fluorophosphate gel. The acidified fluorophosphate gel was worse for preventing dentin permeability (90.8%), followed by the control gel (77.1%), and the E. natalensis extract was the most effective (66.0%). Therefore, E. natalensis presented the most effective action to reduce dentin permeability. Sales-Peres et al. [77] revealed that E. natalensis gel not only reduced dentin permeability, but also resisted posttreatment citric acid challenge without changing its permeability. This effect can be attributed to the naphthoquinone compounds present in twigs and roots of E. natalensis as the compounds result in the dentin tubule obliteration due to the formation of a protective layer on the teeth [77]. Similarly, Sales-Peres et al. [78] evaluated the effect of E. natalensis gel on the reduction of erosive wear with or without abrasion, in enamel and dentin. The authors carried out five-day experimental crossover phases with volunteers wearing palatal devices containing human enamel and dentin blocks. The E. natalensis gel was applied in a thin layer in the experimental group and was not applied in the control group. The E. natalensis gel caused less wear in enamel in the experimental group than in the control group and a statistically significant value was found for erosion and erosion and abrasion in dentin. Therefore, E. natalensis may play a role in the prevention of dentin loss under mild erosive and abrasive conditions [78]. Based on the research findings of Sales-Peres et al. [77,78], E. natalensis extracts may be an alternative health product to protect oral health and prevent dental caries, tooth wear and dentinal sensitivity. These findings support the traditional use of the twigs and roots of E. natalensis as chewing sticks, toothbrush, mouthwash and remedy for toothache in Kenya [5,30], Mozambique [34,35], South Africa [4,8,13,35,38], Swaziland [32] and Tanzania [5,7,21]. The regular use of E. natalensis as chewing sticks, mouthwash or toothbrush might control the formation and activity of dental plaque and therefore reduce the incidence of gingivitis and possibly of dental caries [31]. These findings corroborate the wide application of the twigs and roots of E. natalensis as chewing sticks, toothbrush, mouthwash and remedy for toothache in Kenya, Mozambique, South Africa, Swaziland and Tanzania [4,5,7,21,30,32,38].

Hepatoprotective Effects
Lall et al. [67] evaluated in vitro hepatoprotective activities of E. natalensis ethanolic shoot extracts on human HepG2 cells. The hepatoprotective activities of E. natalensis extracts were tested in vivo using a rat model of isoniazid-and rifampicin-induced hepatotoxicity. Euclea natalensis showed a hepatoprotective effect (50% at 12.5 µg/mL) and the ability to increase T-helper 1 cell cytokines Interleukin 12, Interleukin 2 and Interferon α by up to 12-fold and the ability to decrease the T-helper 2 cell cytokine Interleukin 10 fourfold when compared to baseline cytokine production [67]. These findings provide a further scientific basis to the invention relating to the ethanolic extracts from the shoots of E. natalensis exhibiting immune stimulatory activity and hepatoprotective activity in vitro and in vivo studies [79]. The ethanolic extract of E. natalensis provides an immune stimulatory effect on peripheral blood mononuclear cells, selecting a Th1 immune response over a Th2 immune response. Research by [79] showed that ethanolic shoot extract of E. natalensis showed significant in vitro hepatoprotective activities against D-galactosamine and in vivo studies, the extract was nontoxic, acting as hepatoprotectant against the toxic effect of some of the first line drugs.

Cytotoxicity and Toxicity
Lall et al. [17] evaluated cytotoxicity of crude chloroform extract of the roots of E. natalensis, diospyrin 11 and 7-methyljuglone 12 by exposing different concentrations of samples to green monkey kidney cells (Vero) and a mouse macrophage cell line, J774A.1. Cytotoxicity results for the Vero cell line showed that the crude extract and diospyrin 11 had 50% maximal inhibitory concentration (IC 50 ) values of 64.87 and 17.78 µg/mL, respectively. The concentration of 7-methyljuglone 12 that effected a 90% reduction of growth of Mycobacterium tuberculosis Erdman within J774.1 macrophages was 0.57 µg/mL [17]. Similarly, Lall et al. [18] evaluated cell toxicity of root extracts of E. natalensis by determining the effect of the crude extracts and diospyrin 11 on the monolayers of primary vervet monkey kidney (VK) cells. The dose of the plant samples that inhibited 50% cell growth (ID 50 ) after the incubation period was 0.1 mg/mL and 0.2 mg/mL for acetone and water extracts, respectively. The compound diospyrin 11 exhibited an ID 50 value of 0.02 mg/mL on VK cells. The water extract from the roots of the plant was the least toxic to cell cultures and inhibited the replication of HSV-1 moderately at a concentration of 0.2 mg/mL [18]. More et al. [31] evaluated cytotoxicity of ethanol leaf extracts of E. natalensis using the XTT (sodium 3 -[1-(phenyl amino-carbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitro) benzene sulphonic acid hydrate) assay method. The extracts showed cytotoxicity activity on the Vero cell line with IC50 value of 285.1 ± 4.9 µg/mL [31]. Mahapatra et al. [71] evaluated cytotoxicity activities of the compound 7-methyljuglone 12 isolated from the roots of E. natalensis and its synthetic derivatives using the XTT assay method. Cytotoxicity results for the Vero cell line showed that the compound 7-methyljuglone 12 and synthesized compounds had EC 50 values ranging from 2.5 µg/mL to 36.1 µg/mL [71]. Kishore et al. [80] evaluated cytotoxicity activities of 7-methyljuglone 12 isolated from the root extract of E. natalensis and a series of its derivatives on MCF-7, HeLa, SNO and DU145 human cancer cell lines using the XTT method. Most of the 7-methyljuglone derivatives exhibited significant toxicity on HeLa and DU145 cell lines with IC 50 values ranging from 5.3 µM to 10.1 µM [80]. Lall et al. [67] evaluated in vitro cell cytotoxicity using cell lines (primary peripheral blood mononuclear cells, U937 monocytes and Chang liver cells), acute and sub-acute toxicity were carried out on eight-week-old female Balb/c mice by administering ethanolic shoot extracts of E. natalensis orally. During the study conducted on the cytotoxic effect of E. natalensis on peripheral blood mononuclear cells, U937 monocytes and Chang liver cells, E. natalensis extract showed no cellular toxicity with IC 50 values ranging from 131.3 ± 1.67 µg/mL to 208.9 ± 10.3 µg/mL. An IC 50 value below 50 µg/mL has been considered to be moderately toxic and samples with a toxicity value higher than 100 µg/mL have been considered to be non-toxic [67]. During mechanistic studies, the extract showed a 50% inhibition of mycothiol reductase activity at 38.62 µg/mL. During the acute and sub-acute studies, E. natalensis exhibited no toxic effect and the 50% lethal dose (LD 50 ) was established to be above 2000 mg/kg. The extract was able to reduce the mycobacterial load (1.5-fold reduction) in infected mice. Isoniazid and rifampicin caused significant hepatic damage in rats, and the extract was able to reduce the toxicity by 15% and 40% at 50 mg/kg and 150 mg/kg respectively [67].
Moshi et al. [19] evaluated toxicity of ethanol roots and stem extracts of E. natalensis using the brine shrimp lethality test. The extracts were toxic with LC 50 value of 19.33 µg/mL. These results obtained by Moshi et al. [19] indicate the possibility that E. natalensis may be toxic calls for assessment of target-organ toxicity studies.

Conclusions
Euclea natalensis is an important and frequently used herbal medicines in tropical Africa. The species is widely used for human diseases and ailments such as abdominal pains, antidote for snake bites, diabetes, diarrhoea, malaria, roundworms, stomach problems, toothache and venereal diseases. Recent research on E. natalensis focused primarily on antimicrobial properties of the crude extracts of the species, naphthoquinone and pentacyclic terpenoid compounds isolated from the species. Literature studies revealed that naphthoquinone and pentacyclic terpenoid compounds isolated from E. natalensis such as shinanolone 13 exhibited antibacterial effects [50], shinanolone 13, 20(29)-lupene-3β-isoferulate 14, octahydroeuclein 15 and β-sitosterol 16 exhibited antifungal effects [53], isodiospyrin 3, neodiospyrin 10, diospyrin 11, 7-methyljuglone 12 and shinanolone 13 exhibited antimycobacterial effects [17,39,52,55,65,66], diospyrin 11 and 7-methyljuglone 12 exhibited cytotoxic effects [17]. The compound 7-methyljuglone 12 appears to be the most efficient antimycobacterial of all the compounds that have been isolated from E. natalensis so far. Any future research on E. natalensis should consolidate its ethnomedicinal usage with its phytochemistry and pharmacological effects if ethnopharmacological potential of the species is to be fully realized. Such further research should assess mechanisms of actions, clinical effectiveness and proper dosage for the documented ethnomedicinal uses and associated pharmacological activities. Based on current information, the ethnomedicinal uses and documented pharmacological effects of the species show that there is not enough systematic data on phytochemistry and pharmacological effects for the majority of the ethnomedicinal applications of the species. There is still need for research on phytochemical, bioactive compounds and other medicinal ingredients and minerals that can be used to explain the wide use of E. natalensis as herbal medicine in tropical Africa. Future studies should also focus on the mechanism of biological activities and structure-function relationships of bioactive constituents of the species. Likewise, animal studies and clinical studies are to a large degree missing and should be carried out to determine the potential of this plant to be used in human medicine.