The Genus Diospyros: A Review of Novel Insights into the Biological Activity and Species of Mozambican Flora

Species of the Diospyros L. genus (Ebenaceae family) have been largely used in traditional medicine for the treatment of several diseases, especially infectious ones. To date, active major compounds such as naphthoquinones, triterpenoids, and tannins have been isolated and pharmacologically validated from Diospyros species. The present study summarizes the information available in the literature on the species described in the Flora of Mozambique. To do so, scientific databases (e.g., PubMed, Scopus, Web of Science, and Google Scholar) were searched using various keywords and Boolean connectors to gather and summarize the information. Of the 31 native and naturalized species in the Flora of Mozambique, 17 are used in different regions of Africa and were described for their traditional uses. They were reported to treat more than 20 diseases, mostly infectious, in the gastrointestinal and oral cavity compartments. This work provides an overview of the therapeutical potential of Diospyros species and explores novel insights on the antimicrobial potential of extracts and/or isolated compounds of these Mozambican species.


Introduction
The genus Diospyros L. (Ebenaceae family) contains species that have been recognized and used in traditional medicine (extended ethnomedical use) and have potential new health benefits supported by in vitro biological, in vivo pharmacological, and clinical tests [1][2][3][4]. Furthermore, within certain cultures or communities, various traditional systems have used all plant parts of this botanical genus (leaf, fruit, bark, twig, hardwood, and root) as herbal medicines [1,4].
Beyond their pharmacological value, Diospyros spp. have distinct and complementary important qualities, namely valuable wood, and edible fruits, which provide significant economic benefits and are recognized and utilized in various industrial and commercial sectors [1,4].
Generally, Diospyros spp. are tree shrubs or subshrubs with entire alternate leaves, solitary flowers, and fleshy fruits (berries) with usually two or more seeds. The characteristics of the leaves and flowers of these species are often used to identify fossil casts [5][6][7].
Diospyros species are predominantly distributed between the tropics, and the most notable diversity of this botanical genus occurs in Africa [5,6,8]. As confirmed in The Plant List [9], the WFO Plant List currently contains 1575 species related to the genus Diospyros, of which 734 have accepted scientific names [10]. Regarding the Mozambican flora, the genus is represented by 31 species (Table 1), corresponding to 18 accepted scientific name species, seven accepted subspecies (subsp.), three species that are considered synonyms, and three species that are not yet in the WFO plant list as of 12 February 2022 [10][11][12].   1 Distribution in Mozambique (blue, MD) [10,11]; Common name local (green, MD) [12]: Concerning primary health care (PHC), herbal medicines are used by 80% of the African population, and more than 70% of the population of Mozambique uses such medicines for treating all diseases [13][14][15]. For instance, several Diospyros species with antimicrobial potential have been reported [4,[16][17][18]. Worldwide, the magnitude of infectious diseases (ID), encompassing antimicrobial resistance (AMR), represents a major health problem (approximately 700,000 people die every year) [19,20]. Infectious diseases have a high impact in Africa, particularly in Mozambique [21].
Most of the native and naturalized Diospyros species of Mozambique's flora are generally recognized as traditionally used in different regions of Africa to treat different diseases, with a particular focus on infections affecting the gastrointestinal tract and oral cavity. This work will present a comprehensive overview of the therapeutic potential of Mozambican Diospyros species based on chemical, biological, and toxicological experimental data, particularly addressing its antimicrobial properties and including comparative elements concerning the biological activity of other Diospyros species. Table 2  zombensis. In addition, information is given on the part of the plant used as medicine, the manufacturing process of the traditional formulation, the main traditional therapeutic use, and the country from which the information originates. The results show that 54.8% of the total Diospyros species from Mozambique are referred to for their traditional use (Table 2). Among these, D. rotundifolia (Figure 1), traditionally used to treat diarrhea [22], is a prevailing species of dense undergrowth in the coastal area of the Marracuene District [23]. Table 2 shows the results of the collected ethnomedical data from seventeen Mozambican species, namely D. abyssinica, D. anitae, D. ferrea, D. kabuyeana, D. loureiriana subsp. loureiriana, D. lycioides subsp. sericea, D. mafiensis, D. mespiliformis, D. rotundifolia, D. mafiensis, D. mespiliformis, D. quiloensis, D. rotundifolia, D. squarrosa, D. usambarensis, D. verrucosa, D. villosa, D. villosa var. parvifolia, D. whyteana, and D. zombensis. In addition, information is given on the part of the plant used as medicine, the manufacturing process of the traditional formulation, the main traditional therapeutic use, and the country from which the information originates.

Ethnomedical Use of Diospyros Species of Mozambican Flora
The results show that 54.8% of the total Diospyros species from Mozambique are referred to for their traditional use (Table 2). Among these, D. rotundifolia (Figure 1), traditionally used to treat diarrhea [22], is a prevailing species of dense undergrowth in the coastal area of the Marracuene District [23]. Furthermore, among the Diospyros species present in the Mozambican flora, D. villosa ( Figure 2) is a species with a well-established traditional use of both leaf [24] and root [25]; the latter mainly used as a toothbrush for hygiene purposes [26]. Furthermore, among the Diospyros species present in the Mozambican flora, D. villosa ( Figure 2) is a species with a well-established traditional use of both leaf [24] and root [25]; the latter mainly used as a toothbrush for hygiene purposes [26]. Diospyros species have been reported to be used to treat the signals and symptoms of over 20 diseases. Two of these species (D. abyssinica and D. mespiliformis) have been mentioned most frequently and are used in two to five different countries in Africa (Table 2).
Based on the diverse description in the literature for the human use of the different parts of Diospyros, the results are grouped into infectious diseases (antibacterial, antifungal, anthelminthic, and antiviral); gastrointestinal (diarrhea, dysentery, emetic, flatulence, and other gastrointestinal disorders), oral cavity (oral hygiene, healing of oral wounds, and toothaches); urogenital (anti-hemorrhagic, dysmenorrhea, and infertility); skin diseases (dermatitis, fresh wounds, bedsores, and rashes); musculoskeletal (body pain, bruises, painful fractures, and rheumatism); and others conditions (diabetes, internal injuries, antidotes, hemostatic agents, and snake bites).
Among all the different Diospyros plant parts used in traditional medicine (Figure 3), the root is the most-used part (82%, Figure 3a) and is most used to treat infectious diseases. In the treatment of gastrointestinal disorders, it corresponds to 59%, for oral cavity infections, 41%, and for skin diseases, 18%, as well as for the management of other conditions, comprising 12% (Figure 3b).
The leaf is the second-most used part of the Diospyros species, but it is used in a similar percentage (18%) to the root to treat skin conditions and more commonly (24%) for musculoskeletal bruises, painful fractures, body aches, and rheumatism (Figure 3a,b).  Diospyros species have been reported to be used to treat the signals and symptoms of over 20 diseases. Two of these species (D. abyssinica and D. mespiliformis) have been mentioned most frequently and are used in two to five different countries in Africa (Table 2).
Based on the diverse description in the literature for the human use of the different parts of Diospyros, the results are grouped into infectious diseases (antibacterial, antifungal, anthelminthic, and antiviral); gastrointestinal (diarrhea, dysentery, emetic, flatulence, and other gastrointestinal disorders), oral cavity (oral hygiene, healing of oral wounds, and toothaches); urogenital (anti-hemorrhagic, dysmenorrhea, and infertility); skin diseases (dermatitis, fresh wounds, bedsores, and rashes); musculoskeletal (body pain, bruises, painful fractures, and rheumatism); and others conditions (diabetes, internal injuries, antidotes, hemostatic agents, and snake bites).
Among all the different Diospyros plant parts used in traditional medicine (Figure 3), the root is the most-used part (82%, Figure 3a) and is most used to treat infectious diseases. In the treatment of gastrointestinal disorders, it corresponds to 59%, for oral cavity infections, 41%, and for skin diseases, 18%, as well as for the management of other conditions, comprising 12% (Figure 3b). The majority of documented medicinal uses of Diospyros species are attributed to their effectiveness in treating microbial infections, encompassing bacterial, fungal, and parasitic infections. These include conditions such as diarrhea, dysentery, and various skin and oral cavity infections.

Chemical Composition of Mozambican Diospyros Species
The main classes of chemical constituents identified in Diospyros species from the Mozambican flora are listed in Table 3.
The presence of phenolic acid derivatives, like flavonoids and naphthoquinones (NQs), particularly 1,4-naphthoquinones (1,4-NQs), and terpenoids, mainly triterpenoids (especially lupan, ursane, oleanane derivatives) [3,4,17,62,63] and tetraterpenoids (carote-  The leaf is the second-most used part of the Diospyros species, but it is used in a similar percentage (18%) to the root to treat skin conditions and more commonly (24%) for musculoskeletal bruises, painful fractures, body aches, and rheumatism (Figure 3a,b).   The majority of documented medicinal uses of Diospyros species are attributed to their effectiveness in treating microbial infections, encompassing bacterial, fungal, and parasitic infections. These include conditions such as diarrhea, dysentery, and various skin and oral cavity infections.

Chemical Composition of Mozambican Diospyros Species
The main classes of chemical constituents identified in Diospyros species from the Mozambican flora are listed in Table 3.
Plumbagin has been identified on the root bark of D. abyssinica [28], and isodiospyrin (4), a dimeric 7-methyljuglone derivative [3], has been reported in a hexane extract of D. dichrophylla seeds [66] and in the diethyl ether extract of bark and phylum of almost all Mozambican Diospyros species [4].
Plumbagin has been identified on the root bark of D. abyssinica [28], and isodiospyrin (4), a dimeric 7-methyljuglone derivative [3], has been reported in a hexane extract of D. dichrophylla seeds [66] and in the diethyl ether extract of bark and phylum of almost all Mozambican Diospyros species [4].
In addition, from the methanolic extract derived from D. lycioides twigs, three naphthalene glycosides were identified [72], and carotenoids were identified in the fruit of this species [90]. The presence of galactiol and vitamin E in the D. ferrea leaf was also reported [67]. Condensed tannins (proanthocyanidins and oligopolymeric complex tannins), and particularly hydrolysable tannins (gallotannins, ellagitannins), and have also been identified in Mozambican Diospyros species such as D. villosa [4,25,58] and D. mespiliformis [82,89].
In addition, from the methanolic extract derived from D. lycioides twigs, three naphthalene glycosides were identified [72], and carotenoids were identified in the fruit of this species [90]. The presence of galactiol and vitamin E in the D. ferrea leaf was also reported [67].
So far, the biologically active marker secondary metabolites isolated and studied from several species of the genus Diospyros have mainly been naphthoquinones, triterpenoids, and tannins. Compounds belonging to these chemical classes have been isolated from the twigs, bark, roots, leaves, stems, and fruits of Mozambican species of this genus. Examples include plumbagin, 7-methyljuglone, diospyrin, and isodiospyrin, which have been isolated from the root of several Diospyros species.

In Vitro and In Vivo Biological Activity of Mozambican Diospyros Species and Marker Compounds
In Tables 4-6 (Table 4).
In vivo assays have shown that the hexane fraction of D. mespiliformis leaves has antiinflammatory properties (inhibits stronger the LOX), and that the methanolic extracts of different plant parts showed wound healing effects. On the other hand, the butanol and ethyl acetate fractions activate LOX activity. These results show that D. mespiliformis extract can have pro-inflammatory and anti-inflammatory effects [51].
Lupeol isolated from D. mespiliformis stem bark has shown analgesic activity in both pain inhibition (neurological-first phase) and origin (inflammatory-second phase) in biphasic tests (in vivo) [76].

Antihyperglycemic Activity
Another finding has revealed that the oral administration of a methanolic extract obtained from the leaves of D. ferrea (400 mg/kg) for a duration of 21 days in diabetic rats So far, the biologically active marker secondary metabolites isolated and studied from several species of the genus Diospyros have mainly been naphthoquinones, triterpenoids, and tannins. Compounds belonging to these chemical classes have been isolated from the twigs, bark, roots, leaves, stems, and fruits of Mozambican species of this genus. Examples include plumbagin, 7-methyljuglone, diospyrin, and isodiospyrin, which have been isolated from the root of several Diospyros species.

In Vitro and In Vivo Biological Activity of Mozambican Diospyros Species and Marker Compounds
In Tables 4-6 (Table 4).
In vivo assays have shown that the hexane fraction of D. mespiliformis leaves has antiinflammatory properties (inhibits stronger the LOX), and that the methanolic extracts of different plant parts showed wound healing effects. On the other hand, the butanol and ethyl acetate fractions activate LOX activity. These results show that D. mespiliformis extract can have pro-inflammatory and anti-inflammatory effects [51].
Lupeol isolated from D. mespiliformis stem bark has shown analgesic activity in both pain inhibition (neurological-first phase) and origin (inflammatory-second phase) in biphasic tests (in vivo) [76].

Antihyperglycemic Activity
Another finding has revealed that the oral administration of a methanolic extract obtained from the leaves of D. ferrea (400 mg/kg) for a duration of 21 days in diabetic rats showed significant antihyperglycemic activity [91]. The root of this species is rich in phenolic acids, especially gallic acid, and is therefore traditionally used as a potent antioxidant [70].

Antifungal Activity
Several studies have reported the potential antifungal activity of the root and root bark of most Diospyros species [42,54,92]. However, the antifungal activity of a leaf extract of D. mespiliformis has also been confirmed [47,93].
Various Diospyros medicinal plants are also effective against Candida spp. [1]. The methanolic extract of the D. abyssinica root is active against this microorganism [94]; how-Plants 2023, 12, 2833 13 of 34 ever, in another study, it was only moderately active against the same microorganism [95]. Another medicinal plant, D. mespiliformis, is more active against C. neoformans than against C. albicans. A leaf extract showed anti-C. albicans activity, while a bark extract showed in vitro activity against C. neoformans-isolated strains from South African AIDS patients [96].
D. mespiliformis, traditionally used to treat ringworm, shows remarkable antimicrobial activity against Trichophyton mentagrophytes and Microsporum canis. This result supports the traditional use of this species against dermatophytosis [47]. Aqueous and ethanolic extracts of the leaf and bark of D. mespiliformis showed significant antifungal activity against Aspergillus niger, Aspergillus flavus, and Microsporum gypseum [97].
The decoction of the stem of D. mespiliformis was tested against Plasmodium bergheiinfected mice and demonstrated potent activity, including the inhibition of beta-hematin in an in vitro study [98].
An estimation of the concentration of antioxidant vitamins (i.e., A, C, and E) from crude methanolic extracts obtained from the leaf, bark, and root of D. mespiliformis was also determined using the DPPH [51].    The results of in vitro cytotoxicity tests using normal and tumorous human cells and Artemia salina, as well as in vitro genotoxicity and in vivo acute and sub-chronic toxicity assessment of Diospyros species, are summarized in Table 5.  Most commonly, studies were found to be related to the in vitro assessment of cytotoxicity. For example, the extract of D. lycioides showed cytotoxicity to HeLa cells but was non-toxic to normal cells [38]. The compound diosquinone has been shown to be toxic against most cancer cell lines (human glioblastoma) and hormone-dependent human prostate cancer [84]. In contrast, 7-methyljuglone and isodiospyrin compounds are active against human colon carcinoma cells [61].
non-toxic to normal cells [38]. The compound diosquinone has been shown to be toxic against most cancer cell lines (human glioblastoma) and hormone-dependent human prostate cancer [84]. In contrast, 7-methyljuglone and isodiospyrin compounds are active against human colon carcinoma cells [61].
Preclinical safety assessments of Diospyros species are of paramount importance however, few studies related to Mozambican Diospyros species have been conducted to date. Cantrell et al. (2003) reported that D. dichrophylla is a potent phytotoxicant due to the presence of isodiospyrin (from the inner seed) at a lethal dose of 0.13 g/mL [66]. In anothe study, a hydroethanolic root extract of D. villosa showed possible development of rena dysfunction using an acute toxicity test in mice [111].

Antibacterial Activity
In vitro antibacterial activity data collected from eleven Diospyros species (represent ing 35.5% of the total) are summarized in Table 6. Of the 11 species examined, 47 extract (including AgNPs) showed antimicrobial activity against multiple bacterial strains. The methanolic extract was the most tested. In some of the studies mentioned, biodirected fractionation was also performed, and the antibacterial activity of the obtained fraction and isolated compounds was determined. The results obtained are also shown in Table 6   Preclinical safety assessments of Diospyros species are of paramount importance; however, few studies related to Mozambican Diospyros species have been conducted to date. Cantrell et al. (2003) reported that D. dichrophylla is a potent phytotoxicant due to the presence of isodiospyrin (from the inner seed) at a lethal dose of 0.13 g/mL [66]. In another study, a hydroethanolic root extract of D. villosa showed possible development of renal dysfunction using an acute toxicity test in mice [111].

Antibacterial Activity
In vitro antibacterial activity data collected from eleven Diospyros species (representing 35.5% of the total) are summarized in Table 6. Of the 11 species examined, 47 extracts (including AgNPs) showed antimicrobial activity against multiple bacterial strains. The methanolic extract was the most tested. In some of the studies mentioned, biodirected fractionation was also performed, and the antibacterial activity of the obtained fractions and isolated compounds was determined. The results obtained are also shown in Table 6.   According to the WHO, oral diseases are the most common non-communicable diseases, affecting people throughout life and causing pain, discomfort, disfigurement, and even death [114]. The Global Burden of Disease Study reports that oral diseases are among the leading causes of health problems, estimating that half of the world's population is affected by these diseases [114,115]. The same study provided a comprehensive assessment, and among the results evaluated, permanent tooth decay was the most common cause, representing a major public health problem in many countries [116]. Therefore, preventing and controlling the spread of this health problem is a global challenge, requiring greater efforts and potentially innovative approaches to achieve it. The branches of several Diospyros (particularly D. lycioides, D mespiliformis, and D. villosa) are used as toothbrushes for oral care [41,44,52,104,117], and their plant extracts have been shown to be effective against common oral pathogens, including Streptococcus mutans, S. sanguis, periodontal pathogens (Porphyromonas gingivalis and Prevotella intermedia), Lactobacillus spp., and several strains of Candida spp. [41,44,52,104,117]. In fact, over the past few decades, the scientific community has become increasingly interested in understanding the versatility of medicinal plants from traditional herbal medicine and their guaranteed availability to improve clinical approaches to infectious diseases with the intention of reducing antimicrobial resistance [4].

Naphtoquinones Antibacterial Activity
Plumbagin (1, Figure 4) is recognized as an effective antibacterial agent against both Gram-positive and Gram-negative strains of bacteria. This compound has also shown significant inhibitory activity (MIC < 12.5 µg/mL) against the resistant strain of Mycobacterium tuberculosis H37Rv [3,78,118]. Plumbagin isolated from the bark extract of D. maritima and showed activity against S. aureus and Aeromonas hydrophila (MIC = 0.625 and 5 µg/mL, respectively) [119]. In addition, it has also been obtained from the root of D. mespiliformis and has been described as one of the active marker compounds as well as an effective antibacterial agent against Gram-positive and Gram-negative bacterial strains [50,77,112].
Extensive research has unveiled the mechanism of action of diospyrin and 7-methyljuglone against M. tuberculosis, highlighting their crucial role as non-competitive ATPase inhibitors in key enzymatic reactions [120]. Additionally, emerging evidence has demonstrated the anti-tuberculosis potential of other compounds, such as crassiflorone and plumbagin from D. crassiflora, as well as diospyrone and plumbagin from D. canaliculata, both derived from the stem bark [121].
In a study conducted by Kuete et al. (2010), it was demonstrated that isobavacalcone and diospirone, derived from D. canaliculata, show promise as potential drugs against multidrug-resistant Gram-negative strains. These compounds exhibited enhanced activity when used in combination with efflux pump inhibitors, resulting in MIC values decreased to <10 µg/mL [122,123].
The antiaflatoxigenic activity of D. mafiensis root, another Mozambican medicinal plant, has been linked to the presence of diosquione and 3-hydroxydioquinone, making this herbal drug also an important natural antifungal for preventing fungal growth and aflatoxin accumulation in food [42]. In addition, this species has also been found to have analgesic, antidiabetic, anti-inflammatory, and antioxidant effects, likely correlated with the presence of these kind of constituents.

Antiparasitic Activity
NQs are highly active against pathogens in neglected tropical diseases, including malaria, leishmaniasis, and trypanosomiasis (sleeping sickness). Studies examining Plasmodium sp. have shown that isodiospyrin-derived isodiospyrol A exhibits antimalarial activity (IC 50 = 2.7 µg/mL) [132]. Anti-plasmodial activity has also been reported in the ethanolic extract of leafs of D. monbuttensis (IC 50 = 3.2 nM) [133]. Studies on malaria have proposed a redox cycling mechanism (described for the novel antimalarial-antiparasitic drug atovaquone) to support the in vitro activity of diospyrin and its analogues isolated from D. montana against L. donovani [134]. Plumbagin and its derivative was shown to be active against Leishmania spp., while diospyrin was active against Leishmania donovani [87]. Semisynthetic crassiflorone derivatives display trypanocidal activity against T. brucei and T. cruzi [135]. Antiplasmodial activities with IC 50 values of 16.5 to 29.4 g/mL against chloroquine-sensitive (3D7) and chloroquine-resistant (K1) strains of P. falciparum were observed for the juglone-based 1,4-NQs present in D. sylvatica [136].
Concerning the assessment of anthelmintic activity, it was demonstrated in vitro that D. oocarpa, D. nigrisence, D. candolleana, and D. montana are active on adult earthworms of Pheritima posthuma [137]. Similarly, NQ derivatives, including diospyrin from D. oocarpa, D. nigrisence, and D. candolleana, are antiprotozoal in addition to possessing anthelmintic constituents [138].

Triterpenoids Antibacterial and Antifungal Activities
Betulinic acid isolated from the root of D. lotus presents a broad spectrum against several Gram-positive and Gram-negative bacteria [85,[139][140][141]. Betulin isolated from D. rubra is an active agent against Streptococcus pyogenes, with a MIC of 85 µg/mL, and Corynebacterium diphtheriae, with a MIC range of 64 to 256 µg/mL [88].
Methanolic extract obtained from D. peregrina bark and seed containing triterpenoids has been studied for its antidiarrheal properties [142]. Similarly, the methanolic extract of D. peregrina fruit showed high activity against E. coli (12.6 mm zone of inhibition) and against fungi C. albicans (10.7 mm zone of inhibition) and Penicillium spp. (7.33 mm) [143].
Betulin present in the hexane fraction isolated from the bark of D. paniculata is very efficient against S. dysenteriae, which is responsible for diarrhea (MIC = 30 µg/mL) [144]. However, a study of a reductive green synthesis of nano-sized Ag particles using methanolic root extracts of D. paniculata showed that the maximum activity was displayed against Gram-positive bacteria compared to Gram-negative bacteria. The maximum activity was observed against Penicillium notatum, A. flavus, and Saccharomyces cerevisiae, with moderate activity towards C. albicans and A. niger [145].
In another study of ursane-type triterpenoids obtained from the leaf of D. dendo Welw. Ex Hiern [EtOH−EtOAc (50:50) extract], antimicrobial activity (62% at 10 µg/mL) against Pseudomonas aeruginosa was observed. This Gram-negative bacterium is considered one of the three main causes of human opportunistic infections and has recently been a useful model for the study of biofilm formation, implying antimicrobial resistance to antibiotics [146].

Antiviral Activity
Structure-activity relationships between betulinic acid and its synthetic derivatives inhibiting HIV-1 replication, HIV-1 entry, and HIV-protease or reverse transcriptase (RT) have been verified [147,148]. Betulinic acid was identified as a highly promising antiviral (anti-dengue) present in high proportions in most extracts of distinct species of Diospyros, particularly from the bark of D. glans [83]. Aridanin, isolated from methanol extracts obtained from the leaf, stem, and root of D. conocarpa, presents anti-HIV-1IN activity [149].
In a recent study, the antiviral activity of D. anisandra was demonstrated against the influenza virus AH1N1pdm09. The n-hexane fruit extract exhibited HA inhibitory (HAI) activity, and a fraction of it inhibited the hemagglutination from 12.5 up to 100 µg/mL, which was attributed to the synergistic effect of the different compounds present [150]. Previously, possible antiviral activity against influenza A and B viruses has been attributed to a redox effect of isolated zeylanone epoxide [151].

Antiparasitic Activity
Using in vitro antimalarial assays, betulinic acid 3-caffeate isolated from the dried leaf, twig, and branch of D. quaesita was shown to be moderately active against both chloroquinesensitive and chloroquine-resistant P. falciparum clones [86]. Lupeol and lupenone, isolated from the dichloromethane and ethyl acetate extracts of D. rubra stem, have shown moderate antimalarial activity against P. falciparum [88]. On the other hand, hydroethanolic extracts from the trunk of D. gracilescens and the hexane fraction showed higher activity against promastigote and amastigote forms of L. donovani (IC 50 = 5.84 µg/mL and IC 50 = 0.79 µg/mL, respectively) [87]. Aridanin isolated from methanol extracts of the leaf, stem, and root of D. conocarpa can be sources of new antitrypanosomal active principles [149].

Tannins
Tannins isolated from Mozambican Diospyros species represent an important class of secondary metabolites with remarkable antimicrobial potential against fungi, bacteria, and yeast [152]. Their mechanism of action involves the disruption of microbial enzymes and cell membranes, although their activities are diverse [153]. In addition, recent research has suggested the ability of tannins to generate hydrogen peroxide, which contributes to their important antibacterial properties [154].

Antibacterial and Antifungal Activities
D. melanoxylon bark is another medicinal plant considered to be active against Grampositive and Gram-negative bacteria, which is traditionally used for diarrhea, urinary, and skin troubles and has confirmed claims against E. coli, S. aureus, S. epidermidis, Shigella flexneri, Bacillus licheniformis, Bacillus brevis, Vibrio cholerae, P. aeruginosa, Streptococcus aureus, Candida kruesi, and Bacillus subtilis [155]. Furthermore, it shows promise in the treatment of candidiasis caused by different Candida species (C. viz, C. albicans, C. krusei, C. parapsilosis, and C. tropicalis), with MIC values ranging from 0.375 to 6.0 mg/mL [156]. Extracts derived from the bark of D. melanoxylon are rich in tannins and possess significant potential as antimicrobial agents. In a recent study using strains isolated from humans, it was effective against both Gram-positive and Gram-negative bacteria, suggesting the presence of a broad spectrum of antibiotic compounds or simply general metabolic toxins in the plant methanolic extract [157,158]. In another study conducted in India, acetone ethyl acetate and methanol extracts of D. melanoxylon showed a MIC < 30 µg/mL against Aeromonas hydrophila, Enterobacter aerogenes, E. coli, and Klebsiella pneumoniae [159].
Methanol extract obtained from the bark or seed of D. peregrina, which is rich in tannins and other phenols, was evaluated for its antibacterial potential against the pathogenic bacteria associated with diarrhea. The bark extract demonstrated inhibitory effects against S. aureus, Shigella dysenteriae, E. coli, and P. aeruginosa, while the seed extract inhibited all tested strains except for P. aeruginosa [160]. Similarly, the methanol extract of D. tricolor leaves, known for its abundance of tannins and other phenols, exhibited antibacterial activity against both Gram-positive bacteria (Bacillus cereus and S. aureus) and Gramnegative bacteria (Salmonella typhii and Escherichia coli) [161].
Diospyros kaki Thunb., known as the persimmon tree, is originally from Asia, but it is cultivated in various parts of the world, including Mozambique. Different plant parts are well-known and useful as medicinal plants, and the fruit is known as persimmon. This species has been extensively studied, particularly regarding the antimicrobial activity of the tannins isolated from it. In a study conducted by Liu et al. (2019), the antimicrobial effects of persimmon tannins (PTs) extracted from the fruit of D. kaki against methicillin-resistant Staphylococcus aureus (MRSA) were investigated. The persimmon tannins (MIC = 1000 µg/mL) displayed potential mechanisms of inhibitory activity (i.e., the tannins can change the normal morphology of MRSA and cause severe damage to the cell wall and cell membrane) [152]. In addition, the hydrolysate of condensed tannins (composed of a polymer of flavan-3-ols, such as catechin groups) exhibited high bacteriostatic activity in vitro against the M. avium complex (nontuberculous mycobacteria) that causes opportunistic chronic pulmonary infections [63]. Aqueous extract from the D. kaki fruit was tested in vivo, showing interesting antibacterial activities against Gram-negative strains compared to Gram-positive bacteria, justifying its use in traditional medicine for the treatment and/or management of disorders of the digestive system such as diarrhea [162].
The results of another study showed that the condensed tannins extracted from the unripened fruit of D. kaki displayed antibacterial activity against biofilms containing multiple bacteria. It is estimated that intraoral cavity biofilms consist of at least 800 types of bacteria. Therefore, it is suggested that this medicinal plant has a high potential for preventing dental disease and aspiration pneumonitis in geriatric patients and recovering patients when it is added to mouthwash and toothpaste [163].
The in vitro antibacterial potential of D. blancoi was also found against biofilm formation by S. mutans. Both extracts containing tannins and other phenols showed inhibition ranges of 96% for methanol and 95% for ethyl acetate [164].
Recently, Diospyros species rich in tannins have been applied in the development of nanoparticles. For instance, titanium dioxide (TiO 2 ) nanoparticles containing D. ebenum leaf extract exhibit excellent antibacterial activity and potential against Gram-negative bacteria E. coli [165]. Silver nanoparticles (AgNPs) containing aqueous extract from the fruit of D. malabarica have demonstrated antibacterial activity against S. aureus at 500 µg/mL and against E. coli at 1000 ug/mL, with an average zone of inhibition size of 8.4 ± 0.3 mm and 12.1 ± 0.5 mm and 6.1 ± 0.7 mm and 13.1 ± 0.5 mm, respectively [166]. Similarly, biogenic silver nanoparticles demonstrated excellent antibacterial activity against a broad range of bacteria, with the highest antibacterial activity observed against E. faecalis (17.77 mm) and B. subtilis (20 mm), also demonstrating good hemocompatibility against humans and rat red blood cells [167].

Antiviral Activity
No studies were found on the specific activity of tannins isolated from the native Diospyros species in Mozambique. However, a tannin isolated from D. kaki has been demonstrated to have in vitro antiviral activity against the influenza virus, vesicular stomatitis virus, poliovirus, coxsackievirus, adenovirus, rotavirus, feline calicivirus, mouse norovirus, Sendai virus, and Newcastle disease virus [168]. The results of another study involving D. kaki extracts with tannin contents ranging from 0.08 to ≥0.11 mg/mL demonstrated their capacity to inactivate human noroviruses and bacteriophage MS2, both of which are the cause of gastroenteritis and foodborne illnesses worldwide (i.e., the results suggest that the antiviral effect and astringent effects of tannins are likely related to noroviral genome reduction and MS2 inactivation) [169].

Antiparasitic Activity
Species of the genus Diospyros contain a broad spectrum of antimicrobial agents identified using in vitro and/or in vivo methods against strains capable of causing opportunistic infections as well as neglected parasitic diseases. The anthelmintic activity of a D. peregrina fruit extract containing tannins was compared to the standard drug albendazole. The extract was found to be more potent than the selected standard drug at a concentration of 10 mg/mL [170].
According to the WHO, malaria is one of the most widespread neglected diseases in Africa, caused by the parasite Plasmodium and responsible for severe immune complications and deaths. The anti-Plasmodium activity of extracts from various species of the Mozambican Diospyros species has been reported in the literature. Ethyl acetate extract from D. abyssinica leaves showed moderate activity against chloroquine-resistant Plasmodium falciparum (FcB1), while D. mespiliformis, traditionally used to treat malaria, showed potent antimalarial activity in mice infected with Plasmodium berghei and significant inhibition of beta-hematin using an in vitro assay [98].
The antiparasitic activity against Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei was confirmed in several studies on Diospyros species [99]. For example, an acetate leaf extract of D. abyssinica and the isodiospyrin and diospyrin marker compounds isolated from the bark by bioguided fractionation showed high anti-L. donovani activity (IC 50 = 1.5 g/mL, extract, and IC 50 = 0.5 g/mL, isolated compounds) [65].

Materials and Methods
This review was conducted according to the criteria described in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 statement (http: //www.prisma-statement.org/; accessed on 16 January 2023). For this purpose, the scientific literature data were considered until 10 December 2022.

Search Strategy
The scientific data were collected using the search engines PubMed, Scopus, Web of Science, and Google Scholar, identifying all scientific papers published between 1 January 1970, and 10 December 2022 using the keywords Diospyros AND antibacterial, Diospyros AND antifungal, Diospyros AND antiparasitic, Diospyros AND antiviral, Diospyros AND medicine, Diospyros AND chemical compounds, Diospyros AND biological activity, and Diospyros AND toxicity.

Study Selection
As described in Figure 7, a total of 5528 scientific studies were included in the search and initial data collection based on their title and abstract. After eliminating the duplicates, 2071 studies remained, of which 1852 could not be selected due to a lack of information relevant to this work. After the screening, 279 studies reporting on Diospyros were considered eligible for inclusion in this review.

Conclusions
Species of the genus Diospyros have been studied worldwide, with a significant number exhibiting pharmacological activity. One referenced example, D. kaki, native to East Asia, NaoXinQing, is part of a patented and officially approved traditional Chinese medicine formula for the treatment of stroke. However, there are no studies integrating data on all Diospyros species present in the flora of Mozambique.
More than 70% of Mozambique's population uses medicinal plants for primary health care, and a total of 54.8% of the Diospyros species used in the country's ethnomedicine are also used in other regions of Africa; however, the biological potential of most of them is still largely unknown. For example, 64.5% of these species were not tested for their

Conclusions
Species of the genus Diospyros have been studied worldwide, with a significant number exhibiting pharmacological activity. One referenced example, D. kaki, native to East Asia, NaoXinQing, is part of a patented and officially approved traditional Chinese medicine formula for the treatment of stroke. However, there are no studies integrating data on all Diospyros species present in the flora of Mozambique.
More than 70% of Mozambique's population uses medicinal plants for primary health care, and a total of 54.8% of the Diospyros species used in the country's ethnomedicine are also used in other regions of Africa; however, the biological potential of most of them is still largely unknown. For example, 64.5% of these species were not tested for their Abstract or full text in English;

Conclusions
Species of the genus Diospyros have been studied worldwide, with a significant number exhibiting pharmacological activity. One referenced example, D. kaki, native to East Asia, NaoXinQing, is part of a patented and officially approved traditional Chinese medicine formula for the treatment of stroke. However, there are no studies integrating data on all Diospyros species present in the flora of Mozambique.
More than 70% of Mozambique's population uses medicinal plants for primary health care, and a total of 54.8% of the Diospyros species used in the country's ethnomedicine are also used in other regions of Africa; however, the biological potential of most of them is still largely unknown. For example, 64.5% of these species were not tested for their

Conclusions
Species of the genus Diospyros have been studied worldwide, with a significant number exhibiting pharmacological activity. One referenced example, D. kaki, native to East Asia, NaoXinQing, is part of a patented and officially approved traditional Chinese medicine formula for the treatment of stroke. However, there are no studies integrating data on all Diospyros species present in the flora of Mozambique.
More than 70% of Mozambique's population uses medicinal plants for primary health care, and a total of 54.8% of the Diospyros species used in the country's ethnomedicine are also used in other regions of Africa; however, the biological potential of most of them is still largely unknown. For example, 64.5% of these species were not tested for their Duplicate scientific publications;

Conclusions
Species of the genus Diospyros have been studied worldwide, with a significant number exhibiting pharmacological activity. One referenced example, D. kaki, native to East Asia, NaoXinQing, is part of a patented and officially approved traditional Chinese medicine formula for the treatment of stroke. However, there are no studies integrating data on all Diospyros species present in the flora of Mozambique.
More than 70% of Mozambique's population uses medicinal plants for primary health care, and a total of 54.8% of the Diospyros species used in the country's ethnomedicine are also used in other regions of Africa; however, the biological potential of most of them is still largely unknown. For example, 64.5% of these species were not tested for their Not directly related to medicinal issues and others related but not with species of Mozambican Flora;

Conclusions
Species of the genus Diospyros have been studied worldwide, with a significant number exhibiting pharmacological activity. One referenced example, D. kaki, native to East Asia, NaoXinQing, is part of a patented and officially approved traditional Chinese medicine formula for the treatment of stroke. However, there are no studies integrating data on all Diospyros species present in the flora of Mozambique.
More than 70% of Mozambique's population uses medicinal plants for primary health care, and a total of 54.8% of the Diospyros species used in the country's ethnomedicine are also used in other regions of Africa; however, the biological potential of most of them is still largely unknown. For example, 64.5% of these species were not tested for their Containing irrelevant or incomplete information.

Conclusions
Species of the genus Diospyros have been studied worldwide, with a significant number exhibiting pharmacological activity. One referenced example, D. kaki, native to East Asia, NaoXinQing, is part of a patented and officially approved traditional Chinese medicine formula for the treatment of stroke. However, there are no studies integrating data on all Diospyros species present in the flora of Mozambique.
More than 70% of Mozambique's population uses medicinal plants for primary health care, and a total of 54.8% of the Diospyros species used in the country's ethnomedicine are also used in other regions of Africa; however, the biological potential of most of them is still largely unknown. For example, 64.5% of these species were not tested for their antibacterial properties, namely D. abyssinica subsp. attenuata, D. abyssinica subsp. In summary, out of the 31 native and naturalized species in the flora of Mozambique that are used in different regions of Africa, a total of 17 species have not been studied as antimicrobial agents, of which three species, namely D. dichrophylla, D. whyteana, and D. zombensis, have only been studied at the toxicological level. Of the 14 species that have already been the subject of antimicrobial studies, D. abyssinica and D. mespiliformis are the best studied.
This work provides comprehensive information on the chemical, biological, and toxicological studies of the Diospyros species present in the flora of Mozambique, examining their pharmacological potential in detail. Of the Diospyros plant parts, the root is the bestresearched and documented. The identified studies confirmed ongoing efforts to improve the understanding of the mechanism of action underlying the biological activity, and in particular, the antimicrobial activity of these species, drawing on their traditional use. In addition, several secondary metabolites of Diospyros are currently being investigated for their potential pharmacological applications. However, it is important to emphasize that most of the available data are in vitro assessments of biological activity. Therefore, further efforts are needed to obtain more comprehensive evidence aimed at strengthening the validity and applicability of the results and ultimately contributing to public health benefits, especially in the face of global antimicrobial resistance.