Appraising the therapeutical potentials of Alchornea laxiflora (Benth.) Pax & K. Hoffm., an underexplored medicinal herb: A systematic review

Ethnopharmacological relevance: Alchornea laxiflora (Benth.) Pax & K. Hoffm. (Euphorbiaceae) is an important traditional medicinal plant grown in tropical Africa. The stem, leaves, and root have been widely used in the folk medicine systems in Nigeria, Cameroon, South Africa, and Ghana to treat various ailments, including inflammatory, infectious, and central nervous system disorders, such as anxiety and epilepsy. Material and methods: The scientific name of the plant was validated using the “The Plant List,” “Kew Royal Botanic Gardens,” and Tropicos Nomenclatural databases. The literature search on A. laxiflora was performed using electronic search engines and databases such as Google scholar, ScienceDirect, PubMed, AJOL, Scopus, and Mendeley. Results: To the best of our knowledge, no specific and detailed review has been reported on A. laxiflora. Consequently, this review provides an up-to-date systematic presentation on ethnobotany, phytoconstituents, pharmacological activities, and toxicity profiles of A. laxiflora. Phytochemical investigations disclosed the presence of important compounds, such as alkaloids, flavonoids, phenolics, terpenoids, and fatty acids. Furthermore, various pharmacological activities and traditional uses reported for this botanical drug were discussed comprehensively. Conclusion: This systemic review presents the current status and perspectives of A. laxiflora as a potential therapeutic modality that would assist future researchers in exploring this African botanical drug as a source of novel drug candidates for varied diseases.


Introduction
Indigenous herbal medicines are the first-line treatment for most third-world countries (Mahomoodally, 2013). According to the World Health Organization (WHO), about 80% of the world population employs herbal medicine for their primary health care using plant extracts (Mahomoodally, 2013;Nabatanzi et al., 2020). Various factors encourage herbal medicines, such as acceptability, poverty, cost-effectiveness, accessibility, and unavailability of modern health facilities (Hossain et al., 2014). However, there are concerns about the toxic effects of certain botanical drugs if used unchecked and irrationally (Firenzuoli and Gori, 2007;Okaiyeto and Oguntibeju, 2021). Globally, 28,187 plant species have been recorded as constituting medicinal use in 416 families of angiosperm plants. Euphorbiaceae is among the top three families with a significantly higher proportion of medicinal plants (Phumthum et al., 2019).
Recently, it has been established that medicinal plants are rich sources for new drug development, and traditional medicinal data has a quite good success rate in new therapeutics (Ochieng et al., 2022). Africa represents about a quarter of the world trade of biodiversity, and it is surprising that only a few drugs have been commercialized compared to other countries (Maroyi, 2016;Ali et al., 2017;Nabatanzi et al., 2020). The reason could be the lack of documentation, the secretive practices of local healers and folklore medicine practitioners, or lack of interest by first-world countries (Geethangili and Ding, 2018). A. laxiflora is one of the least explored plants possessing diverse ethnomedicinal and non-medicinal uses, as reported from different cultures and localities in Africa for centuries. However, it has gained the scientific interest of researchers in the last 2 decades regarding its pharmacological activities. A few studies have been conducted to identify and isolate the bioconstituents, and only limited reports are available for pharmacological studies. Although this plant has a wide distribution throughout Africa, it is worth noting that only Nigeria and Cameroon were the countries with the highest number of reports considering plant occurrence, traditional uses, and pharmacological activities.
A couple of reviews broadly summarized the traditional and pharmacological uses of the Alchornea genus, primarily from Alchornea cordifolia (Schumach. & Thonn.) Müll. Arg. and Alchornea floribunda Müll. Arg. (Boniface et al., 2016;Agbo et al., 2020). However, no specific and detailed review has been reported in the literature on A. laxiflora. This review is intended to present detailed information systematically on the ethnobotany, phytochemistry, and pharmacology of A. laxiflora. Furthermore, this review will explore the therapeutic potential and evaluate future research opportunities pertaining to A. laxiflora. Figure 2 summarizes the crucial information on A. laxiflora.

Methods
The literature search was done from various search engines and databases such as PubMed, Google Scholar, ScienceDirect, AJOL, Scopus, and Mendeley. We examined the literature published before June 2021 on ethnomedicinal uses, phytochemistry, pharmacology of extracts, and isolated compounds of A. laxiflora. Following the general guidelines on scientific nomenclature for plants to avoid any ambiguity and errors (Rivera et al., 2014), the species names, families, plant authority, and synonyms were verified using books, journal articles, and Webpages such as the "Kew Royal Botanic Gardens" (mpns.kew.org), the Missouri Botanical Garden's Tropicos Nomenclatural database (www.tropicos.org), and "The Plant List" (www.theplantlist.org). The search terms "Alchornea laxiflora" or "A. laxiflora extract" or "A. laxiflora compounds" were used with no specified time limit. All articles with potential full-texts and titles/abstracts were included, and no language restrictions were applied. All the relevant references were checked for additional and unpublished citations. Most ethnobotanical data were collected from Nigeria, Cameroon, South Africa, and Zimbabwe. The pharmacological research literature on A. laxiflora was critically assessed for the general requirements of the pharmacological research of botanical drugs as suggested by Heinrich et al. (2020), and only the literature that met the general requirements was considered in this presented review.

Botanical description and distribution
A. laxiflora is a deciduous understorey tree or shrub that grows up to 6 m tall and is often found in places such as lowland tropical forests, wetlands, riverine vegetation, mixed deciduous woodlands, sub-montane forests, and semi-deciduous tropical rainforests (Mwavu and Witkowski, 2008;Obodai and Nsor, 2009;Tegene, 2018;Verlhac et al., 2018). The leaves are simple and alternate in arrangement, elliptic-lanceolate to oblonglanceolate in shape, with dimensions of up to 17 × 8 cm. Moreover, the leaves are thinly structured, light green in color, turning to yellow, or red color in the dry season with three-veined venation from the base and shallowly crenateserrate margination (Figure 3). The young leaves appear purple in color. The plant is monoecious, with male and female inflorescence on separate branches (Akinpelu et al., 2015). The flowers are unisexual on the same plant with conspicuous reddish bracts. The fruit is 5-7 mm in diameter with two-to-four-lobed capsules, which are thinly woody and blackish-brown (Hutchinson and Dalziel, 1937;Burkill, 1985). This plant has multiple traditional uses, but no attempts have been made to domesticate it. It was found to be key in resprouting woody species in natural or manmade disturbed forests (Mwavu and Witkowski, 2008). In Uganda, A. laxiflora was reported to be one of the several novel plant species used by Chimpanzees to make nests under anthropogenic pressure of habitat loss (McCarthy et al., 2017).

Ethnobotany
Traditionally, all parts of A. laxiflora have utility in folk medicine for various purposes in different regions of Africa. Interestingly, maximum reports of traditional uses were from Nigeria and Cameroon, DR Congo, South Africa, and Ghana. However, no literature reports are noticed from Tanzania, Kenya, Frontiers in Pharmacology frontiersin.org 03 Kenya, Malawi, Mozambique, Northern Provinces, Rwanda, Sierra Leone, Sudan, Swaziland, Tanzania, Uganda, Zambia, Zaïre, and Zimbabwe. Other than medicinal uses, A. laxiflora has deep-rooted effects on the environmental and cultural aspects of Africa. A diversity of folk medicine applications of A. laxiflora are emmenagogue, promoting dental hygiene, easing toothache, and managing sickle cell diseases, as well as being antidiabetic, anti-inflammatory, antioxidant, anti-infectious, antianemic, antifungal, and hepato-protective ( Figure 2).

Whole plant
In the South and Southwestern regions of Nigeria, this woody plant is an important component of rural architecture. Natives used to construct life fences (Fongod et al., 2014). In South Africa and DR Congo, A. laxiflora is reported to be used as an antivenom for snake bites (Molander et al., 2014). Similarly, the whole plant has been reported for the treatment of malaria, pile, dysentery, eczema, cough, and high fever in the Ekiti state of Nigeria (Jayeoba et al., 2012;Adeniran, 2015).

Leaves
Leaves are the most frequently used plant part in folk medicine preparations, taken orally in most instances as an infusion, decoction, or juice, followed by the stems, branches, and roots. Leaves crushed in water are sometimes applied externally to treat skin diseases (Ajibesin et al., 2007). Similarly, the leaves of A. laxiflora are squeezed and mixed with milk or cheese (wara). A cup of the resultant mixture is suggested to be taken twice a week as a remedy for anemia by the Yoruba tribe of southwest Nigeria (Fabeku and Akinsulire, 2008). For managing uterine contraction and prevention of miscarriages, traditional healers in the south of Nigeria use A. laxiflora leave concoction prepared by squeezing leaves in water, filtering them, and mixing them with calabash chalk or calabash clay, taken twice a day after conception for 6 months (Bafor et al., 2018). Moreover, in the food industry, A. laxiflora leaves are used as wraps to preserve Kola nuts (Garcinia kola) and other perishable food items (Ihejirika et al., 2015).

Stem (bark and branchlets)
The stem branches are used as Chewing sticks (local toothbrushes) for cleaning teeth in Nigeria (Farombi et al., 2003). Stem bark and branches have also been used in traditional medicine for various purposes, notably for malaria, anemia, emmenagogue, ringworm, venereal disease, typhoid fever, antioxidant, infertility in females, infectious diseases, tumor, inflammation, teething problems, and toothache in South Africa, Ghana, and Nigeria (Dzoyem and Eloff, 2015;Kaur et al., (2012); Obodai and Nsor, 2009).

Root
In the Ogun and Osun states of Nigeria, the roots and fruits are used as ethnomedicine to treat fibroids (Oyeyemi et al., 2019). In the Edo state of Nigeria, the root bark of A. laxiflora is boiled with an egg from local chicken and eaten as a remedy for hemorrhoids (Ugbogu and Chukwuma, 2019). The traditional practitioners in the Ekiti state of Nigeria also prescribe root decoction for lowering blood pressure and reducing incidences of heart failure (Adeniran and Falemu, 2017).
Traditional use of A. laxiflora is limited to Africa, with lots of similarities in therapeutic applications, possibly due to the shared cultural exchange of its ethnobotanical use. Table 1 shows the ethnomedicinal uses of A. laxiflora in different African countries, regions, and communities, together with the plant parts used.

Phytochemistry
Although various phytochemical screening studies have suggested the presence of multiple classes of chemical constituents such as alkaloids, saponins, tannins, flavonoids, phenols, steroids, cardio-active glycosides, and reducing sugars, more studies are required to fully characterize the phytochemistry. However, the flowers and fruits of this plant species have not been studied extensively to identify phytoconstituents, relating to their low degree of non-usage in traditional medicines. The leaves, stems, and barks are highly exploited parts of the plant for the isolation and identification of phytoconstituents. The leaf has a higher diversity of phytochemicals compared to other plant parts. All the isolated compounds from A. laxiflora are mentioned in Table 2, and their chemical structures are presented in Supplementary Figures S1-S7.

Flavonoids
Flavonoids are the most common group of natural polyphenolic substances found in all fruits and vegetables. The flavonoids reported in A. laxiflora are in the form of flavanol and glycoside. From the ethyl acetate soluble fraction of the crude methanolic leaf extract of A. laxiflora, one novel acetylated flavonoid quercetin-3,4′-diacetate and three known flavonoid glycosides, quercetin, quercitrin, and rutin, were isolated (Ogundipe et al., 2001b). Concurrently, two novel sulfated flavonoids were isolated for the first time in the genus Alchornea and the family Euphorbiaceae, namely, quercetin-Frontiers in Pharmacology frontiersin.org
This literature review reveals that A. laxiflora is rich in flavonoids, phenolic compounds, terpenoids, fatty acids, steroids, and alkaloids. To date, 132 compounds have been identified and structurally elucidated from the extracts of A. laxiflora, including 13 flavonoids, 19 phenolics, 22 terpenoids, 43 fatty acids, 6 alkaloids, and other secondary metabolites. Nonetheless, most of these compounds have been identified from the leaves, stems, and roots of this plant. Consequently, it is suggested to utilize inflorescence, flowers, and fruits to identify and isolate chemical constituents. Moreover, further research is required to establish the therapeutic applications of isolated compounds of this plant.

Pharmacological activity
Traditionally, the whole plant, leaves, roots, stem, and fruits of A. laxiflora are used to treat various complications in different regions of Africa ( Figure 2). In particular, the Cameroonian traditional medicine system documented different applications of this plant as remedies for various health issues in Cameroonian traditional pharmacopeia (Sandjo et al., 2011). A wide range of pharmacological activities of the A. laxiflora extracts and its isolated phytochemicals have been reported using different in vitro and in vivo methods in the last 2 decades (Tables 3, 4 and Figures 4-6).

Antibacterial and antifungal activity
The most common ethnomedicinal use of A. laxiflora includes the treatment of various infectious diseases such as typhoid, diarrhea, urinary tract infections, and venereal diseases. Hence, the application of A. laxiflora as an anti-infective agent prompted (Ogundipe et al., 2001b) to carry out bioactivityguided isolation of the active constituent of A. laxiflora. Ogundipe et al. (2001b) Figure 4). Different extracts (hexane, ethyl acetate, and butanol) of A. laxiflora leaves were screened against S. aureus, E. coli NCIB 86, B. subtilis NCIB 3610, and P. aeruginosa NCIB 950 using the agar well-diffusion method with streptomycin as a control. The mean zone of inhibition (ZOI) ranged between 10.1 and 17.1 mm, equivalent to the solvents (hexane, ethyl acetate, and butanol) used in the study (Oloyede et al., 2010), thereby getting negative results. Similar findings were reported (Essiett and Ajibesin, 2010) for antimicrobial evaluation of ethanolic extract of leaves against six human pathogenic bacteria-S. aureus NCIB 8588, B. subtilis NCIB 3610, E. coli NCIB 86, Proteus vulgaris (P. vulgaris) NCIB 67, P. aeruginosa NCIB 950, and Klebsiella pneumoniae (K. pneumoniae) NCIB 418-and two clinical fungal isolates, C. albicans and A. flavus. The obtained results revealed that the extract exhibits moderate inhibition against P. aeruginosa NCIB 950, P. vulgaris NCIB 67, and A. flavus with ZOI of 15 ± 3.6 mm, 11 mm, and 9 mm, respectively. The highest MIC value of 250 µg/ml was observed against P. aeruginosa NCIB 950 and A. flavus. However, no activity was observed with other tested strains (four bacterial and one fungal) (Table 3).      Frontiers in Pharmacology frontiersin.org Akinpelu et al. (2015) reported a broad-spectrum antimicrobial activity of hydroalcoholic extract from A. laxiflora leaves. The antibacterial and antifungal activity of the A. laxiflora extract was evaluated against a panel of bacterial (39) and fungal (17) isolates. The extract at a concentration of 25 mg/ml inhibited all the bacterial isolates, with the ZOI ranging between 12 and 24 mm and MIC ranging between 0.78 and 25 mg/ml. Similarly, at the concentration of 35 mg/ml, the A. laxiflora extract inhibited 15 isolates out of 17 fungal isolates, with ZOI ranging between 11 and 23 mm and MIC ranging between 8.75 and 35.00 mg/ml. The highest antibacterial, antifungal activity of A. laxiflora was observed against Shigella species (24 ± 0.50 mm; MIC 3.13 mg/ml) and Trichophyton tonsurans (T. tonsurans) (23 ± 0.50 mm; MIC 2.19 mg/ml), respectively.
The different extracts (aqueous, ethyl acetate, and ethanol) of A. laxiflora leaves were tested against the clinical strains of Salmonella typhi (S. typhi) and Salmonella paratyphi (S. paratyphi) isolated from human stool. The extract showed dose-dependent inhibition in both the tested strains in different concentrations, likely 20, 40, and 60 mg/ml. However, ethyl acetate extract at the concentration of 60 mg/ml showed the highest antibacterial activity against S. typhi and S. paratyphi with ZOI of 24 and 19 mm, respectively (Osuntokun and Olajubu, 2015).
Similar results were reported in another study by Wansi et al. (2017), in which the extract of methanol leaves exhibited preferred activity against S. typhi and Shigella flexneri (S. flexneri) with MIC values of 512 μg/ml and 1024 μg/ml, respectively. Osabiya et al. (2017) demonstrated that aqueous and ethanol extracts from the leaves of A. laxiflora inhibited six bacterial strains, namely, B. subtilis, S. aureus, E. coli, Enterococcus faecalis (E. faecalis), K. pneumoniae, and S. typhi. The MIC ranged between 2.5 and 40 mg/ml. However, it was a noteworthy result that the ethanol extract exhibited more potent inhibitory activity at a concentration of 60 mg/ml against S. aureus (19.33 ± 0.58 mm) and K. pneumoniae (18.33 ± 0.58 mm) compared with the antibiotic chloramphenicol (11.67 ± 0.57 mm and 10.01 ± 0.00 mm). Furthermore, in vitro antibacterial activity of essential oils from leaves of A. laxiflora was carried out against B. cereus ATCC 10872, B. subtilis ATCC 6633, S. aureus ATCC 25923, P. aeruginosa ATCC 9027, and E. coli ATCC 25922. The mean ZOI ranged between 7 and 9 mm in contrast to the standard antibiotic ampicillin, with ZOI ranging between 6 and 8 mm (Otuechere et al., 2019).
On account of the traditional use of A. laxiflora leave decoction and infusion for the treatment of digestive and gastric disorders, Ngnameko et al. (2019) investigated the anti-Helicobacter pylori (H. pylori) activity of A. laxiflora leave extract. The extract was active against H. pylori at a MIC of 20 mg/ml. Lately, in vitro antibacterial activity of hexane, chloroform, ethyl acetate, methanol, ethanol, and aqueous extracts of the leaves, roots, and stem bark of A. laxiflora on the skin, gastrointestinal, respiratory, and urinary pathogens was also conducted. The extracts were tested against four Grampositive bacteria, namely, B. cereus ATCC 11778, E. faecalis ATCC 29212, S. aureus ATCC 25923, and Staphylococcus saprophyticus (S. saprophyticus) ATCC 15305, and four Gramnegative bacterial strains, namely, E. coli ATCC 25922, K. pneumoniae ATCC 13883, Moraxella catarrhalis (M. catarrhalis) ATCC 23246, and Proteus mirabilis (P. mirabilis) ATCC 43071 using ciprofloxacin as a standard antibacterial agent. All the extracts were effective against most of the tested Gram-positive strains, with MIC ranging between 50 and 63 μg/ ml. In addition, bioactivity-guided fractionation of methanolic extract of the A. laxiflora stem results in the isolation of ellagic acid, 3-O-methylellagic acid, 3-O-β-D-glucopyranosyl-βsitosterol, 3-O-acetyl-oleanolic acid, and 3-O-acetyl-ursolic acid. All the compounds displayed antibacterial activity against tested strains with MIC values as low as 4 μg/ml (Siwe-Noundou et al., 2019) (Table 3).
Based on the literature survey, certain gaps were identified in the reported studies. For example, Osabiya et al. (2017) did not mention the strain collection numbers of investigated bacterial strains, which makes it difficult to establish a comparison with other studies. Similarly, limitations were observed in the studies reported by Ngnameko et al. (2019), Osuntokun and Olajubu (2015), and Wansi et al.

Frontiers in Pharmacology
frontiersin.org

FIGURE 4
Anti-microbial activity of isolated flavonoids from A. laxiflora, derived from Ogundipe et al. (2001b).  (Matuschek et al., 2014). Furthermore, the assessment of anti-microbial activity using only the disc diffusion method is a preliminary approach because it did not ascertain the exact concentration-causing antimicrobial effect. Oloyede et al. (2010) reported the ZOI of different extracts determined by the disc diffusion method only. Unfortunately, they did not use agar or broth dilution tests to determine the MIC values. Moreover, the use of the agar diffusion method to determine the antimicrobial activity of plant extracts is considered inadequate owing to the lack of diffusion of non-polar molecules into the aqueous agar matrix, insensitivity, and non-reproducibility of the results in different laboratories. Therefore, serial microplate dilution methods using INT or rezurasin as indicators of growth are the preferred methods to determine realistic and reproducible MIC values (Eloff, 2019). Only a few studies reported the outcomes of rapid INT colorimetric assay. Plant extracts displaying MIC value ≤100 μg/ml are considered to possess noteworthy antimicrobial activity (Bueno, 2012). Nonetheless, multiple studies included in this review have reported MIC values higher than the accepted limit of ≤100 μg/ml. It is often not determined whether the antimicrobial activity is caused by general toxicity to all cells or a selective activity against the microorganisms.
Frontiers in Pharmacology frontiersin.org 7.51 ± 0.24, 52.63 ± 0.22, and >100 μg/ml, respectively. However, the ethyl acetate fraction exhibited the most promising activity against both strains of P. falciparum. Further, the fractionation of ethyl acetate extract led to the isolation of 34 phytoconstituents, including polyunsaturated fatty acids (PUFA), phenolics, and flavonoids. Likewise, prophylactic, suppressive, and curative effects of ethanol A. laxiflora leave extract (200-600 mg/kg, p. o.) were tested in vivo using Plasmodium berghei (P. berghei) infected mice. The extract showed promising antimalarial activity (Table 4; Figure 5). Similar results were reported by Okokon et al. (2017a) with the A. laxiflora root extract. The root extract and fractions also exerted moderate activity against CQsensitive (Pf 3D7) and CQ-resistant (Pf INDO) strains of P. falciparum, with ethyl acetate fraction exerting the highest activity with an IC 50 value of 38.44 ± 0.89 and 40.17 ± 0.78 μg/ ml in Pf 3D7 and Pf INDO strains, respectively. Recently, an in vivo antiplasmodial activity of methanol and chloroform leave extract of A. laxiflora was reported (Table 4; Figure 5). Briefly, the extract was administered orally (200-600 mg/kg) to P. berghei-infected mice. After 5 days of the treatment study, methanol extract exhibited significantly higher prophylactic, suppressive, and curative activity than the chloroform extract. These studies confirmed the ethnopharmacological use of A. laxiflora as a promising indigenous antimalarial drug (Oluyemi and Blessing, 2019) (Table 4).

Larvicidal activity
Morah and Uduagwu (2017) investigated the larvicidal activity of ethanol and petroleum ether extracts from the leaves of A. laxiflora against the third and fourth instar Anopheles mosquito larvae. Larva mortality was observed for both petroleum ether and ethanol extract at all concentrations assayed (0.08, 0.1, 0.15, 0.2 mg/ml), with the lowest activity at 0.08 mg/ml and the highest mortality rate observed at 0.2 mg/ml. Moreover, petroleum ether extract exhibited the highest 78% mortality compared to 68%, by ethanol extract at a similar dose of 0.2 mg/ml. In contrast, the larvicidal activity was attributed to the presence of methyl palmitate, icosyl oleate, and diisooctyl phthalate (Table 4; Figure 5).

Anti-HIV potential
Various extracts of A. laxiflora leaves, stem, and roots were evaluated for their Anti-HIV activity using HIV-1 integrase strand transfer assay. HIV-1 integrase inhibitory activity dwelt in most of the root extracts of A. laxiflora. The methanolic extract of A. laxiflora root exhibited noteworthy HIV-1 integrase inhibitory activity at an IC 50 value of 0.21 ng/ml compared to chicoric acid taken as a reference (IC 50 = 6.82 μM) without any significant cytotoxicity against HeLa cells. The ethanolic root extract and ethyl acetate root extract also exhibited marked HIV-1 integrase inhibitory activity with IC 50 values of 67.07 and 6.034 μg/ml, respectively. Moreover, the inhibitory activity of five compounds (ellagic acid, 3-O-methylellagic acid, 3-O-β-Dglucopyranosyl-β-sitosterol, 3-O-acetyl-oleanolic acid, and 3-Oacetyl-ursolic acid) isolated from the methanolic stem extract of A. laxiflora was also investigated on HIV-1 integrase, although all the isolated compounds were non-cytotoxic. However, they did not exhibit significant anti-HIV-1 integrase activity. Only ellagic acid laid out the best HIV-1 integrase inhibitory activity with an IC 50 value of 90.23 μM. The IC 50 values of other isolated compounds (3-O-methylellagic acid, 3-O-β-D-glucopyranosylβ-sitosterol, 3-O-acetyl-oleanolic acid, and 3-O-acetyl-ursolic acid) were either >100 μM or could not be determined (Siwe-Noundou et al., 2019) (Table 4; Figure 5).

Antidiabetic activity
The use of A. laxiflora leave decoction for treating diabetes is common among the Badagry people of Nigeria. This practice was validated through in vitro and in vivo studies. Ogbole et al. (2016) showed that methanolic extract obtained from A. laxiflora leaves inhibits α-amylase moderately with an IC 50 value of 295.60 μg/ ml. The anti-diabetic properties of A. laxiflora have recently been studied using an in vivo alloxan-induced diabetic rat model. When methanolic extract of A. laxiflora leaves was administered orally at a dose of 500 mg/kg, it showed a significant (p < 0.05) anti-hyperglycemic effect by lowering blood glycemic levels in diabetic rats (Nimenibo-uadia, 2018) (Table 4).

Analgesic and anti-inflammatory activity
In order to substantiate the folklore medicinal use of A. laxiflora roots infusion or decoction as treatment of malaria and associated pain-related symptoms, Okokon et al. (2017a) investigated the analgesic activity of the crude ethanol extract and other solvent fractions of A. laxiflora using mice against the standard drug acetylsalicylic acid (ASA; 100 mg/kg) by acetic acid-induced writhing, hot plate, and formalin-induced hind paw licking methods. The administration of A. laxiflora root extract ((75, 150, and 225 mg/kg, p.o.) and extract fractions (150 mg/kg, p.o.) demonstrated a significant (p < 0.05-0.001) and dosedependent analgesic activity in all used pain models. The ethyl acetate fraction exerted the highest analgesic activity compared with the standard drug ASA. The aqueous and methanol extracts from A. laxiflora leaves were appraised for analgesic effect in mice. The hot plate and tail immersion tests were used to evaluate the central analgesic effects, whereas the acetic acid-induced abdominal writhing assay was used to evaluate peripheral analgesic activity. The extract increased the mean reaction Frontiers in Pharmacology frontiersin.org time to pain significantly in the hot plate and tail immersion tests. Additionally, the number of abdominal writhes also decreased substantially, endorsing the folklore use of this plant (Nwonu et al., 2018a). The anti-inflammatory activity of acetone extract from A. laxiflora leaves was investigated in vitro. In the study, A. laxiflora extracts exerted dose-dependent inhibition of NO production by LPS-activated malignant macrophage cell line RAW264.7 at doses 6.25, 12.5, 25, and 50 μg/ml. At the concentration of 25 μg/ml, the extract exhibited the highest percent inhibition (96.53%). Simultaneously, the A. laxiflora extract significantly inhibited 15-LOX activity with an IC 50 value of 46.03 μg/ml compared to quercetin used as a reference drug (35.85 μg/ml) (Dzoyem and Eloff, 2015). Furthermore, Ngoungoure et al. (2019) reported that methylene chloride/methanol (1:1; v/v) extract obtained from leaves of A. laxiflora demonstrated similar results. The A. laxiflora extract exerted 68.10% NO inhibition (IC 50 = 66.57 μg/ml) and 54.58% 15-LOX inhibitory activity (IC 50 = 90.42 μg/ml) (Table 4).

Anti-diarrheal activity
To substantiate the ethnomedicinal use of A. laxiflora leaves in Cameroon to treat some gastrointestinal disorders, Wansi et al. (2017) conducted an in vivo study to appraise the antidiarrheal activity of methanolic and aqueous extracts of A. laxiflora (125, 250, and 500 mg/kg) using S. flexneri-induced infectious, castor oil-induced secretory, and magnesium sulphate-induced osmotic diarrhea in rats. The methanolic extract showed promising antidiarrheal activity in all animal models. The A. laxiflora methanolic extract significantly prolonged the latency period at all doses in the model of osmotic diarrhea, with the 250 mg/kg dose giving the highest 71.6% prolongation in latency time (Table 4).

Hepatoprotective activity
In the Bamun folk medicine (Cameroon), A. laxiflora is reported to treat hepatitis and other liver-related disorders. As toxic hepatitis is often associated with the oxidative destruction of lipids and proteins, Njayou et al. (2008) carried out ex vivo rat liver microsomal lipid peroxidation (enzymatic and nonenzymatic) and protein oxidation inhibitory potential of methanol-methylene chloride extract of A. laxiflora leaves. On the whole, the A. laxiflora extract inhibited the biochemical process in a dose-dependent manner (10, 100, and 200 μg/ml) with the significant inhibition of microsomal lipid peroxidation (95.90 ± 0.57% and 79.17 ± 1.57%; enzymatic and nonenzymatic, respectively) and protein oxidation (95.60 ± 0.59%) at 200 μg/ml concentration.
The ethyl acetate extract of A. laxiflora was studied for its possible hepatoprotective effect against CCl 4 -induced hepatotoxicity in rats to rationalize some folklore use. The ethyl acetate extract of A. laxiflora at 100 mg/kg body weight significantly counteracted the CCl 4 -induced liver damage by lowering the elevated marker enzymes, namely, ALT, AST, ALP, and LDH levels in the blood to 18.872,7.054,22.864,and 180.321,respectively,compared to CCl 4 elevated levels of 35.712,12.513,27.509,and 480.312 for ALT, AST, ALP, and LDH, respectively. Further, histopathological analysis of the liver showed that the ethyl acetate extract may protect the liver from centrilobular necrosis, vacuolization, and macrovesicular fatty change set up by CCl 4. The hepatoprotective activity was ascribed to four isolated flavonoids, namely, quercetin, quercetin-3,7.3′,4′-tetrasulphate, quercetin-3-O-β-D-glucopyranoside, and quercitrin (Oloyede et al., 2011) (Table 4; Figure 6). In a separate study, A. laxiflora was tested for its ability to alleviate sodium arsenate-induced liver damage in Wistar rats. The hexane extract of the A. laxiflora leaves could significantly reverse the liver damage caused by sodium arsenate (Esosa et al., 2013). Similarly, the methanolic extract of A. laxiflora leaves significantly modulated the levels of liver biochemical parameters GGT, GST, ALT, and AST (Uhunmwangho et al., 2016). The hepatoprotective activity of A. laxiflora roots has recently been investigated using sodium arsenate-induced hepatotoxicity as a model. The hexane root extract could significantly lower the liver enzymes (ALT, AST, and ALP), liver metabolizing enzymes (NAD, GST, and Cytochrome b5), and other biochemical parameters (total protein, albumin level, and globulin) (Uhunmwangho et al., 2018). These findings backed up the folklore's claim that this botanical drug can help with liver problems. (Table 4). Oladiji et al. (2014) investigated the anti-anemic potential of aqueous leave extract of A. laxiflora at doses 100, 200, and 300 mg/kg, p.o. on hematological indices (PCV, Hb, RBC, MCV, MCH, and MCHC) of iron-deficient rats. The extract could significantly increase the hematological indices at all doses, compared with the reference drug ferrous sulfate and iron-sufficient rats, attesting to its folklore medicinal use in the treatment of anemia. Similar results were reported by Bada et al. (2017) with an ethanolic extract of A. laxiflora leaves. Another study was conducted on the anti-sickle cell anemic activity of A. laxiflora. The Anti-sickling activity of methanolic extract from A. laxiflora leaves was reported by Bamimore and Elujoba (2018). The extract had 98.8% sickling inhibitory action when given at a level of 8 mg/ml (Table 4; Figure 5).

Tocolytic and fertility-promoting activity
Traditional African medicine uses the leaf of A. laxiflora to treat various reproductive system diseases, including preterm Frontiers in Pharmacology frontiersin.org labor, miscarriage, menstrual disorders, postpartum discomfort, fibroids, and infertility. To appraise the ethnomedicinal use of A. laxiflora leaves to prevent preterm labor or miscarriage, Bafor et al. (2015) investigated the effect of methanolic extract of A. laxiflora leaves on female reproductive structures. Briefly, nonpregnant female mice were orally administered methanolic leave extract (100 and 1,000 mg/kg) for 6 days, using progesterone (10 mg/kg s.c.) as a positive control. The results revealed that the A. laxiflora extract at a lower dose (100 mg/kg) exerted progesterone-like activity on the ovaries, uterus, and cervical glands asserting its folklore use in maintaining pregnancy. In another investigation, ex vivo uterine contraction modulatory activity of methanolic extract of A. laxiflora was conducted using uterine tissue preparation isolated from female albino mice. The extract significantly inhibited spontaneous, oxytocin, and potassium chloride-induced uterine contractions, possibly via calcium and potassium ions channel interaction. Furthermore, the authors also isolated three important bio-constituents, namely, 3-deoxy-arabino-hept-2-ulosonic acid, 17hydroxyingenol, and pheophorbide A, possibly contributing to the methanolic A. laxiflora extract activity (Bafor et al., 2018). Uhunmwangho et al. (2016) studied the effect of the methanolic A. laxiflora leave extract on CCl 4 -induced reproductive toxicity. The oral administration of the extract at a graded dose (0.1, 0.5, 1.0, 10.0, and 50 mg/kg body weight) in male Wistar rats for 7 days, significantly reversed the toxic effects of CCl 4 and aided in male fertility by significantly increasing the percentage motility of sperm and inhibiting sperm morphological aberrations compared with positive control (normal saline) (Table 4).

Anticonvulsant and sedative activity
The leaves of A. laxiflora are traditionally used in the form of decoction or maceration to treat epilepsy and sleeplessness. Hence, the in vivo anti-convulsant and sedative activity of A. laxiflora was evaluated by Bum et al. (2009). The aqueous extract of the leaves of A. laxiflora was investigated for anticonvulsant and sedative activity in male Swiss mice using MES, PTZ, NMDA, INH, PIC, and STR-induced convulsions or turning behavior and diazepam sleepinduced animal models. The aqueous extract at a dose of 60 mg/kg protected 100% of mice against NMDA-induced turning behavior, and at a dose of 120 mg/kg, it protected 75% of mice against STR-induced seizures. The A. laxiflora extract failed to provide any significant protection against MES, PTZ, INH, and PIC-induced seizures. Moreover, in the diazepaminduced sleep test, A. laxiflora was insignificant in modifying the sleep duration of the control group, indicating the nonsedative activity of A. laxiflora. However, a recent study by Nwonu et al. (2018b) demonstrated the anxiolytic and sedative effects of aqueous and methanol extracts of A. laxiflora leaves using a staircase climbing test in mice. The results showed that both the methanol and the aqueous extracts of A. laxiflora leaves had significant sedative activity at a high dose (800 mg/kg) by significantly decreasing staircase climbing (Table 4).

Anti-Alzheimer activity
The cholinergic deficit is implicated in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease and associated progressive memory loss and cognitive function impairments. The cholinergic deficit is the result of a reduction in central nervous system ACh activity due to the AChE-related breakdown of ACh. The extracts from different parts of A. laxiflora have been screened for AChE and BuChE inhibitory activity. The first report is that of Dzoyem and Eloff (2015), reporting the AChE inhibition activity of the acetone extract of A. laxiflora leaves using the modified Ellman method. The extract exhibited significant but weak inhibitory activity with an IC 50 value of 364.12 ± 2.39 μg/ml compared to eserine with an IC 50 value of 4.94 ± 0.015 μg/ml. Similarly, the methylene chloride: methanol (1:1; v/v) extract at a single concentration of 200 μg/ml exhibited 36.02 ± 0.18% AChE inhibitory activity with >200 μg/ml, IC 50 value compared to galantamine (100 μg/ml; IC 50 value 24.65 ± 2.12 μg/ml); a standard drug (Ndam Ngoungoure et al., 2019). Elufioye (2017) investigated the effects of various extracts (hexane, ethyl acetate, and aqueous) obtained from A. laxiflora leaves, stem bark, and root bark in inhibiting both AChE and BuChE. The results reported that the A. laxiflora stem bark and root bark extracts showed selective AChE inhibitory activity with percent inhibition ranging from 10.69% to 34.20% (Table 4).

Antipsychotic activity
In vivo antipsychotic effects of aqueous and methanol extracts of A. laxiflora leaves were evaluated by Nwonu et al. (2018c). According to their findings, oral administration of the aqueous and methanol extracts in mice at graded doses (100, 200, 400, 800, and 1,600 mg/kg, p.o.) significantly reduced apomorphine-induced climbing and stereotypic behavior in mice at all tested doses, compared with chlorpromazine, a psycholeptic agent (Table 4). 0.1 and 1.0 μg/ml significantly altered the aminochrome-induced (75 μM) cell death and mitochondrial membrane potential reduction in both cell lines, implicating the potential usefulness of A. laxiflora in Parkinson's disease. However, further studies are warranted in isolating the active constituents and detailed elucidation of the mechanism of the action for safe and effective utility in Parkinson's disease (Table 4).

Anxiolytic activity
The anti-anxiety efficacy of aqueous and methanol extracts of A. laxiflora in mice was tested using the elevated plus-maze and the staircase behavioral paradigms to indicate the ethnomedicinal usage of A. laxiflora as an anxiolytic drug (Nwonu et al., 2018b). A. laxiflora exerted a significant anxiolytic effect on the elevated plus maze and staircase animal model against the standard drug diazepam (0.1 mg/kg, p.o.). Albino mice were divided into seven groups of six animals each. The control group (I) received 10% Tween-80 (10 ml/kg, p.o.), whereas the test groups (II-VI) received A. laxiflora aqueous or methanol leave extract in graded doses (100, 200, 400, 800, 1,600 mg/kg, p.o.) and standard drug group (VII) received standard drug diazepam (1 mg/kg i.p.) 30 or 60 min before the experiments. In the elevated plus maze model, the A. laxiflora methanol extract significantly increased the percent entry and percent time spent in open arms at lower and higher doses (100, 200, 800, and 1,600 mg/kg). In contrast, almost all doses significantly decreased the index of open-arm avoidance, attesting to anxiolytic activity. Similar results were observed for the aqueous extract of A. laxiflora leaves at doses 400 and 1,600 mg/kg, p.o., validating its anti-anxiety effect. In the staircase paradigm also, the methanol and the aqueous extracts of A. laxiflora leaves exhibited anti-anxiety activity by decreasing staircase rearing (aqueous extract: all doses; methanol extract: 400, 800, and 1,600 mg/kg, p.o.) and increasing staircase step-climbing (aqueous extract: 200 and 800 mg/kg p.o.; methanol extract: 100 and 200 mg/kg, p.o.) behaviors significantly (Table 4).

Antioxidant activity
In the Ugba region of Nigeria, the leaves of A. laxiflora are traditionally used to wrap food items for preservation. Farombi et al. (2003) were the first to report the antioxidant activity of hexane and methanol extracts from A. laxiflora leaves and roots using the ferric thiocyanate method, horseradish peroxidase catalyzed oxidation of ABTS, β-carotene linoleate model system, and rat liver microsomal lipid peroxidation assay. The antioxidant activity was observed in the following order: hexane root extract (76.4%) > methanol root extract (63%) > methanol leave extract (40%) > hexane leave extract (38%) at 0.05% concentration. The hexane root extract's antioxidant activity was compared with that of BHA (80%), a standard antioxidant. Another report indicated the antioxidant activity of hydroethanolic extract and solvent fractions of A. laxiflora leaves by the DPPH spectrophotometric assay method. All the test samples showed less scavenging activity (EC 50 12.97, 24.34, and 106.74 μg/ml for ethyl acetate, n-butanol, and crude ethanol extract, respectively) relative to the reference standard ascorbic acid (EC 50 4.78 μg/ml). Moreover, bioassay-guided fractionation of n-butanol fraction led to the isolation of two flavonoids, namely, taxifolin glycoside and quercitrin, suggesting their involvement in observed antioxidant activity (Adeloye et al., 2005).

Anti-cancer activity
Although A. laxiflora plant parts are traditionally used as an alternative medicine for cancer treatment, there is a paucity of experimental and clinical data on the anticancer activity of Frontiers in Pharmacology frontiersin.org A. laxiflora (Dzoyem and Eloff, 2015). In the brine shrimp bioassay against Artemia salina, the aqueous, ethanol, and methanol extract of A. laxiflora was found toxic with LC 50 values of 41.01, 8.91, and 142.40 μg/ml, respectively, indicating potential anti-cancer properties (Ogbole et al., 2016;Osabiya et al., 2017) ( Figure 5). The methanolic extract of A. laxiflora root, stem, and leaves had significant anticancer activity against drug-sensitive leukemia CCRF-CEM cell line with IC 50 values of >80, 49.21 ± 11.16, and 43.67 ± 4.06 μg/ml, respectively (Kuete et al., 2016). In two separate studies, Okokon et al. (2017aOkokon et al. ( , 2017b (Table 4). Though multiple studies have focused on the pharmacological role of A. laxiflora, some parts of A. laxiflora, such as stem branchlets, fruits, and seeds, have not yet been studied. Indeed, fruits of this plant are used in traditional medicine for curing infertility and fibroid treatment in women. Hence, it is highly recommended to conduct further research in this domain. Moreover, most of the research focuses on in vitro pharmacology of A. laxiflora. Thus, researchers are strongly recommended to conduct in vivo pharmacological and clinical studies to understand the molecular mechanism of action of this botanical drug.

Toxicity studies
Only a few researchers have evaluated the toxicity of A. laxiflora. Farombi et al. (2003) investigated the acute toxicity of the hexane and methanol extract of A. laxiflora roots and leaves by administering it in Wistar rats by oral route (100-5,000 mg/kg). After 14 days of monitoring, the extract did not affect mortality. In rats given the drug orally, the LD 50 was greater than 5,000 mg/kg.
The protective effects of hexane leaf extract of A. laxiflora against sodium arsenate-induced (2 mg/kg, p.o.) liver toxicity in Wistar rats was investigated by Esosa et al. (2013). The rats were treated with sodium arsenate for 2 days and suffered elevation in serum and liver biochemical indices (ALT, AST, ALP, GGP, and TB). Pretreatment with the extract of A. laxiflora hexane leaves at 10 mg/kg as prophylaxis significantly reversed these changes. Though, posttreatment of animals with the extract after a sodium arsenate treatment did not completely normalize the biochemical indices, suggesting that A. laxiflora extract may protect against sodium arsenate-induced hepatic toxicity.
The toxicity of the methanol extract of A. laxiflora leaves was evaluated by brine shrimp lethality bioassay. The methanolic extract displayed significant lethality with an LC 50 value of 142.40 μg/ml (Ogbole et al., 2016). In the same line, Osabiya et al. (2017) reported the high toxicity potential of ethanol and aqueous leaf extracts of A. laxiflora by brine shrimp lethality bioassay. The aqueous and ethanol extracts showed strong toxicity with LC 50 values of 41.01 and 8.91 μg/ml, respectively ( Figure 5).
In two separate studies, Okokon et al. (2017aOkokon et al. ( , 2017b) examined the possible development of toxicity caused by ethanol extracts of A. laxiflora leaves and roots by administering various doses of extracts intraperitoneally in albino mice. The LD 50 values were 2,236 and 748.33 mg/kg for leaves and root extract, respectively, indicating that the ethanolic extract might be toxic at higher doses when administered intraperitoneally.
The acute toxicity of the A. laxiflora aqueous and methanolic leave extract was assessed using albino mice. In this study, the aqueous extract was well tolerated by the animals up to 1,600 mg/kg (oral, intraperitoneal), whereas the methanol extract was safe up to 400 mg/kg and 1,600 mg/kg, intraperitoneally and orally, respectively (Nwonu et al., 2018b;2018c;2018a).
In a 21-day study, the effect of ethanolic extract from A. laxiflora was assayed on hematological indices and organ body weight of Wistar rats. The extract had an anemia-ameliorative effect by increasing the RBC, WBC, platelets, PCV, and Hb levels. However, there was an increase in the heart and lung weights at higher doses (200 and 300 mg/kg, p.o.), showing that the plant should be used cautiously when used orally at higher doses for the treatment of anemia .
Essential oils from A. laxiflora leaves obtained by hydrodistillation were also assessed for toxic effects at doses 100, 200, and 400 mg/kg on albino rats. Oral administration of essential Frontiers in Pharmacology frontiersin.org oils at higher doses (200 and 400 mg/kg) elevated serum ALT activity, depletion of serum bilirubin, and liver hypertrophy, suggesting possible derangement of hepatic functions at higher doses (Otuechere et al., 2019). A few reports have highlighted the toxicological aspects of A. laxiflora. Although in vitro and in vivo models have been utilized, only acute effects have been studied. Sub-acute, chronic, mutagenic, and teratogenic effects need to be assessed to support the safe use of this plant. Moreover, the toxicity analysis of chemical constituents is recommended to be carried out rather than its crude extract.

Clinical studies
Chewing A. laxiflora sticks are common oral cleaning practices in Nigeria (Odugbemi and Akinsulire, 2008), considered a good source of fluoride, an essential element for preventing dental caries. To rationalize A. laxiflora use as chewing sticks as a viable alternative in providing the required fluoride in poor communities, Emeke et al. (2019) determined and compared the salivary fluoride retention after the A. laxiflora stick used with a non-herbal fluorinated product by using a double blind cross over experimental study with 20 participants. Salivary fluoride concentration was determined after 0, 10, 30, 45, and 60 min after chewing stick use. In the results, the baseline salivary fluoride concentration was 25.95 ± 4.58 ppm, whereas after A. laxiflora use, the salivary fluoride concentration was 228.0 ± 032.80 ppm. The difference in mean salivary fluoride concentration was statistically significant (p < 0.001), indicating that chewing sticks are a cost-effective and efficient means of caries prevention. Although multiple pharmacological effects are assigned to A. laxiflora, a smaller number of clinical trials have been conducted. Hence, more clinical studies are required to establish the pharmacological significance or clinical applications of A. laxiflora.

Other applications
The ethanol extract of A. laxiflora leaves was evaluated against a field to store pests of maize grains, namely, maize weevil (Sitophilus zeamais). The extract displayed significant insecticidal activity in stored maize seeds, suggesting an ecofriendly alternative to synthetic pesticides (Ileke et al., 2020) ( Figure 5). The A. laxiflora leave extract has been reported to prevent the corrosion of mild steel in an acidic medium (Olasehinde et al., 2015). The A. laxiflora stem bark and leave extract has been used as a reducing and capping agent for synthesizing platinum and bimetallic platinum-copper nanoparticles for catalytic oxidative desulfurization of model oil (Olajire et al., 2018;. Similarly, Ekennia et al. (2021) biosynthesized quasi-hexagonal zinc oxide nanoparticles using the aqueous leave extract. Moreover, the synthesized nanoparticles exhibited good photocatalytic activity against Congo red dye solution and mushroom tyrosinase inhibitory activity.

Conclusion
This review highlights crucial information about the traditional use, phytochemistry, and pharmacological activities of crude extracts, as well as the phytochemicals of A. laxiflora. Diverse ethnomedicinal uses are linked with various parts of A. laxiflora, including the treatment of malaria, diabetes, sickle cell anemia, inflammatory conditions, skin disorders, and venereal diseases; gastrointestinal problems such as hepatitis, stomachache, dysentery, and piles; and neurological disorders such as anxiety, depression, insomnia, and epilepsy. Although several pharmacological activities of A laxiflora have been reported based on ethnomedicine uses, some must be validated pharmacologically. Meanwhile, most pharmacological activities have been reported using crude extracts rather than isolated compounds. As a result, further research is required to determine the links between isolated chemicals and medicinal uses. Phytochemical investigations revealed the presence of alkaloids, flavonoids, terpenoids, phenolic compounds, and fatty acids. The compounds 3acetyloleanolic acid, 3-acetoxyursolic acid, ellagic acid, and quercetin and their derivatives could be drug candidates for treating HIV, cancer, and microbial infections because of their potent biological activities. This study suggests that in-depth investigations on the mechanism of action, the pharmacological activity of isolated compounds, and toxicological analysis of biologically active extracts and active compounds of A. laxiflora are all worthwhile endeavors. Research in these areas could support possible medicinal uses and future development into therapeutic modalities.

Data availability statement
The raw data supporting the conclusion of this article will be made available by the authors without undue reservation.

Author contributions
NJ and BC planned the review outline, content, and structure. NJ, MT, SK, BC, YA, and PW wrote the manuscript and created the figures. HC, AK, HD, MB, and KC contributed to editing the manuscript, tables, and figures. All authors approve the manuscript for publication.
Frontiers in Pharmacology frontiersin.org