Alchornea floribunda (Müll. Arg.)-A review of its phytochemistry and biological activities

Alchornea floribunda is an evergreen plant that grows up to 32 feet tall and belongs to the family of Euphorbiaceae. It is found mainly in African forest undergrowth. In Nigeria, its leaves, stems and roots are widely used in folkloric medicine to manage ailments and diseases. The pharmacological activity of A. floribunda depends mainly on the part used. A previous study showed that the leaves have antiinflammatory, antimicrobial, antioxidant and anti-cancer activities, while its roots and stem possess antibacterial activity. Thus, this review takes a critical look at previously reported findings and information about the phytochemistry, biological activities and various isolated bioactive constituents from the leaf, root and stem of Alchornea floribunda.


INTRODUCTION
Alchornea floribunda is a shrub commonly found in African forest and is a member of the Euphorbiaceae family. Alchornea has many other species including Alchornea coelophylla, Alchornea cordifolia, Alcornea hirtella, and among others [1]. Alchornea floribunda leaves, stems and roots are used in Nigerian tradomedical practice for the treatment of many ailments including eczema, hepatitis, pains, infectious diseases and inflammatory disorders [2][3][4]. The water extract of the leaf and root bark is used in the management of human African trypanosomiasis (HATs) in Congo, while the decoction from the leaf is used in Cameroon for the management of parasitic and bacterial infections [5,6].
Alchornea floribunda is used in folkloric medicine for the management of various ailments and contains anti-inflammatory, antioxidant, antimicrobial, anti-cancer properties and as an aphrodisiac agent [2,[7][8][9][10]. Alchornea floribunda from tropical Africa is reputed to have hallucinogenic properties [11]. The powdered leaves decoction of A. floribunda when given to dogs increased its sensitivity of the sympathetic nervous system to epinephrine [12]. Alchornea floribunda leaves are eaten in the Congo as an antidote for poison, and the root sap or leaf is administered as a salve to irritated or wounded skin [13].

Phytochemistry
Several phytochemical analyses of different extracts of A. floribunda leaves, stem and roots have been carried out, and the extracts have been shown to contain a wide range of phytochemicals and compounds. Solvent extracts (n-hexane and methanol) and column fractions (chloroform and ethyl acetate) of the leaves of A. floribunda were found to contain terpenes, sterols, flavonoids, tannins, carbohydrate, glycoside, saponins and alkaloids [26].
Voukeng and co-workers [27] previously reported the phytochemical constituents of the stem bark and leaves of methanol extracts. The leave extract contained triterpenes, sterols and saponins while the stem bark had alkaloids, triterpenes, polyphenols and saponins. The phytochemical analysis of the solvent fractions (ethyl acetate and n-hexane) from the methanol extract of the leaves indicated that steroids and terpenoids were present in the n-hexane fraction while saponins, tannins and flavonoids were found in the ethyl acetate fraction [2]. Bio-assay guided purification of the ethyl acetate fraction of the methanol leaves extract by Okoye and Osadebe yielded a novel flavonoidal glycoside 3, 5, 7, 3'-tetrahydroxyflavone-3-O-α-L-rhamnoside [28]. Gas Chromatography-Mass Spectrometric analysis of the analysis of the non-polar fraction (n-hexane fraction) of the Alchornea floribunda leaves extract revealed the presence of unsaturated and saturated fatty acids and fatty acid esters [19]. Furthermore, the purification of the ethyl acetate sub-fraction of the methanol leave extract using semi-preparative reverse phase HPLC yielded seven known flavonoid glycosides [29].
Phytochemical investigation of the methanol stem barks and roots extracts led to the isolation of yohmbine alkaloid and three imidazopyrimidine alkaloids [30][31][32]. Several studies have been conducted with a view to isolating and characterising the bioactive secondary metabolites of the leaves, roots and stems of A. floribunda. These metabolites included steroids, flavonoids and alkaloids (Table  1; Figure 1).

Biological properties
In the continual search for new drugs, natural products have been found to be superior to combinatorial chemistry for the discovery of novel drug 'lead' compounds that could be developed into new products [33]. Plant extracts, fractions and pure compounds remain a starting point for the discovery of bioactive compounds for combating numerous diseases affecting animals and human beings [34]. Alchornea floribunda possesses immunomodulatory, antiinflammatory, antibacterial and antioxidant activities. Acute toxicity study of the extract was determined using Lorke's method by administering graded doses of methanol leaves extract in vivo and the animals were observed for any sign of toxicity and death for 24 h and 14 days respectively [2,4]. The result showed that the extract had a safety margin below 2000 mg/kg (p.o.) and 567.70 mg/kg (i.p.). Lower dose of the extract of A. floribunda is recommended for in vivo assays. An in-vivo acute and chronic inflammation study on the methanol extract and solvent fractions of the leaves of A. floribunda using animal model has been reported [2]. The administration of 200 mg/kg dose of the extract showed moderate inhibition of egg albumen induced oedema (54.69 %) 4 h post-administration of the extract compared to 53.13 % of the control (aspirin).
The ethyl acetate (EF) and n-hexane (HF) solvent fractions at a dose of 200 mg/kg elicited a stronger inhibition of oedema (67.19 and 81.25 %, respectively) 4 h post-administrations compared to 53.13 % inhibition for aspirin (100 mg/kg) as a standard drug. In the chronic anti-inflammatory study, solvent fractions reduced formaldehyde induced arthritis in rats. The result showed that HE (200 mg/kg) showed 36.79 % significant inhibition of leucocytes migration in vivo but could not stabilise hypotonicity and heat lysis of human erythrocyte (200 and 400 μg/mL) in vitro. This suggested that the anti-inflammatory properties of the extract and solvent fractions is attributed to the presence of secondary metabolites like terpenoids and steroids in nhexane fraction; flavonoids, tannins and saponins in ethyl acetate fraction [2].
Membrane stabilizing activity is a method of determining the preliminary in vitro antiinflammatory activities of plant extracts, fractions or isolated compounds [41,42]. Inflammation results from the release of lysosomal constituents because of degranulation of their cell membranes. The released lysosomes cause cell death. Stabilization of the membranes will inhibit the release of lysosomes and, thus, offers a mechanism for anti-inflammatory activity.
Stigmastane steroids (1-3) obtained from the nhexane leaves extract were assayed in vivo and in vitro for their anti-inflammatory activity. The compounds at a dose of 20 mg/kg (i. p.) showed significant inhibition of the egg albumen-induced acute inflammation with 1 having higher inhibition unlike 2 and 3 (50.9, 34.4 and 32.2% respectively) comparable to the inhibition of the standard drugs, indomethacin (39.90%) and prednisolone (48.0%) at a dose of 20 mg/kg 3 h post administration of the compounds and the drugs [8]. Similarly, the compounds at doses of 50 and 100 μg/ear exhibited significant inhibition of xylene-induced oedema which is dose dependent, 2 h post administration. The results showed that the isolated compounds showed better inhibition of the ear oedema than the standard drugs with compounds 1 and 2 at 100 mg/kg having three-or four-times inhibition than the drugs.
Steroids are more lipophilic than indomethacin and prednisolone and penetrate the skin lipoidal layers easily. This accounts for the increased topical anti-inflammatory activity of the compounds viz-a-viz the drugs. The in-vitro antiinflammatory study of compounds 1-3 showed significant inhibition of the heat-induced lysis of the human red blood cells which increased with increase in the dose administered, but none of the compounds had effect on hypotonicityinduced lysis. It has been reported that steroids exert their anti-inflammatory activity by their actions on the lysosomal membrane, which is like the human red blood cells [43]. Activityguided purification of the ethyl acetate fraction of the methanol leaves extract yielded a new favonol glycoside (4).
The in vivo ant-inflammatory activities of two major ethyl acetate column fractions (EFA and EFB) and the isolated compound were investigated using egg albumen induced rat paw oedema. The column fractions (EFA, 100 mg/kg) exhibited a pronounced and significant inhibition of oedema with inhibition of 78.4% 3 h post administration unlike EFB (100 mg/kg) that exhibited moderate inhibition of oedema (40.5%, 3 h). Compound 4 exhibited a dose dependent inhibition of acute inflammation in a dose dependent manner, with 50 mg/kg (%inhibition = 51.4) having higher inhibition than the standard drug (aspirin, 100 mg/kg; %inhibition = 45.9) 3 h post administration [2].

Immunomodulatory activity
The immune system is the body's natural guard against foreign agents and protects it against a wide variety of pathogens [44]. A healthy and efficient immune system is needed to fight the emergence of dreaded diseases like AIDs and Ebola virus that reduce the immune responses. Study has shown that extracts or fractions from A. floribunda are used in the treatment of immuno-inflammatory diseases [45]. In an in vitro assay, flavonoid glycosides (5-11) isolated from the leaves of Alchornea floribunda were assayed for their immune regulatory activities in relation to the expression of type-1 cytokines (IFNγ and IL-2) by CD4 + and CD8 + T cells using flow cytometric method. These compounds demonstrated their ability to modulate the intracellular expression of IFNγ and IL-2 in culture of splenic T lymphocytes stimulated with and without these compounds in the presence of 2 μM monensin. FACS data analysis of the result showed that simulation with the compounds (6.25-25 mg/mL) increased the level of CD8 + /IFNγ + and CD4 + /IFNγ + T compared to the untreated cells in the controls. The result showed increase in the ratio of CD8 + and IFNγ + from 57.85 to 72.45% compared to 57.85% for the untreated control. Also, the ratio of CD4 + and IFNγ + T increased from 3.21 to 7.21% compared to 2.57% for the untreated control with no secretion of intracellular IL-2 by treated T cells [29].

Antioxidant activity
Free radicals have been implicated in many diseases due to cellular damage caused by free radicals in vivo. Oxidative stress is responsible for many degenerative diseases like cancer, heart disease, aging, diabetics and immune system disorder [46]. Plant polyphenols have been reported to have good radical scavenging ability [47,48]. The in vitro antioxidant activity of compounds 12-15 isolated from the purification of the ethyl acetate sub-fraction of the methanol leaves extract was investigated. In vitro antioxidant assay was determined using 2, 2diphenyl-1picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP) and hydrogen peroxide scavenging activity assays. The compounds showed significant in vitro antioxidant effect (EC50 = 88-19 μg/mL) relative to the positive control (ascorbic acid, EC50 = 6 μg/mL) in the DPPH model; EC50 value range of 88-48 μg/mL compared to 66 μg/mL of the positive control in FRAP model and EC50 of 18-8 μg/mL compared to the control (8 μg/mL) in H2O2 assay.
Compound 14 had the highest radical scavenging activity in the DPPH and H2O2 models but had no antioxidant effect in the FRAP assay [4]. The in vivo antioxidant assay of the methanol extract and solvent fractions was investigated by determining their effects on serum catalase enzyme, serum superoxide dismutase enzyme and serum malondialdehyde in experimental animals. Ethyl acetate fraction (200 mg/kg) significantly elevated the level of catalase enzyme activity with a significant reduction in the level of serum malondialdehyde. Administration of 200 and 400 mg/kg doses of ethyl acetate fraction caused a significant increase in the serum superoxide dismutase and catalase enzyme, but only 400 mg/kg dose caused an in increase in serum superoxide dismutase enzyme unlike the n-butanol fraction that significantly caused an increase in both enzymes.

Antibacterial/antimicrobial activity
The demand for new anti-infectious drugs, especially those with activity against Gram negative and recalcitrant bacteria, is urgent [49]. More than thirteen million lives across the globe are lost each year due to infectious diseases [50]. Over the last ten years, this figure has doubled resulting from the emergence of multidrug-resistant strains. Regrettably, only few new antibiotics have been introduced to the market in the last four decades. The Global cost of antimicrobial resistance to GDP is estimated to be between $ 2.1 trillion -$ 124.5 trillion dollars [51]. Natural products made by plants and microorganisms represent an unparalleled starting point for the treatment of infectious disease [52]. Okoye and Ebi [26] have reported wide range of data on the antibacterial and antifungal activities of leaves extracts and solvent fractions of Alchornea floribunda using the agar well diffusion method against Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella keitambii, Aspergillus niger and Candida albicans. The result showed that only fraction A2 (terpenoid rich fraction) obtained from the purification of the n-hexane extract showed pronounced antimicrobial activity with inhibition zone diameters (IZD) of 42, 34 and 32 mm respectively for Pseudomonas aeruginosa, Salmonella keitambii and Bacillus subtilis respectively compared to ciprofloxacin (drug) with IZD of 45, 56 and 40 mm against P. aeruginosa, S. keitambii and B. subtilis respectively. Purification of the terpenoid-rich fraction (A2) yielded 14 sub-fractions, with only four sub-fractions (AF7, AF9, AF12 and AF13) having weak antimicrobial activities against S. keitambii and P. aeruginosa (IZD value range: 13-18 mm). The antibacterial activities of aqueous, methanol, ethanol, ethyl acetate, chloroform and n-hexane extracts of the leaves, stems and roots of A. floribunda using the microdilution assay have been reported [3] [53].

Anti-trypanosomal and anti-malaria activities
Human African trypanosomiasis (HATs) is a disease caused by protozoan Trypanosoma; and transmitted to humans through the bite of Tsetse flies. It ranks high in the list of neglected tropical diseases (NTDs) and causes considerable morbidity and mortality in livestock [54,55]. Malaria is also a protozoan disease caused by Plasmodium and transmitted to humans via the bite of the female Anopheles mosquito. It is the leading cause of child deaths in Nigeria and West Africa. Over 192,284 deaths resulting from malaria were recorded in 2015 [56]. Conventional drugs for the treatment of these parasitic diseases have some adverse effects and the search for new anti-protozoan agents is urgent [57]. Musuyu Muganza et al [58] have demonstrated that the aqueous roots and leaves extracts of A. floribunda have both antitrypanosomal and anti-malaria activities in an in vivo study. Their findings showed that the extracts could clear the parasitaemia levels of T. cruzi, T. brucei brucei, and L. infantum with IC50 values of 37.26, 19.65, and > 64 μg/mL respectively. The extracts also exhibited remarkable activity against K1 strain of P. falciparum (K1 strain) with IC50 value of 20.80 μg/mL.

CONCLUDING REMARKS
This review of A. floribunda reveals that various parts of the plant have different biological activities. The phytochemical constituents depend on the part of the plant investigated. While alkaloids are present in the stem-bark and roots, flavonoids and terpenoids are predominant in the leaves. These secondary metabolites exhibit anti-inflammatory, antioxidant, Immunomodulatory, antimicrobial and antiprotozoan activities.

Conflict of interest
No conflict of interest is associated with this work.

Contribution of authors
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