Phytochemical analysis, antioxidant and anti-inflammatory potential of FERETIA APODANTHERA root bark extracts

Plants have in one way or the other provided the basic need of mankind – food, shelter, clothing, protection from disease causing agents and treatment of various infections and illnesses since early days of human history [1]. Medicinal plants comprise of phytochemicals that improves the physiological balance of human beings and the knowledge of these healing properties has been passed down through generations. Traditional medicine is used by many African countries like Cameroon, Mali, Nigeria and Zambia to meet their health care needs [2, 3]. A vast range of medicinal plants around the globe has not yet been investigated to ascertain the claims made by traditional folks about their usefulness in treating diseases. Therefore, future research can be focused on medicinal plants.

Inflammation is usually a body response to tissue damage and to a number of systemic malfunctions including asthma, atherosclerosis, arthritis, physical injury and infection amongst many others [4]. Pro-inflammatory cytokines such as interleukin (IL)-1β, tumour necrosis factor (TNF)-α, and vascular endothelial growth factor (VEGF) mediate key points in inflammation [5]. Inflammation may be caused by foreign bodies, physical injury, allergens, radiation, stress, trauma, frostbite and alcohol amongst other things. Inflammation serves to isolate and eliminate infectious agents, induce repair and give protective response. Unfortunately, inflammation can lead to pain and discomfort that can persist for a long period of time [6]. Inflammation is manifested as pain, redness, loss of function of cells, swelling and heat. Based on its duration, inflammation is classified into acute and chronic inflammation.

Feretia apodanthera Del, is from the family Rubiaceae, a bush delicious shrub with winding or twisting branches, growing from 2 to 6 m tall [7], the leaf plate are elliptic to ovalate in shape with sometimes apiculate apex and rounded or cuneate base. The fruits are about 3-7 mm in diameter. It is commonly known as kuru-kuru by Hausa/Fulani - Northern Nigeria [8]. Traditionally, the root decortion of F. apodanthera is used to enhance erectile function of the penis and to treat stomach upset by the Hausa tribe of Kano State of Nigeria [8, 9].

Extracts from different part of the plant including the back, root and leaves have been used to treat renal and urinary infections, stomach ache, nausea, syphilis, infective wounds and other disease conditions [10, 11]. Feretia apodanthera is also used to enhance erectile function of the penis in some parts of Nigeria [8]. Mental conditions had been calmed using its extract. Taiwe et al. [12] reported that the extract of F. apodanthera was effective against cognitive errors, oxidative stress and seizures and in repairing memory impairment. Harman and tetrahydroharman isolated from methanol extract of F. apodanthera showed antimalarial activities and also a low cytotoxicity [13]. Recently, Feretia apodanthera extracts have also been shown to have high antioxidant activity similar to quercetin and relatively high flavonoid content [11]. The presence of these metabolites may be responsible for the therapeutic effect exhibited by this plant. Investigations with extract of Feretia apodanthera in rats also showed a decreased the activity of nuclear factor kappa β and nitric oxide which have been implicated in inflammation [14, 15].

Many plant antioxidant potentials are related to their therapeutic potentials [21, 22]. The qualitative phytochemical screening of the extracts of Feretia apodanthera revealed the presence of saponins, triterpenes, tannins, flavonoids, cardiac glycosides and steroids which might be responsible for the obvious anti-inflammatory activities of the extracts of the plant. This is in line with the report of Ahmadiani et al., [23] who stated that flavonoids as well as tannins possess anti-inflammatory effects. Flavonoids such as Quecetin are revealed to be useful in acute inflammation [24, 25]. Some flavonoids have a significant inhibitory potential against a wide array of enzymes such as phosphodiesterases, phospholipase A₂, protein tyrosine kinases, protein kinase C, and others [26]. Manthey et al., [27] also reported that a number of flavonoids act by inhibiting key enzymes that are central in synthesizing prostaglandins processes. Triterpenoids may perform its anti-inflammatory action by reducing the cells that expresses inducible nitric acid synthase (iNOS), eg lupeol [28] or by inhibiting the production of nitric oxide by decreasing iNOS expression [29]. This shows that the high potential of these phytochemical compounds to inhibit inflammation processes is due to a decline in pro-inflammatory cytokines and iNOS production [30, 31]. Alkaloids is said to reduce the intensity of oedema caused by carrageenan by inhibiting vascular permeability induced by histamine [32].

The significant (p < 0.05) anti-inflammatory effect of the diethyl ether, aqueous and ethanol extracts may be due to the presence of flavonoids, triterpenes and tannins while the reduced anti-inflammatory effect of n-hexane is probably due to the absence of tannin, saponin and flavonoids. Ethanol extract showed the highest anti-inflammation potential, this may be due to a higher intensity of triterpenes and flavonoids, along with the presence of saponin, tannin, cardiac glycosides, steroids and alkaloids. This is in agreement with the work of Han and Bakovic [31] which suggests that triterpenoids are biologically active in producing anti-inflammatory effects and with the research done by Hamaleinen et al., [33] that showed that flavonoids inhibits inflammation by inhibiting signal transducer and activator of transcription 1 (STAT-1) and nuclear factor kappa beta (NF-kβ) activations.

One of the primary methods for evaluating radical scavenging activity is by evaluating the ability of the antioxidants present in an extract to reduce purple coloured 1,2-diphenyl 2-picryl hydrazyl (DPPH] radical to yellow coloured diphenylpicrylhydrazine [31, 34]. A higher DPPH radical-scavenging activity is associated with a lower IC₅₀ value [32]. Therefore, the ethanol extract had the highest DPPH reducing activity based on its relatively low IC₅₀ values which was comparable with vitamin C as no significant (p < 0.05) difference was observed between their IC₅₀ (Table 3). A positive result by the aqueous and ethanol extracts in this test indicates that they contain antioxidants that can scavenge free radicals [35]. Ethanol extract was followed by the aqueous extract, the n-hexane extract and the diethyl ether extract showed the least DPPH reducing activity due to its relatively high IC₅₀ values. Previous studies on the DPPH radical scavenging of the aqueous and aqueous-acetone extract of F. apodanthera by Coulibaly et al. [11] showed lower IC₅₀ values than all the extracts. This may be due to the difference in sample or extraction variation.

Carrageenan induced rat paw oedema is a well-established animal model for evaluating the anti-oedematous effect of drugs or compounds [36]. From the data presented on the anti-inflammation activity of the crude extracts, oedema was mildly inhibited by 400 mg/kg of the aqueous, ethanol, diethyl ether and n-hexane extracts from the first three hours (varying from 14.47 to 33.65%). This is in line with the report by Stark et al. [37] that showed that the root bark of several African plants from the family Rubiaceae possess anti-inflammatory and analgesic effects. When compared to the standard ketoprofen, a significantly (p < 0.05) higher inhibition of carrageenan induced inflammation was exhibited by the extracts in the fourth and fifth hours (77.83 to 93.35%) for all the extracts except for n-hexane which had a reduced inhibition of oedema in the fifth hour. This shows that while ketoprofen may act by inhibiting the secretion of serotonin, histamine and bradykinin, the extracts produce its anti-inflammatory effect in the second phase by limiting production of prostaglandin and cyclooxygenase (COX-2), making Feretia apodanthera a promising plant for further studies. This is in line with the studies that reveals that formation of oedema caused by carrageenan is in two phases; the first hour after carrageenan injection (first or early phase) involves the release of serotonin, histamine and bradykinin while the second or late phase (2–5 h) with increased oedema formation that remains up to the fifth hour involves the release of prostaglandins [38] COX-2 also induction in the hind paw [39].

For FTIR, the analysis time was less than five minutes and it required a minute quantity of the sample. The spectrum revealed the presence of functional groups such as aromatics, ethers, esters, alcohol, alkanes, ketones, alkenes, alkynes, amines and carboxylic acid [40]. Fink [41] in his report stated that the presence of keto carboxylate elicits antioxidant effect by scavenging hydrogen peroxide; compounds with aromatic rings and alcohol groups as seen in phenols have been known to modulate inflammation at different levels by decreasing the production of reactive nitrogen and oxygen species, limiting the activity of iNOS and COX, suppressing inflammatory chemokines and cytokines synthesis as well as controlling pathways for NF-κβ signaling [42].

GCMS analysis of Feretia apodanthera showed the presence of 2-Pentanone, 4-hydroxy-4-methyl-; Hexadecanoic acid, methyl ester; trans-9-Octadecenoic acid, pentyl ester; Methyl cyclohexanepropionate; Palmitoleic acid; 10-Undecenoyl chloride and Heptasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13-tetradecamethyl- (Table 6). Hexadecanoic acid, methyl ester for example, is reported to reduce the production of nitric oxide- a key mediator in inflammation processes [35]- in cells by inhibiting arachidonic acid (Table 7) which is a precursor in the biosynthesis of prostagladins and therefore, inhibit the levels of pro-inflammatory mediators such as interleukin-10 (IL-10), prostaglandin (PGE₂) and tumor necrosis factor-alpha (TNFα) [43]. Palmitoleic acid which was also identified in the extract was shown to have inflammatory lowering effects [44]. Heptasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13-tetradecamethyl- have been identified in highly medicinal plants such as moringa leaves [45].