Chemotaxonomic Study of Six Nigerian Ficus Species ( Moraceae )

The current study was designed to investigate the chemical fingerprints in six Nigerian Ficus species based on the phytoconstituents for chemotaxonomic classification. The powdered leaf samples of the plants were extracted separately with absolute ethanol and chloroform and were then concentrated in vacuole to yield the crude ethanol and chloroform extracts respectively. The total flavonoid and tannin contents of each plant species were estimated via spectroscopic techniques. The results revealed the presence of tannins in chloroform and ethanol extracts of F. exasperata, F. sycomorus and chloroform extracts of F. glumosa, F. mucuso and F. vogelli. Flavonoids were present in chloroform extract of all the studied species and ethanol extract of F. mucuso, F. exasperata and F. sycomorus, while anthraquinone was absent in all. Also, saponins and alkaloids tested positive in F. glumosa, F. vogelli, F. cordata, F. sycomorus and F. mucuso (alkaloid absent), while steroids and triterpenes were present in F. glumosa, F. mucuso, F. vogelli and F. exasperata only. The presence of flavonoids, tannins, saponins, steroids and the absence of anthaquinone in all the Ficus species studied indicated that they are generic features. The study revealed that Ficus species contained valuable phytomarkers which are of pharmaceutical importance and suitable for authentication and differentiation among the studied species.


Introduction
Plants synthesized a wide variety of bioactive compounds that are used in treatment of ailment in both humans and animals and this dates back to antiquity (Paulsamy and Jeeshna, 2011).The science of chemotaxonomy is used for the classification of plants on the basis of their chemical constituents which relied on the chemical similarity of taxon (Atal, 1982;Rasool et al., 2010).Three broad categories of compounds used in chemotaxonomy are primary metabolites; secondary metabolites and semantides.Primary metabolites are the compounds that are involved in the fundamental metabolic pathways (Singh, 2011).
Products of primary metabolism are usually in high volume but low value and include such substances as carbohydrates, amino acids, fatty acids and proteins which are vital for growth, development and maintenance of life processes (Rehana and Nagarajan, 2014).The products of secondary metabolism, in contrast, are usually in low volume but high value and include such substances as alkaloids, flavonoids, saponins, tannins, phenolics, steroids, terpenoids (Rehana and Nagarajan, 2014).Species in the Moraceae family have important economic and medicinal value.Ficus species are rich sources of bioactive secondary metabolites such as flavonoids stilbenes, triterpenoids and xanthones (Ngadjui et al., 2005;Han et al., 2006;Jayasinghe et al., 2008;Lee et al., 2009).Plants produce these chemicals to protect themselves against diseases, stress and grazing but recent research demonstrates that many of such phytochemicals can protect humans against disease.But since these secondary metabolites occur in low volume, there is therefore need to screen such medicinal plant species in order to reveal their active principles and possibly isolate and characterize them.While chromatographic techniques are unavoidably important in the separation of complex herbal mixtures, thin layer chromatography (TLC) is often the first step in identifying compounds present in such samples (Karthika et al., 2014).In this present study, the phytochemical constituents of six Ficus species (Ficus exasperata, Ficus cordata, Ficus sycomorus, Ficus glumosa, Ficus mucuso and Ficus vogelii) were investigated using Thin Layer C Chromatographic (TLC) and spectroscopic techniques.
The diversity in the genus Ficus has resulted in a number of taxonomic problems leading to misidentification of the

Thin layer chromatographic analysis
The ethanol and chloroform extracts of the six Ficus species were manually spotted using capillary tubes on precoated TLC Silica gel plates 60 F254 (MERCK, Germany) (20 × 20 cm with 0.2 mm thickness (Wagner et al., 1996).The plates were activated at 105 °C for 15 min and then transferred into a chromatography tank already saturated with appropriate solvent systems.After the separation of phytochemical constituents, the spraying reagents such as Dragendorff reagent, 1% aluminium chloride, 1% ferric chloride, 50% vanillin sulfuric acid and 70% sulphuric-acetic acid in ethanol were used to identified alkaloids, flavonoid, tannins, saponin and steroid respectively.The retardation factor (Rf) was calculated from the equation: Retardation factor (Rf) = Distance moved by solute/Distance moved by solvent

Results
The results of phytoconstituents of chloroform and ethanolic leaf extracts of six Ficus species are as shown in Table 1.All the studied Ficus plants tested positive for flavonoids, tannins, saponins and steroids.However, anthraquinone was absent in all the plant samples; alkaloids were specific to F. glumosa, F. vogelli, F. cordata and F. sycomorus while triterpenes were present in F. glumosa, F. mucuso, F. vogelli and F. exasperata.Cardiac glycosides tested positive in F. mucuso, F. vogelli, F. exasperata and F. sycomorus while F. cordata, F. vogelli, and F. exasperata tested positive for xanthoproteins.Phlobatannins was present only in F. vogelli and F. sycomorus.
TLC fingerprints (Fig. 1) confirmed the presence of various phytochemicals in the studied Ficus species, as indicated in Table 1, after spraying with appropriate reagents (Table 2).Different Rf values (Table 3) were produced by different phytochemicals in different solvent system.Alkaloid with Rf value 0.88 is peculiar to F. vogelli, F. sycomorus, for flavonoids, while species like F. vogelli, F. glumosa, F. cordata and F. sycomorus all show congruence in having similar spots at Rf 0.70 and 0.80.Similar pattern of relationship is observed in the types of tannins, saponins and steroids with only few exceptions.
Quantitative analysis of flavonoids and tannins contents in the leaves of the studied Ficus species are as shown in Table 4.It was observed that chloroform extracted flavonoid better in all the studied Ficus species except for F. cordata and F. vogelli while ethanol showed a better solvent of extraction for tannin with exception of F. mucuso.The highest total flavonoid and tannins content were found in chloroform extracts of F. glumosa and F. mucuso respectively.Figures 2a and 2b show the extent of relationship among the Ficus species based on the ethanolic and Choroform extracts.The Ficus species were clustered differently based on the phytoconstituent as well as on the basis of types of extracts.
species.These misidentifications have gross implications for the effective use of the species for their medicinal benefits.Therefore, it is envisaged that this study will provide diagnostic chemical characters for taxonomic delimitation of each of these Ficus species.

Preparation of the extract
Each plant material was air dried and milled into powder.The powdered materials (20 g each) were separately extracted with absolute ethanol or chloroform for 48 hrs respectively and filtered.The filtrate for each sample material was concentrated in vacuole at 35 °C to yield the ethanol and chloroform extracts of each sample.

Estimation of tannins content
Tannic acid standard method (Van-Burden and Robinson, 1981) was adopted for the estimation of total tannin contents.The reaction mixture, consisted of extract (0.2 ml), 0.2 ml ammonium ferric citrate (3.5 g/l) and 0.2 ml of 20% (v/v) ammonia, was incubated for 15 min at room temperature.The absorbance was read at 500 nm against the reagent blank.The concentration of tannins in the extract was extrapolated from the standard curve and expressed as milligram tannic acid equivalent per g of extract (mg TAE/g extract).

Total flavonoids content
Aluminum chloride reaction method (Sun et al., 1999) was adopted for the estimation of total flavonoid concentration.The reaction mixtures consisted of extract (0.2 ml) in ethanol, distilled water (2.8 ml), 5% (w/v) NaNO2 (0.3 ml), 10% (w/v) AlCl3 (0.3 ml) and 4% (w/v) NaOH (4 ml).The reaction mixture was allowed to stand for 15 min and absorbance was read at 500 nm.The flavonoid content of the extracts was expressed as mg/g (QE) Quercetin equivalent.showed the presence of pharmaceutically useful classes of phytochemicals.Phytochemicals are naturally occurring and biologically active plant compounds (Halliwell and Gutteridge, 1992) that give plants their therapeutic and pharmaceutical properties (Evans, 2002).Alkaloids are group of naturally occurring products that contain mostly basic nitrogen atoms, though usually toxic to some organisms but have pharmacological agents.Most alkaloids are used as active drugs in treatment of various diseases to treat diseases such Flavonoids constitute one of the most important groups of polyphenols used as natural antioxidant in food, medicinal and non-nutritive plant materials because of their ability to scavenge free radicals (Kim et al., 1990).Flavonoids had been reported to possess many useful pharmacological properties such as anti-inflammatory, oestrogenic, anti-allergic and antitumor activities (Larson, 1998;Tapas et al., 2008).
Saponins have been variously attributed with a diverse range of pharmacological properties such as immunostimulant, antioxidant, pesticidal, insecticidal, molluscicidal, allelopathic, protectants (Hostettmann and Marston, 1995).Some saponins are used in confectionery, as beverages and cosmetics (Price et al., 1987;Petit et al., 1995;Uematsu et al., 2000).Moreover, saponins and polyphenols had been documented to possessed hypocholestrolemic effects in both animals and humans which reduces incidence of atherosclerosis (John and Chapman, 1995;John, 1996).
Tannins, widely distributed among many plant species, are found in plant vacuoles and surface waxes (Devi et al., 2012).Studies have reported anti-inflammatory, antioxidant; antiviral, antibacterial, anti-parasitic, anticancer, antiseptic and antidiuretic properties of tannins (Souza et al., 2006;Banso and Adeyemo, 2007).The metabolite is also regarded to possess potential malaria suppressive effects through sequestration of iron and as botanical chelators (Etkins and Ross, 1982;Etkins, 1996).Tannin constituents had been reported to prevent lipid peroxidation by inhibiting cyclooxygenase activity (Zhang et al., 2004) Steroidal compounds are of importance and play a vital role as anti-inflammatory (Cos et al., 2004;Ghaderi et al., 2014), antibacterial, antiviral and aphrodisiac agents.Certain steroids are also used in treatment of sexual dysfunction (Sunday et al., 2012).
Cardiac glycosides are also pharmacological important phytochemical used in treatment of heart related diseases e.g.Digitalis and Strophanthus species (Seigler, 1998).Antibacterial and antifungal properties due to presence of terpenoids had also been reported (Amaral et al., 1998).
The TLC profiling of both the chloroform and ethanolic extracts of the studied Ficus species confirmed the presence of number of phytochemicals.It can be inferred from the TLC profiles that chloroform (a nonpolar solvent) extracted more varieties of phytoconstituents from the leaves of Ficus species than ethanol (a polar solvent).
The occurrence and variation of secondary metabolites in Ficus species are not only important in chemotaxonomy but also in understanding the species-function relationship.Takhajatan (1973) reported that both qualitative and quantitative information on secondary metabolites are useful in taxonomic classification of plant.The presence of flavonoids, tannins, saponins, steroids and the absence of anthaquinone in all the Ficus species studied indicated that they are generic features.
Moreover, there is a general overlap in the types of secondary metabolites present in all the Ficus species studied.This could possibly explain the difficulty in devising a good infrageneric classification for the genus as earlier reported by several authors.The observations recorded in the study align with the current subgeneric, sectional and sub sectional classification schemes produced by Ogunkunle and Oladele (2008) based on foliar epidermal morphology of the members of the genus.
It was evident from this study that Ficus species contained secondary metabolites with antioxidant activity (flavonoids and tannins).This corroborates the report by Sirisha et al. (2010) that Ficus species are rich source of naturally occurring antioxidant.Therefore, the leaves of studied Ficus species are potential source of natural antioxidant that could possibly boost the antioxidant defense system in humans when consumed.
The patterns of clustering observed in the dendrogram clearly contradict the sectional classification of Croner (1965) reproduced by Sonibare et al. (2005).The species were clustered in a way that does align with classification based on morphometric analysis as done by Sonibare et al., (2004).

Conclusions
The present study revealed useful information on phytoconstituent and chemotypes variation for a better identification and classification of some Ficus species.Researches are in progress to clarify the identity of these phytoconstituents and to provide information on authentication as well as on the taxonomic relationship existing among the Ficus species.

Table 1 .
Phytochemicals constituents of chloroform and ethanolic leaf extracts of different Ficus species

Table 2 .
Solvent system/spraying reagent used in chromatographic procedure of Ficus leaf extracts

Table 3 .
Rf values of TLC solvent systems for leaf extract of different Ficus species

Table 4 .
Flavonoids and tannins content of Ficus extracts