METABOLIC PROFILE ASSESSMENT OF PHYTOCHEMICALS IN CEIBA PENTANDRA (L.) GAERTN, FROM ZARIA CITY, NIGERIA

Ceiba pentandra (Silk cotton) is a multipurpose tree in the province of Zaria city, Kaduna State, Nigeria, which is being exposed to uncontrolled exploitation by the natives. Samples of the plant were collected from standing trees located at Zaria city: Kofar Gayan, Rimin Doko, Kwarbai, Kofar Doka and Kofar Kibo. The study was aimed at assessing the secondary metabolites in C. pentandra plant extracts of leaves, stem barks and roots This was achieved through subjecting the extracts to phytochemical screening and gas chromatography/mass spectrometry fingerprinting (metabolic profiling) of phytochemical markers. The results of the screening revealed flavonoids, saponins, steroids, tannins, triterpenoids and reducing sugars to be present in moderate variation, across the extracts. The metabolic fingerprinting indicated high variation among the phytochemicals obtained from the plant extracts. Where, twentyseven (27) secondary metabolites were predicted from the methanol leaf extracts, twenty-five (25) from the methanol stem bark extracts and twenty-seven (27) from the methanol root extracts, out of which only one of the metabolites (2-hydroxypropyl ether) was predicted in all the extracts. Therefore, for a clearer and broader identification of these phytochemical markers, Nuclear Magnetic Resonance spectrometry (NMR) should be employed to determine the detailed chemical structures of the identified metabolites, which will help in determining their functions and how they can be used to improve the biodiversity of these tree species.


INTRODUCTION
Over the centuries humans have relied on plants for basic needs such as food, clothing, and shelter, all produced or manufactured from plant matrices (leaves, woods, fibers) and storage parts (fruits, tubers) (Saurabh et al., 2015). Plants have also been utilized for additional purposes, namely as arrow and dart poisons for hunting, poisons for murder, hallucinogens used for ritualistic purposes, stimulants for endurance, and hunger suppression, as well as inebriants and medicines. The plant chemicals used for these latter purposes are largely the secondary metabolites, which are derived biosynthetically from plant primary metabolites (e.g., carbohydrates, amino acids, and lipids) and are not directly involved in the growth, development, or reproduction of plants. (Anurag et al., 2014) Nearly 80% of the world's population relies on traditional medicines for primary health care, most of which involve the use of plant extracts (Sandhya et al., 2006).
A plant cell produces two types of metabolites: primary metabolites involved directly in growth and metabolism (carbohydrates, lipids and proteins), (Anurag et al., 2014) and secondary metabolites which are substances produced by plants as defense chemicals. Their absence does not cause bad effects to the plants. They include alkaloids, phenolics, steroids, essential oils, lignins, resins and tannins etc. Secondary metabolites are compounds biosynthetically derived from primary metabolites and are compounds that are not required for normal growth and development, and are not made through metabolic pathways common to all plants (Bourgaud et al., 2001). Secondary metabolites are accumulated by plant cells in smaller quantities than primary metabolites (Anurag et al., 2013).
Secondary metabolites from plants, which are distinguished from primary metabolites such as nucleic acids, amino acids, carbohydrate, fat, etc. (Justin et al., 2014) are extremely diverse; thousands of them have been identified in several classes. Each plant family, genus, and species produces a characteristic mix of these chemicals, and they can sometimes be used as taxonomic characters in classifying plants (Thrane, 2001). Many scientific sources state that their role is not crucial for living cells in normal growth, development, and reproduction (Justin et al., 2014), but they act in defense purposes to protect a plant from any possible harm in the ecological environment (Stamp, 2003), and other interspecies protection (Samuni-Blank et al., 2012). Therefore, they are usually synthesized in plants for particular needs, while the primary metabolites have generally the shared biological purposes across all species. Plants have been evolving to adapt to the environment with genetic encoding of useful and diverse syntheses for secondary metabolites (Waterman, 1992).
Silk cotton (Ceiba pentandra) tree was first introduced into the study area, Zaria (named as 'Rimi' in Hausa language) from Indo-Arabian sub-continent by the slave merchants who exchanged its products (mattresses and horse saddles) for slaves (Al-Amin, 2013). The tree's seed was brought through the trade, dispersed into the area and eventually germinated and grown as a tree through such trade. Its uses in the area helped in the initial propagation, planting and protection when sooner, the people of Zaria realized several uses of the tree products for animal forage, medicinal uses and several other utility uses. This made the tree species a "Hotcake" for the natives (Al-Amin, 2013).
The silk cotton tree colony is relentlessly being depleted by several human activities like; deforestation, the use of the timber as domestic fuel and construction processes among others. As the destruction is going unrestricted, the effect is obtrusively manifesting at least in the last two decades (Al-Amin, 2013). Unfortunately, no or less effort is being made to study this phenomenon more or less to stop it. The resulting negligence on the tree colony has incited this present research to find and provide possible solutions for the amplification of conservational (both Ex situ and In situ) actions of the plant in order to protect, conserve and beautify the environment.

MATERIALS AND METHODS Study Area
Zaria city is located at 11° 5' 7.9476''N and 7° 43' 11.8020'' E in Kaduna State on the central high plains of Hausa-land in northern part of Nigeria (Al-Amin, 2013). The major soil type in the area is the tropical ferruginous; while along the wide gentle sloping valley bottom lands are the dark vertisol (Fadama soils). Zaria falls within the northern guinea savanna vegetation. The climax vegetation of the area was thought to be tropical deciduous, however, because nearly all the vegetation of the area had been degraded due to intense urban agriculture, fuel wood harvesting and urbanization processes, tree climax vegetation is absent except in the remnant silk-cotton tree colony around the southern suburb (Al-Amin, 2013).

Collection, Identification and Preparation of Plant Materials for Phytochemical Analyses
The fresh leaves, young stems and roots of C. pentandra species were collected from the above mentioned sampling points. The plant samples were identified at the Herbarium of Biological Sciences Department, Ahmadu Bello University, Zaria, with a voucher specimen (No. 7059). Collected samples were transported to Biological Sciences Department, Bayero University, Kano for laboratory analysis. The plant samples were air-dried indoors, finely powdered using a sterilized grinder for the extraction procedure.

Preparation of Extracts
The extraction scheme was performed according to the standard method adopted by Okoye and Osadede (2009). One hundred grams (100g) of powdered plant materials were subjected to solvent extraction for a period of two days by cold maceration in 99% methanol with periodic shaking. The crude methanol extract was drained, filtered and concentrated almost to dryness under reduced pressure at 40°C using rotary evaporator (Rotavapo R210/215, Buchi).

Phytochemical Screening of Extracts
The methanol extracts obtained from the above method were subjected to preliminary phyto-chemical screening following standard protocols (Poongothai et al., 2011).The extracts were screened for the presence of tannins, flavonoids, saponins, steroids, triterpenoids and reducing sugar. A portion of the extract (0.5mg) was treated with saturated solution of ferric chloride; a green-black or blue-black colour indicated the presence of tannin (Shamsuddeen et al., 2009). Flavonoids detcetion was carried out in accordance with the method of Shamsuddeen et al. (2009), where piece of magnesium ribbon was added to 4mg/ml of each extract which was followed by the addition of concentrated hydrochloric acid (HCl), drop-wise. Appearance of Crimson to magenta colour indicated the presence of flavonoids. 0.5g of each extract was shaken in 10ml of distilled water. Production of persistent foam which lasted for 15 minutes indicated the presence of saponin (Shamsuddeen et al., 2009).One milligram (1mg) of the extract was dissolved in 10ml of chloroform and equal volume of concentrated sulphuric acid was added by the side of the test tube. The upper layer turns red and sulphuric acid layer showed yellow with green fluorescence. This indicated the presence of steroids (Poongothai et al., 2011).Ten milligram (10mg) of the extract was dissolved in 5ml of chloroform; 1ml of acetic anhydride was added following the addition of 2ml of conc. H2SO4.Formation of reddish violet colour indicates the presence of triterpenoids (Poongothai et al., 2011).Test for detection of reducing sugars was carried out in accordance with the method of Poongothai et al. (2011) where 1ml of stock solution of each extract was diluted with 2ml of distilled water, followed by the addition of Fehling's solution (A+B) and then warmed. Brick red precipitates at the bottom of the tubes indicate the presence of reducing sugars.

Gas Chromatography-Mass Spectrometry Analysis of Extracts
GC-MS analysis of the extracts was performed using GCMS-QP2010 PLUS (Shimadzu Japan system). The sample (2 ml) was injected into a RTX-5 column (60m x 0.25mm internal diameter, film thickness 0.25 μm) of GC-MS. Helium was used as carrier gas at a constant column flow of 1.58 ml/min. at 108.0 kpa inlet pressure. Temperature programming was maintained from 80°C to 250°C with constant rise of 5°C/min. and then held isothermal at 250°C 10 min.; further the temperature was increased by 30°C/min. up to 310°C and again held isothermal at 320°C for 22 min. The injector and ion source temperature were 270°C and 230°C, respectively. Each of the extracts was dissolved in methanol, and in turn injected with a split ratio of 1:20. Mass spectra were taken at 70 eV; a scan interval of 0.5 seconds and the total GC/MS running time 70 minutes. Interpretation and identification of the spectrum of the GC-MS was obtained using the database of library in GC-MS system of the National Research Institute for Chemical Technology (NARICT), Zaria.

DISCUSSION
Massive loss of valuable plant species in the past centuries and its adverse impact on environmental and socioeconomic values has triggered the conservation of plant resources. Appropriate identification and characterization of plant materials is essential for the successful conservation of plant resources and to ensure their sustainable use (Arif et al., 2010).
The results of the phytochemical assessment from the five sampling points (Kofar Gayan, Rimin Doko, Kwarbai, Kofar Kibo and Kofar Doka) indicate relative variation among the extracts from the above mentioned sampling points which may have resulted from continuous cutting down of the trees for firewood and other domestic needs. It may also have been due to stress posed by environmental hazards such as pollution, drought, ultra violet 434 exposure, herbivory and pathogenic attack, (Ali and Alquraini, 2006).
Phytochemicals are biologically synthesized chemical compounds in plants, which serve in protecting the plant cells against environmental hazards such as: pollution, stress, and drought, ultra violet exposure, herbivory and pathogenic attack (Ali and Alquraini, 2006). Phytochemicals, also referred to as secondary metabolites, provide health benefits to humans; acting as synergistic agents, hence, allowing more efficient nutrient use by the body (Andre et al., 2010). This study identified flavonoids, saponins, steroids, tannins, triterpenoids and reducing sugar (Tables 1, 2 and 3).
Overproduction of oxidants (reactive oxygen species and reactive nitrogen species) in the human body is responsible for the pathogenesis of some diseases. The scavenging of these oxidants is thought to be an effective measure to depress the level of oxidative stress of organisms. It has been reported that intake of plant parts is inversely associated with the risk of many chronic diseases, and antioxidant phytochemicals in the plants are considered to be responsible for these health benefits. They often possess strong antioxidant and free radical scavenging abilities, as well as antiinflammatory action, which are also the basis of other bioactivities and health benefits, such as anticancer, anti-aging, and protective action for cardiovascular diseases, diabetes mellitus, obesity and neuro-degenerative diseases (Zhang et al., 2015).
The presence of the range of phytochemicals in the different plant parts could be related to the use of the plant for medicinal purposes. Bairwa et al. (2010) reported protective activity of ethyl acetate fraction of methanol extract of stem bark of C. pentandra against induced liver damage in rats. C. pentandra root and bark extracts have been reported to have a hypoglycaemic effect (Djomeni-Dzeufiet et al., 2006).
Tannins were found present in the leaves, bark, stem and the roots of C. pentandra extracts (Tables 1, 2 and 3). Tannins possess a characteristic feature of tan, i.e. they have the potential to convert things into leather (Sarker and Nahar, 2007), provide protection to the plant against microbial pathogens, harmful insects and other herbivores (Ali and Alquraini. 2006).Tannins present in the extracts of C. pentandra have being also reported by Akinpelu and Onakoya (2006) as the main component for the treatment of intestinal disorders like diarrhea and dysentery. Triterpenoids found in these plant extracts serve as a major component of many essential oils, resins or oleoresins (Firn, 2010), and are widely used in herbal medicine (Edeoga et al., 2005). Many sesquiterpene lactones with moderate antimicrobial activity have been isolated from the root bark of C. pentandra (Rao et al., 1993). Flavanoids presented in the Tables 1, 2 and 3have a number of nutritional functions and have been described as biological response modifiers', mostly act as antioxidant and some have anti-inflammatory properties or as free radical scavengers (Kar, 2007). Flavonoids were also described as phyto-constituents serving as flavouring ingredients of spices and vegetables (Osuntokun et al., 2017). Isoflavone glucosides isolated from the bark of C. pentandra showed inhibitory effects on cyclooxygenase-2-catalyzed prostaglandin biosynthesis indicating an analgesic potential (Noreen et al., 1998). The steroids otherwise termed as steroid glycosides or cardiac glycosides are plant phytoconstituents having therapeutic applications as arrow poisons or cardiac drugs (Firn, 2012), they are also reported to have the ability to promote nitrogen retention in osteoporosis and in animals with wasting illness Madziga et al. (2010). Saponins as presented in Tables 2 and 3 possess hypolipidemic and anticancer activity, and therefore, necessary for cardiac glycosides activities, steroidal saponins are used in the commercial production of sex hormones for clinical use (Sarker and Nahar, 2007). The result of these phytoconstituents in C. pentandra further suggested the reason for the usage of the plant in curing many diseases such as colic in man, dressing of sores for tumors, whitlow, inflammatory diseases, cancer, fatigue, lumbago, gonorrhea, dysentery, anti-microbial and anti-fungal effects (Edeoga et al., 2001). Since these phytochemicals are present in all the targeted organs in the plant, the use of root and stem bark of the plant should be discouraged to ensure sustainable usage of the plant, as the use of root in particular can result in destructive usage. Variation in biosynthesis of these metabolites may be resulted from both genetic and environmental factors, which play important roles in the development of phenotypic variations in plants as reported by Salim et al. (2011).
Some of the compounds revealed in the GC-MS analysis includes: Squalene; a triterpene which is an intermediate for phytosteol biosynthesis in plants extracts (Table 4) and is highly appreciated in the cosmetic and pharmaceutical industry due to its remarkable properties; easy incorporation in cosmetic emulsions as effect of its light consistency and excellent spreadability, non-greasy texture, high stability, also due to the fact that the plant origin squalene is odourless and colorless with no harmful substances, as such serve as an potentiating for chimiotherapic drugs (Ioana et al., 2014). Linalool or Linalyl acetate is a naturally occurring aromatic compound (Table 5), which is regarded as substantial criteria in the authenticity control of essential oils and also adopted for quality assurance by the flavor and fragrance industry (Kreis and Mosandi, 1992). Decanoic, Pentadecanoic, Hexadecanoic, Octadecanoic and Eicosanoic acids are saturated fatty acids (Tables 4-6) and serve as important component in the cosmetic industry (CHEMIK, 2014). They are primarily used as intermediates in the manufacture of corresponding alkali salts, which are in turn, used as emulsifiers, emollients, and lubricants in a variety of cosmetic creams, cakes, soaps and pastes (Mary, 1987), these compounds extracted from the leaves of Sesuviumportulacastrumrevealed their ability to have the potential antimicrobial agents against tested human pathogenic microorganisms (Chandrasekaran, and Santhilkumar, 2011). Anthranilic acid and its derivatives as presented in Table 4 have applications in various sectors, including; food and perfumery industries, and are used in the synthesis of bioactive molecules (Wiklund and Bergman, 2006). It also exhibits anticancer, antimicrobial, insecticide, antiviral and anti-inflammatory activities (Pajor and Sun, 2013).