Qualitative and Quantitative Determination of Phytochemical Contents of Indigenous Nigerian Softwoods

The phytochemical contents of some milled Nigerian softwood chips were carried out in a quest to evaluate their potentials as sources of alternative medicine as well as uses in other industrial applications. The qualitative and quantitative analysis were ascertained. Tannin was found in all the Nigerian softwoods examined with the highest quantities obtained in Sterculia oblonga (1240mg/100 g) and Barteria nigritiana (1230mg/100 g). Highest quantities of alkaloid were obtained in Cordia millenii (11.2%) and Sterculia oblonga (10.4%). Barteria nigritiana (14.2%) andMoringa oleifera (12.2%) recorded more flavonoid content than other individual softwoods. Saponin wasmore inAnogeissus leiocarpus (12.5%) andDichrostachys cinerea (9.8%). Oxalate was found to be higher inCombretodendronmacrocarpum (5.84 g/100 g) andGlyphaea brevis (3.55 g/100 g).Pentaclethramacrophylla (890mg/100 g) and Moringa oleifera (880mg/100 g) contained more cyanogenic glycosides. Sacoglottis gabonensis (4.68mg/g) and Pentaclethra macrophylla (4.04mg/g) showed the highest contents of phenol, whilemore lipids (8%and 7.2%)were found inAnogeissus leiocarpus andKaempferia galanga, respectively.The results showed that these Nigerian softwoods grains could be a source for the exploitation of these phytochemicals beneficial in the pharmaceutical and alternative medicine industries.


Introduction
Nigeria abounds in diverse forest and natural resources.Forest trees are found in various parts of the country where they are lumbered and sold locally for house building and various construction purposes.Phytochemical studies of softwood stem vis-à-vis Nigerian timber components have been inadequate as the focuses of most research are on leaves and fruits of these plants.Some of these softwood stems contain phytochemicals locally extracted using alcohol or in aqueous form by traditional drug peddlers, showing that they have undocumented medicinal values.The medicinal value of plant lies in the phytochemical (bioactive) constituents of the plant which shows various physiological effects on human body.Therefore, through phytochemical screening one could detect the various important compounds which may be used as the bases of modern drugs for curing various diseases [1].Chemical compounds produced as a result of metabolic reaction during plant growth are known as phytochemicals.Harborne [2] and Okwu [3] refer to such metabolic chemicals as "secondary metabolites" which include alkaloids, flavonoids, coumarins, tannins, terpenes, terpenoids, phenols, gums, polysaccharides, and glycosides.Human bodies under stress condition produce less enzymatic antioxidants (e.g., superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase) and nonenzymatic antioxidants (e.g., ascorbic acid (vitamin C), tocopherol (vitamin E)) but more reactive oxygen species (ROS) (e.g., superoxide anion radicals, hydroxyl radicals, and hydrogen peroxide) [4].An imbalance could result from the above causing damage to the body cell [5][6][7][8] and other health challenges [9].Preventive medicine has been immensely enhanced by the use of these natural plant antioxidants.Plants contain lots of free radical scavenging molecules some of which include alkaloids, amines, betalains, vitamins, terpenoids, phenolic acids, lignins, stilbenes, and tannins 2 New Journal of Science as well as other secondary metabolites with high level of antioxidant activity [4].Most phytochemicals are antioxidant agents which essentially reduce the damages caused in tissue during physiological processes.This research is aimed at determining the qualitative and quantitative phytochemical components in Nigerian softwood as indices of producing plant secondary metabolites which have medicinal values as well as application in industries.

Materials and Methods
2.1.Materials.Soft wood samples were locally collected at saw mill in different states in Nigeria and identified as reported elsewhere [10,11].

Wood Sample Preparation.
Twenty-four identified softwoods were prepared before analysis in a finely grounded chip form as reported by Ejikeme et al. [11].

Phytochemical Constituents of Nigerian Softwood Samples.
Methodologies for the determination of phytochemical used in this research were adapted from those reported by Keay et al. [10] and Ejikeme et al. [11], respectively.

Qualitative Analysis of the Phytochemicals of
the Wood Sample 2.4.1.Test for Tannins.Analysis used was the method reported by Ejikeme et al. [11].Each wood powder sample (0.30 g) was weighed into a test tube and boiled for 10 minutes in a water bath containing 30 cm 3 of water.Filtration was carried out after boiling using number 42 (125 mm) Whatman filter paper.To 5 cm 3 of the filtrate was added 3 drops of 0.1% ferric chloride.A brownish green or a blue black colouration showed positive test.

Test for Phlobatannins.
Analytical method is according to Ejikeme et al. [11].To each sample (0.30 g) weighed into a beaker was added 30 cm 3 of distilled water.After 24 hours of extraction, aqueous extract (10 cm 3 ) of each wood sample was boiled with 5 cm 3 of 1% aqueous hydrochloric acid.Deposit of red precipitate showed positive test.

Test for Saponin.
Methodology is as reported by Ejikeme et al. [11].Distilled water (30 cm 3 ) was added to wood powder samples (0.30 g) and boiled for 10 minutes in water bath and filtered using Whatman filter paper number 42 (125 mm).A mixture of distilled water (5 cm 3 ) and filtrate (10 cm 3 ) was agitated vigorously for a stable persistent froth.The formation of emulsion on addition of three drops of olive oil showed positive result.

Test for Steroid.
Analytical method used is according to Ejikeme et al. [11].Each sample (0.30 g) weighed into a beaker was mixed with 20 cm 3 of ethanol; the component was extracted for 2 hours.To the ethanolic extract of each sample (5 cm 3 ) was added 2 cm 3 acetic anhydride followed with 2 cm 3 of concentrated tetraoxosulphate (VI) acid.A violet to blue or green colour change in sample(s) indicates the presence of steroids.

Test for Terpenoids.
Methodology is as reported by Ejikeme et al. [11].Each wood powder sample (0.30 g) was weighed into a beaker and extracted with 30 cm 3 and component extracted for 2 hours.A mixture of chloroform (2 cm 3 ) and concentrated tetraoxosulphate (VI) acid (3 cm 3 ) was added to 5 cm 3 of each extract to form a layer.The presence of a reddish brown colouration at the interface shows positive results for the presence of terpenoids.
2.4.6.Test for Flavonoids.The test for flavonoid adopted is as reported by Sofowara [12] and Harborne [2].Each sample (0.30 g) weighed into a beaker was extracted with 30 cm 3 of distilled water for 2 hours and filtered with Whatman filter paper number 42 (125 mm).To 10 cm 3 of the aqueous filtrate of each wood extract was added 5 cm 3 of 1.0 M dilute ammonia solution followed by the addition of 5 cm 3 of concentrated tetraoxosulphate (VI) acid.Appearance of yellow colouration which disappeared on standing shows the presence of flavonoids.

Test for Alkaloids.
Test for flavonoid used is as reported by Hikino et al. [13].Extraction of component from 2 grams of each wood powder sample was carried out using 5% tetraoxosulphate (VI) acid (H 2 SO 4 ) (20 cm 3 ) in 50% ethanol by boiling for 2 minutes and filtered through Whatman filter paper number 42 (125 mm).The filtrate was made alkaline using 5 cm 3 of 28% ammonia solution (NH 3 ) in a separating funnel.Equal volume of chloroform (5.0 cm 3 ) was used in further solution extraction in which chloroform solution was extracted with two 5 cm 3 portions of 1.0 M dilute tetraoxosulphate (VI) acid.This final acid extract was then used to carry out the following test: 0.5 cm 3 of Dragendorff 's reagent (Bismuth potassium iodide solution) was mixed with 2 cm 3 of acid extract and precipitated orange colour infers the presence of alkaloid.
2.4.8.Test for Glycoside.Glycoside test was conducted according to the method reported by Hikino et al. [13].To 2.00 g of each sample was added 20 cm 3 of water, heated for 5 minutes on a water bath and filtered through Gem filter paper (12.5 cm).The following tests were carried out with the filtrate: (a) 0.2 cm 3 of Fehling's solutions A and B was mixed with 5 cm 3 of the filtrate until it became alkaline (tested with litmus paper).A brick-red colouration on heating showed a positive result.
(b) Instead of water, 15 cm 3 of 1.0 M sulphuric acid was used to repeat the above test and the quantity of precipitate obtained compared with that of (a) above.High precipitate content indicates the presence of glycoside while low content shows the absence of glycoside.
To the reagent prepared above, 10 g of phosphomolybdic acid (H 3 PMo 12 O 40 ) and 25 cm 3 of orthophosphoric acid (H 3 PO 4 ) were added.Two-hour reflux of the mixture was carried out, cooled, and diluted to 500 cm 3 with distilled water.One gram of each wood powder (sample) in a conical flask was added to 100 cm 3 of distilled water.This was boiled gently for 1 hour on an electric hot plate and filtered using number 42 (125 mm) Whatman filter paper in a 100 cm 3 volumetric flask.Addition of 5.0 cm 3 Folin-Denis reagent and 10 cm 3 of saturated Na 2 CO 3 solution into 50 cm 3 of distilled water and 10 cm 3 of diluted extract (aliquot volume) was carried out after being pipetted into a 100 cm 3 conical flask for colour development.The solution was allowed to stand for 30 minutes in a water bath at a temperature of 25 ∘ C after thorough agitation.With the aid of a Spectrum Lab 23A spectrophotometer optical density was measured at 700 nm and compared on a standard tannic acid curve.Dissolution of 0.20 g of tannic acid in distilled water and dilution to 200 cm 3 mark (1 mg/cm 3 ) were used to obtain tannic standard curve.
Varying concentrations (0.2-1.0 mg/cm 3 ) of the standard tannic acid solution were pipetted into five different test tubes to which Folin-Denis reagent (5 cm 3 ) and saturated Na 2 CO 3 (10 cm 3 ) solution were added and made up to the 100 cm 3 mark with distilled water.The solution was left to stand for 30 minutes in a water bath at 25 ∘ C. Optical density was ascertained at 700 nm with the aid of a Spectrum Lab 23A spectrophotometer.Optical density (absorbance) versus tannic acid concentration was plotted.
The following formula was used in the calculation: where  is concentration of tannic acid read off the graph.

Determination of Alkaloids.
Quantitative determination of alkaloid was according to the methodology by Harborne [2].Exactly 200 cm 3 of 10% acetic acid in ethanol was added to each wood powder sample (2.50 g) in a 250 cm 3 beaker and allowed to stand for 4 hours.The extract was concentrated on a water bath to one-quarter of the original volume followed by addition of 15 drops of concentrated ammonium hydroxide dropwise to the extract until the precipitation was complete immediately after filtration.After 3 hours of mixture sedimentation, the supernatant was discarded and the precipitates were washed with 20 cm 3 of 0.1 M of ammonium hydroxide and then filtered using Gem filter paper (12.5 cm).Using electronic weighing balance Model B-218, the residue was dried in an oven and the percentage of alkaloid is expressed mathematically as % Alkaloid = Weight of alkaloid Weight of sample × 100. (2) 2.5.3.Determination of Flavonoid.Flavonoid determination was by the method reported by Ejikeme et al. [11] and Boham and Kocipai [15].Exactly 50 cm 3 of 80% aqueous methanol added was added to 2.50 g of sample in a 250 cm 3 beaker, covered, and allowed to stand for 24 hours at room temperature.
After discarding the supernatant, the residue was reextracted (three times) with the same volume of ethanol.Whatman filter paper number 42 (125 mm) was used to filter whole solution of each wood sample.Each wood sample filtrate was later transferred into a crucible and evaporated to dryness over a water bath.The content in the crucible was cooled in a desiccator and weighed until constant weight was obtained.The percentage of flavonoid was calculated as % Flavonoid = Weight of flavonoid Weight of sample × 100.
(3) 2.5.4.Determination of Saponin.Saponin quantitative determination was carried out using the method reported by Ejikeme et al. [11] and Obadoni and Ochuko [16].Exactly 100 cm 3 of 20% aqueous ethanol was added to 5 grams of each wood powder sample in a 250 cm 3 conical flask.The mixture was heated over a hot water bath for 4 hours with continuous stirring at a temperature of 55 ∘ C. The residue of the mixture was reextracted with another 100 cm 3 of 20% aqueous ethanol after filtration and heated for 4 hours at a constant temperature of 55 ∘ C with constant stirring.The combined extract was evaporated to 40 cm 3 over water bath at 90 ∘ C. 20 cm 3 of diethyl ether was added to the concentrate in a 250 cm 3 separator funnel and vigorously agitated from which the aqueous layer was recovered while the ether layer was discarded.This purification process was repeated twice.60 cm 3 of n-butanol was added and extracted twice with 10 cm 3 of 5% sodium chloride.After discarding the sodium chloride layer the remaining solution was heated in a water bath for 30 minutes, after which the solution was transferred into a crucible and was dried in an oven to a constant weight.
The saponin content was calculated as a percentage: 2.5.5.Determination of Oxalate.Oxalate quantitative determination was carried out using the method reported by Ejikeme et al. [11] and Munro and Bassir [17].Exactly 20 cm 3 of 0.3 M HCl in each wood powder sample (2.50 g) was extracted three (3) times by warming at a temperature of 50 ∘ C for 1 hour with constant stirring using a magnetic stirrer.For oxalate estimation, 1.0 cm 3 of 5 M ammonium hydroxide was added to 5.0 cm 3 of extract to ensure alkalinity.Addition of 2 drops of phenolphthalein indicator, 3 drops of glacial acetic acid, and 1.0 cm 3 of 5% calcium chloride to make the mixture acidic before standing for 3 hours was followed by centrifugation at 3000 rpm for 15 minutes.After discarding the supernatant, the precipitate was washed three times using hot water by mixing thoroughly each time centrifugation.Then, to each tube, 2.0 cm 3 of 3 M tetraoxosulphate (VI) acid was added and the precipitate dissolved by warming in a water bath at 70 ∘ C. Freshly prepared 0.01 M potassium permanganate (KMnO 4 ) was titrated against the content of each tube at room temperature until the first pink colour appears throughout the solution.The solution was allowed to stand until it returned colourless, after which it was warmed on an electric hot plate at 70 ∘ C for 3 minutes, and retitrated again until a pink colour appears and persists for at least 30 seconds.Titration reaction of oxalate in sample was calculated as Ratio of reacting ions = 1 : 1 where  1 is molarity of KMnO 2.5.8.Determination of Phenols.Defatting of 2 g wood powder sample was carried out for 2 hours in 100 cm 3 of ether using a soxhlet apparatus.The defatted sample (0.50 g) was boiled for 15 minutes with 50 cm 3 of ether for the extraction of the phenolic components.Exactly 10 cm 3 of distilled water, 2 cm 3 of 0.1 N ammonium hydroxide solution, and 5 cm 3 of concentrated amyl alcohol were also added to 5 cm 3 of the extract and left to react for 30 minutes for colour development.The optical density was measured at 505 nm.0.20 g of tannic acid was dissolving in distilled water and diluted to 200 mL mark (1 mg/cm 3 ) in preparation for phenol standard curve.Varying concentrations (0.2-1.0 mg/cm 3 ) of the standard tannic acid solution were pipetted into five different test tubes to which 2 cm 3 of NH 3 OH, 5 cm 3 of amyl alcohol, and 10 cm 3 of water were added.The solution was made up to 100 cm 3 volume and left to react for 30 minutes for colour development.The optical density was determined at 505 nm [10].

Discussion
Softwoods such as Sterculia oblonga, Barteria nigritiana, Albizia adianthifolia, Afzelia bella, and Combretodendron macrocarpum, whose qualitative and quantitative determination in this study were shown to contain tannins, could be exploited for their industrial usage.Tannin is one of the major active ingredients found in plant based medicines [22]; it serves as caustics for cationic dyes (tannin dyes) used in the dyestuff industry as well as in the production of inks (iron gallate ink).Other uses of tannin are for wine, fruit juice, and beer clarification in food industries [23] Tannins are used in the dyestuff industry as caustics for cationic dyes (tannin dyes), and also in the production of inks (iron gallate ink), textile dyes, antioxidants in beverages, and coagulant in rubber production [24] as well as possessing antiviral, antibacterial, and antitumor activity [22,25,26].Tannin has been reported to selectively inhibit HIV replication [27].
Therefore, the above listed Nigerian softwoods have potential in the provision of tannin.According to Braunwald et al. [28] cardiac glycoside has been used in treatment of congestive heart failure due to its direct action which increases the force of myocardial contraction.They also explained that in the vascular system cardiac glycoside acts directly on the smooth muscles.Their effects on neural tissues and indirect effect on electrical activities of the heart and vascular resistance as well as capacitance are equally reported [28].Moringa oleifera, Afzelia bipindensis, Tetrapleura tetraptera, Combretodendron macrocarpum, and New Journal of Science Cordia millenii in this study were shown to contain glycosides which could be exploited for their medicinal properties.
Flavonoids are known to have antioxidant effects and have been shown to inhibit the initiation, promotion, and progression of tumors [29]; reduction of coronary heart disease has been reported to be associated with intake of flavonoid [30].This research has shown that Barteria nigritiana, Moringa oleifera, Combretodendron macrocarpum, Cordia millenii, Afrormosia laxiflora, and Sacoglottis gabonensis contain appreciable quantity of flavonoid.Apart from the antioxidant properties of flavonoid, other biological functions it possesses include protection against platelet aggregation, microorganisms, hepatotoxins, viruses, tumors, ulcers, free radicals, inflammation, and allergies [31].
Phenols are antioxidants in human and plants [32].Some softwoods in this research such as Sacoglottis gabonensis, Pentaclethra macrophylla, Moringa oleifera, and Anogeissus leiocarpus may be exploited for their saponin content.Haslam [33] points to current interest in the potential for amelioration of diseases simply by improving the dietary intake of nutrients with antioxidant properties, such as vitamin E, vitamin C, -carotene, and carotenoids, and plant phenolics such as tannins and flavonoids.
Plant lipids are important in paints production, pharmaceutical, and cosmetic raw materials as well as being sources for production of animal feeds.From the foregoing, extraction of lipids from nonfoliage parts of softwood (e.g., stems) is ideal for use as listed above.Thus stems of Anogeissus leiocarpus, Kaempferia galanga, Dichrostachys cinerea, and Allanblackia floribunda from the result of this research are ideal sources for lipid extraction.
Appreciable quantities of saponin are found in Anogeissus leiocarpus, Kaempferia galanga, Dichrostachys cinerea, and Allanblackia floribunda as shown in Tables 2 and 3 in this research.Saponins protects against microbial attack in plants; it is also useful in treating yeast and fungal infections [1].
According to Sodipo et al. [34] most phytochemicals serve as natural antibiotics, which assist the body in fighting microbial invasion and infections.Alkaloids, for instance, consist of chemical compounds that contain mostly basic nitrogen atoms which occur naturally, mainly, in plants but may be produced by bacteria, fungi, and animals.In this research, appreciable quantities of alkaloid were obtained in Cordia millenii, Sterculia oblonga, Amphimas pterocarpoides, and Khaya ivorensis (Table 3).Extraction of alkaloid from these softwoods could serve as a cheap and steady means of providing this secondary metabolite in industries where they are greatly needed.According to literature, alkaloids have a wide range of pharmacological activities including antimalarial (e.g., quinine), anticancer (e.g., homoharringtonine) [35], antibacterial (e.g., chelerythrine) [36], and antihyperglycemic activities (e.g., piperine) [37].Alkaloids have equally been exploited for their importance in traditional pharmaceutical usage.Other alkaloids possess psychotropic (e.g., psilocin) and stimulant activities (e.g., cocaine, caffeine, and nicotine) and have been used as recreational drugs [37].Although alkaloids carry out many metabolic activities in humans and other animals, they almost uniformly evoke a bitter taste [38].

Conclusion
Nigerian softwoods apart from their timber uses are also potential for traditional medicine.There have been well known usages of most of their foliage parts such as seeds and leaves in traditional medicine, but the stem has had less usage in the phytochemical application in industries and medicine.This research result has established through the investigation of their phytochemistry that they have potential in the industries particularly for pharmaceutical usages.

Table 1 :
Table of the botanical and local names as well as location of various Nigerian softwoods.

Table 2 :
Table of the qualitative determination of the phytochemical components of Nigerian softwoods.

Table 3 :
Table of quantitative phytochemicals of Nigerian softwoods.