Plant Secondary Metabolites Profile of Some Economic Tree Species Using Methanolic Extraction

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Published on International Journal of Agriculture & Agribusiness
Publication Date: April, 2020

Awotedu, B. F., Awotedu, O. L., Chukwudebe, E. P. & Ogunsiji, A. O.
Forestry Research Institute of Nigeria, P.M.B 5054, Jericho hills, Ibadan, Oyo State, Nigeria
Moist Forest Research Station, Benin City, Edo State, Nigeria

Journal Full Text PDF: Plant Secondary Metabolites Profile of Some Economic Tree Species Using Methanolic Extraction.

Abstract
Plant chemicals possesses non-essential nutrients that are not required for human nutrition, these chemicals are produced naturally for protection of the plant against diseases. This study assessed the phytochemical (Plant secondary metabolites) constituents of five tree species: Eucalyptus camaldulensis, Afzelia aficana, Adansonia digitata, Cedrela odorata and Mansonia altissima. The phytochemical screening and analysis of the selected tree species were investigated using methanolic extraction described by different standard analytical techniques and the following phytochemicals were evaluated; Alkaloids, Saponin. Tannin, flavonoids. Phenol, Cardiac glycosides and Terpenoids. The result for the qualitative screening shows the presence of all the secondary metabolite in some tree species like Afzelia africana and Cedrela odorata, while tannin, terpenoids and cardiac glycosides was absent in Eucalyptus camaldulensis, Adansonia digitata and Mansonia altissima respectively. While for the quantitative analysis, results for Alkaloid for all the investigated tree species ranges from 3.48-5.12%, flavonoid 2.50-4.47%, Saponin 2.84-9.00%, Tannin 2.05-3.90%, Phenolics 6.42-15.22%, Cardiac glycoside 1.02-1.54%, Terpenoids 1.72-2.01% respectively. All the plant species examined in this study shows that it possesses promising medicinal potentials. This study has revealed the richness of these tree species because of the availability of the secondary metabolites thereby suggesting the tree species has a disease preventive plants, which also helps in the management of diverse medical conditions.

Keywords: Eucalyptus camaldulensis, Afzelia africana, Adansonia digitata, Cedrela odorata, Mansonia altissima & Trees.

1. INTRODUCTION
Medicinal plants, all over the world have served as a very rich source of raw materials for traditional healers and in modern orthordox medicine both in Asia and Africa (Tsakala et al., 2006). The medicinal and pharmaceutical properties of these tree species are due to the type of chemical substance they produce and store. These include compounds that are utilized by man as food and other animals and also other compounds that exert physiological effects on them. These plant chemical compounds that possess important therapeutic properties can be used in the treatment of different human complications. Immunolatory responses in the body are usually boosted by phytochemicals because they provide immunity against many illnesses/diseases. Some secondary metabolites are known to reveal the physiological and medicinal potentials which are alkaloids, flavonoids, tannin, phenols, saponin and phytosterols e.t.c (Yadav et al., 2017). In developed countries, about twenty-five percent of drugs are produced from bioactive agents of medicinal plants (Diallo et al., 1999). These plants bioactive agents have been reported to have therapeutic activities such as antimicrobial, antidiabetic, antioxidant and antidiarrhea activities (Newman and Cragg, 2007; Ogbulie et al., 2007). Right from time immemorial, plants and the products derived from plant parts such as stem bark, leaves, fruits and seeds have been parts of phytomedicine, thus indicating that any part of a plant may contain useful active compounds (Soladoye and Chukwuma, 2012).
Eucalyptus camaldulensis (Dehnh.) is a big tree which belongs to the Myrtaceae family. Common names include red gum, red chewing gum, river chewing gum. It is a fast growing tree and evergreen that can tolerate drought for long periods and withstand high temperature up to 60oC. The plant is considered a large aromatic genus which contains about 900 species. Eucalyptus species have been used widely in treating asthma, burns, fever, sore throat, arthritis, influenza, malaria, wounds and pharyngitis. It is also used in soap and cosmetic industries. Eucalyptus is a dark family (Myrthas).
E. camaldulensis which is a perennial tree usually grows up to 30-45m in height, also possess single stem and large trunk. They can last up to 1000 years (Ghasemian et al., 2019). It can be found around edge of rivers with seasonal or continuous water flow.
Afzelia africana (Smith) belongs to the family Ceasalpiniaceae. The English name is Mahogany. The tree is widely distributed in Asia and Africa (Keay, 1989). It is used as food and planks and widely used as folklore remedies among many tribes in Africa.
Adansonia digitata L. which is one of the tallest trees in the world belongs to Malvaceae family. It is also known as baobab tree and it is native to tropical Africa. Adansonia is a very rich tree that has a multi-purpose uses which offers protection, clothing and medicine, food and raw materials or humans. African baobab also known as dead rat tree is regarded as the “Queen of all carbon storage trees”. All plant organs are consumable. The tree is older than 4000 years. A. digitata is a large deciduous tree, that grows up to 20-30 m tall in height with 2-10m in diameter at adult age. The trunk has a vast girth. Smooth bark, reddish brown to grey, soft and possesses longitudinal fibers. Baobab is highly branched and it produces multi-lateral root system that grows until 50 m from the trunk. At times, the roots tips are in the form of tubers. In traditional dishes, the tubers, twigs, seeds, flowers, fruits and leaves are all edible common ingredients used in remote areas in Africa.
Cedrela odorata L. also called “cedro amargo” belongs to Meliaceae family. The tree is monoecious semi-deciduous ranging in height from 10-30m (33 -98ft). The trunk has a thick grey grown bark with longitudinal irregular grain. Pinnately compound leaves, grouped towards the end of the branches, 15-50cm long, with pairs of scythe-shaped leaflets, lanceolate to oblong, 7-15cm with the base obliquely truncated and asymmetric.
Mansonia altissima (A. Chev) is an evergreen tree that belongs to the family Sterculiaceae. It is a semi-deciduous forest species that can grow up to 45m in height and trunk to 2.5m girth (Maku et al., 2014). It is grown throughout west tropical Africa. It occurs particularly in disturbed areas or light gaps in lowland moist forest. The crown is small, ovoid and dense. The bole is straight and can be branchless for up to 30m. The common names include: Aprono (Ghana), Bete (Ivory Coast), Ofun (Nigeria). There are numerous number of tree species whose importance has not been well explored and proven scientifically and thus, requires investigations of their phytochemical profile for medicinal credence, Hence, there is more requirements to explore the applicability of these plant resources which are rich in phytochemicals and may have beneficial effect on health.
2. MATERIALS AND METHODS
Plant Collection and Identification
In this study, the five medicinal tree species (Eucalyptus camaldulensis, Afzelia africana, Adansonia digitata, Cedrela odorata, Mansonia altissima) were collected from a mother tree within the premises of Forestry Research Institute of Nigeria, Ibadan, Oyo State, Nigeria. It was identified and authenticated at the taxonomy unit of the institute. Then for like 2-3 times the leaves part was washed in a running clean tap, the plant leaves washed was left for drying. After drying, a mechanical blender was used to ground the sample into powdered form. Then the powdered sample was stored in air tight container for future use with proper labelling.

Preparation of Extract
Methanol was used as the extraction solvent. 50g of each of the leaves sample was soaked in 250ml of methanol (95%, boiling point; 64.6oC) for three days during which it was agitated on a mechanical shaker at 220rpm. After three days, during which, it was agitated on a mechanical shaker at 220 rpm, then, the resulting mixture was filtered and the filtrate concentrated under vacuum using the rotary evaporator, and the crude extract recovered in a petri dish, it was then kept in a desiccator at room temperature to remove residual solvent before they were analyzed (Awotedu et al., 2020)

Phytochemical Evaluation
Qualitative screening and Quantitative phytochemical analysis were done to identify and quantify the chemical components present in the leaves of some medicinal tree species. The bioactive constituents assayed includes; alkaloids, saponin, flavonoids, tannin, phenolics, terpenoids and cardiac glycosides. The phytochemical screening and analysis was evaluated following standard techniques described by different authors.

Qualitative Screening
Test for Flavonoid
5 ml of diluted ammonia was mixed with aqueous filtrate of the plant extract. Concentrated sulphuric acid of about 1 ml was put together with the above mixture. Formation of yellow colored precipitate indicated the presence of flavonoids (Manoj et al., 2015).

Test for Tannins
1gm of the extract was boiled with water of 20 ml and then filtered. Few drops of 0.1% solution of ferric chloride was added and a greenish brown colour was observed which indicates the presence of tannin (Manoj et al., 2015)

Test for alkaloids:
The plant extract was dissolved in chloroform and the solution was extracted with dil. H2SO4 and acid layer taken and tested for presence of alkaloids:

Dragendroff’s test: To 5 ml of acid layer of test solution, 5ml of Dragendroff’s reagent (potassium bismuth iodide solution) and 5ml of dil. HCl were added. An orange-red precipitate indicated the presence of alkaloids.
Mayer’s test: To the 5ml of acid layer of test solution, 5ml of Mayer’s reagent (potassium mercuric iodide solution) was added. Whitish or cream colored precipitate indicated the presence of alkaloids.
Wagner’s test: To the 5ml of acid layer of test solution, 5ml of Wagner’s reagent (iodine in potassium iodide) was added. Reddish-brown colored precipitate indicated the presence of alkaloids (Gibbs, 1974).

Test for Cardiac glycosides:
5 ml of sulphuric acid was added to 0.1 g of the extract in a test tube and was boiled for 15min in a water bath, it was cooled and neutralized with 20% potassium hydroxide solution. Equal parts of 10 ml Fehling’s solution A and B was added for 5min. A dense brick red precipitate shows that glycoside was present (Manoj et al., 2015).

Test for Saponins
1g of sample extract was added to 10 ml of distilled water. For 10 minutes, the mixture was shaken vigorously. Few drops of Olive-oil was added and stirred very well. Formation of an emulsion of the mixture was observed (Manoj et al., 2015)

Test for Phenols:
10 ml of distilled water was added to test sample of 1g. For 3 mins, the mixture was heated in a water bath and filtered. One of the fitrate test tubes was diluted with distilled water in ratio 1:4. A greenish or blue colour indicated the presence of phenols (Manoj et al., 2015).

Test for Terpenoids:
Salkowski’s test:
4 ml of chloroform was added to 1 gm of the plant extract, sulphuric acid of 6 ml was also added and a reddish brown colour was observed, which shows the presence of terpenoids (Manoj et al., 2015)

Quantitative Analysis
Determination of saponin
Saponin content was determined gravimetrically following the procedure previously used by Ezeonu and Ejikeme, (2016). 5g of pulverized sample for each collection time was extracted using 20% aqueous ethanol (100ml) for 4hrs in a water bath set to 550C with constant mixing, the solution was filtered to recover the residue which was then repeatedly re-extracted going through the previous process the extracts obtained were combined and concentrated over water bath to 40 ml at 90oC, then, diethyl ether (20 ml) was added in a separating funnel with vigorous shaking; the ether layer was dispose of, while the aqueous layer was retained after repeated purifications. Then, n-butanol (60 ml) was introduced, the mixture extracted twice using 10 ml NaCl (5 %). The NaCl layer was discarded while the residual mixture was further concentrated for 30 minutes in a water bath, the mixture decanted into a crucible and dried to a constant weight in an oven. % Saponin concentration was estimated using the formula:

Determination of flavonoids
Quantitative flavonoid level was determined using the procedure described by (Ejikeme et al., 2014). Five grams of pulverized samples for each collection time was extracted with 50 ml of 80% aqueous methanol for 24hrs at ambient temperature, the mixture was filtered while the filtrate was discarded and the residue re-extracted thrice with 50 ml of ethanol; then the filtrate was combined and decanted into a crucible with the liquid evaporated to dryness in a water bath after which the crucible with its content was cooled in a desiccator and weighed to constant weight. The % flavonoid was calculated using the formula

Determination of phenol
The total phenols in the three samples was determined by the method previously used by Kyung et al., (2007) with little modifications. 10g of ground sample was treated with 30ml of 80% methanol in a 250ml beaker at 25°C in the dark to extract the phenolic. The mixture was transferred to warring blender and homogenized three times for an hour with successive addition of 30ml of 80% methanol. The homogenized mixture was then filtered using a Whatman No 1 filter paper into a volumetric flask of 100ml, then made up to mark with 80% methanol. 1ml of phenolic extract was added to 1ml of folin-ciocaltal reagent followed by incubation for 5mins at 22oC and then addition of 5ml of 20% sodium carbonate. 0-10ppm of gallic acid standard solutions were prepared from 100ppm garlic acid standard and treated similarly like the samples and then read at 735nm on the spectrophotometer. % total phenolic was estimated using gallic acid as standard.

Determination of alkaloids
The gravimetric method (Harborne, 1973) was used in estimating the alkaloid contents of the samples. 2g of pulverized sample was extracted with 200 ml of 10% acetic acid prepared in ethanol for 4hrs, the resulting solution was filtered and the filtrate concentrated to a fourth of the original volume, this was then followed by addition of concentrated NH4OH (about 15 drops) to the extract until there is complete precipitation. The mixture was allowed to settle for 3hrs after which the supernatant was dispensed with and the precipitates washed using 20 ml of 0.1M NH4OH, filtered, the residue dried and weighed. The % alkaloid was calculated as:

Determination of glycosides
The total glycoside level was determined using the procedure described by (Onwuka, 2005). 10g of samples collected at different times were extracted with ethanol after which 10ml of extract was transferred using a pipette into a 250ml conical flask, then addition of 50ml of chloroform, with the mixture agitated on a vortex mixer for an hour. The resultant solution was separated by filtration into a fresh 100ml conical flask, where 10 ml of pyridine and 2 ml of 2% sodium nitro-prusside were introduced and well agitated for 10 minutes. The development of brownish yellow colouration was achieved by the addition of 3ml of 20% sodium hydroxide. Glycoside standards (0-5 mg/ml) were prepared from stock, and taken through the analytical procedure described above. The absorbance of the samples and those of standards were read at a wavelength of 510nm using a spectrophotometer. Percentage glycoside of the samples was calculated from the concentration extrapolated from the standard curve absorbance against concentration.

Determination of tannins
The tannin content was determined according to the AOAC (1999) methods. Two grams of pulverized sample was weighed into a beaker, 100ml distilled water was introduced and the resulting mixture placed in water bath to boil for an hour with constant agitation to achieve uniform mixing, then, the mixture was separated by filtration into 100 ml volumetric flask using Whatman filter paper. Colour development was achieved through the addition of 5 ml of Folin-Denis reagent, 10 ml of saturated Na2CO3 and 5 ml of distilled water with 10ml of extract, after which the resultant solution was allowed to stand for 30 minutes at a temperature of 250C in a water bath with thorough shaking. Different concentrations of tannic acid (0-1mg/ml) was prepared from tannic acid stock and taken through the same procedures as the samples, then the absorbance of both samples and standards were taken at 700nm using spectrophotometer. Tannin amount was computed from the concentration value extrapolated from the calibration curve using the formula:

Determination of Terpenoids
The extract (1 g) was macerated with 50 ml of ethanol and filtered. To the filtrate (2.5 ml), 2.5 ml of 5% aqueous phosphormolybdic acid solution was added and 2.5 ml of concentrated H2SO4 was gradually added and mixed. The mixture was left to stand for 30 min and then made up to 12.5 ml with ethanol. The absorbance was taken at 700 nm (Ekwueme et al., 2015)

Data analysis
Data generated were analyzed statistically using SPSS version 20 and MS Excel, results are shown as means ± standard deviation of triplicate measurements.

3. RESULTS AND DISCUSSION
Table 1 presents the procedures and observations of the phytochemical screening of five tree species (Eucalyptus camaldulensis, Afzelia africana, Adansonia digitata, Cedrela odorata, Mansonia altissima).

Table 1: Phytochemical Screening Test Procedures and Observations

Table 2 presents the result of the phytochemical screening (Qualitative) of leaves investigated in some of the tree species. The qualitative phytochemical screening shows the presence of a varieties of plant secondary metabolites such as alkaloids, flavonoids, saponin, tannin, phenolics, cardiac glycosides and terpenoids.

Table 2: Qualitative phytochemical screening inference of the leaves of some tree species

Table 3 presents the results obtained for the phytochemical analysis(quantitative) of the five tree species evaluated. All the five species had the presence of some secondary metabolites in varying quantities. Only two trees species which are Afzelia africana and Cedrela odorata out of five tree species examined contained all the phytochemicals investigated.

Table 3: Quantitative phytochemical analysis (mg/100g) of the leaves of some tree species

Trees, apart from having an economic advantage can also be useful in combating various diseases because it contains some bioactive compounds with biological and metabolic activity which are usually responsible for the characteristic colour of plant, odour, pungencies, while others give the plant its particular medicinal or poisonous virtue (Wahood et al., 2013). The result of the phytochemical screening obtained in this study in Table 2 shows that secondary metabolites are present in all the five trees species evaluated. This is contrary and at the same time in consonance with those reported for other tree species. Alkaloids and flavonoids are present across the five plants. These secondary metabolite (alkaloids and flavonoids) produced by plants serves as chemical defence against pest and diseases attacks. Flavonoids also possess hypoglycemic property used for the treatment of anti-microbial, anti-allergic, anti-inflammatory, anti-cancer and anti-diabetes (Ghamba et al., 2012). The results gotten for alkaloids and flavonoids for all the five tree species is in consonance with (Usie et al., 2016) who indicated the
presence of alkaloid and flavonoids on Tamarindus indica leaves. This research also shows that Saponins are present in the plants, this may be due to inflammatory properties it possesses as reported by (Ekwueme et al., 2011). Plant which have saponin according to Adegboye et al., (2008) are reported to be very effective for treating rheumatism, syphilis, treatment of abscesses, ulcer, septic wounds etc. In our findings, tannin is present in all the plant extracts except for Eucalyptus camaldulensis. The presence of tannin in the other four tree species which are A. africana, A. digitata, C. odorata and M. altissima are effective as antidotes for poison (Norton, 2000). Tannins is a group of phenolic substance found in almost every plant parts, which has been reported by different researchers to have some physiological effects like anti-parasitic, anti-secretolytic, anti-irritant, anti-phlogistic, and anti-microbial. Therapeutically, plants containing tannin are used to treat diverse range of diseases. (Ijoma et al., 2017) in its findings also indicated the presence of tannin in Dialium indium leaves which is synonymous to our findings. The presence of phenol in the extract of E. camaldulensis, A. africana, A. digitata, C. odorata and M. altissima shows the tree species has high medicinal values. These compound (Phenol) have been investigated for their medicinal potentials against oxidative damage leading to various cardiovascular diseases, cancer, inflammation and so many degenerative diseases. Tumour cells which includes leukaemia cells, usually have higher reactive oxygen species (ROS) than normal cells because the cells are particularly sensitive to oxidative stress Mandal et al., 2010. Phenol are chemical compounds consisting of hydroxyl function, the number and site of hydroxyl groups on the phenol group are closely related to their toxicity to microbes (James and Nnacheta, 2008). Polyphenolic compounds are known to possess phytochemical and antioxidant properties (Djeridane et al., 2006). The findings of (Ogunmefun et al., 2017) that researched on Tectona grandis leaves corroborates with our study that also reveals the presence of phenol. The cardiac glycoside as reported in this research shows that all the tree species investigated reveals the presence of cardiac glycoside except for Mansonia altissima. (Usie et al., 2016) in his report shows the absence of Cardiac glycoside in Tamarindus leaves. The result gotten by (Usie et al., 2016) favours M. altissima leaves which is also absent and it is contrary to other tree species which are all present. Also (Ogunmefun et al., 2017) results on Tectona grandis leaves shows the absence of cardiac glycoside. Terpenoids are present in all the investigated tree species except Adansonia digitata. (Ijoma et al., 2017) reported in his research work that the leaves of Dialium indium reveals the presence of terpenoids which is in line with our findings. Terpenoids, among the plant secondary metabolites functions as phytoalexins in plant direct defence which herbivores partake and their natural enemies (McCaskill and Croteau 1988). Most of the plants produce volatile terpenes which attracts some specific insects for pollination and expel certain animals feeding on such plants as food (Degenhardt 2003). Terpenes plays a significant role as growth regulators and as signal compounds of plants as revealed by preliminary investigations.
Table 3 shows the quantitative analyses for Eucalyptus camaldulensis, Afzelia africana, Adansonia digitata, Cedrela odorata and Mansonia altissima. For the five tree species examined, alkaloid ranges were estimated from 3.48-5.63%, flavonoid 2.50-4.47%, saponin 2.84-9.00%, tannin 2.05-3.90%, phenolics 6.42-15.22%, cardiac glycoside 1.02-1.82%, terpenoids 1.72-2.94% respectively.
The phytochemical content for E. camaldulensis and Afzelia africana are significantly different from each other except flavonoids and saponin, cardiac glycoside and terpenoids that are not significantly different. Meanwhile, all the phytochemicals examined for Adansonia digitata and Mansonia altisimma are all significantly different from each other. In Cedrela odorata, alkaloids, flavonoids and Saponin are not significantly different from each other except tannin, phenolics, C. glycosides and terpenoids that are significantly different at p<0.05. Alkaloid is most abundant in A africana followed by M altissima, A altissima, E. camaldulensis and C. odorata. In Flavonoids, M altissima has the highest amount followed by C. odorata, A. africana, E. camaldulensis, and A. digitata while in Saponin, A. digitata has the highest concentration followed by Eucalyptus camaldulensis, C. odorata, A africana and M. altissima. For Tannin, M. altissima has the highest content followed by A. digitata, C. odorata and A. Africana but absent in Eucalyptus camaldulensis. Phenolics was revealed the highest in A. digitata while a similar value was reported for both A. Africana and C. odorata, followed by E. camaldulenis and M. altissima. Cardiac glycoside recorded the highest quantity in C. odorata followed by A. africana, A. digitata, E camaldulensis while it is absent in Mansonia altissima. The highest quantity of terpenoids was recorded for C. odorata, followed by Mansonia altissima, Afzelia africana, Eucalyptus camaldulensis and absent in Adansonia digitata. Phytochemicals in the leaf extract shows varying amounts. Studies shows that all the plants investigated have the tendency to treat diverse ranges of ailments. In this present study, phenolic has the highest range across the five plants while Ijoma et al., 2017 reported that saponin has the highest range in Dialium indium leaves conducted. The phenolic compounds detected have been studied to possess potent antioxidant activity. (Adedapo et al., 2009) related to the activity of the phytochemical possessing antioxidant to the mechanism of action one antimicrobial. Also the result gotten for phenolics in this study is 7.54 which is higher and contrary to the one obtained by Namrata et al., 2018 for Eucalyptus globulus extract that is 1.014. While for Alkaloid, the result gotten by Namrata et al., 2018 which is 24.499 is astronomically higher than that gotten from this studies which is 4.27. Cardiac glycoside result (1.54%) obtained in my study is higher than that gotten by (Ijoma et al., 2017) whose range is 1.009%. Secondary metabolites in plants such as phenolic compounds are necessary for plant reproduction, growth, prevents chronic illness such as neurodegenerative disease, cardiovascular diseases, diabetes and certain types of cancer which serves as protecting agents against pathogens (Scalbert, 2005). Phenolic content in plants could be used as immune enhancers, hormone modulators, anti-inflammatory and has specific ability to block some enzymes that causes inflammation and diseases (Okwu and Omodamino, 2005: Okwu 2004).

4. CONCLUSION
This study has shown that the five tree species studied contained the bioactive constituents which are alkaloids, tannins, flavonoid, saponin, cardiac glycosides, terpenoids and Phenol. These phytochemicals enabled these plants to be of great medicinal importance to humans as they are used in the management and treatment of many health related diseases. Phytochemical screening and analysis can be beneficial for drug discovery and development. Our study revealed that important medicinal components are present in the studied species. Hence, this plant can be used as a good source for beneficial drugs and its quantified values can be used as a tool for a drug to obtain a quality control profile.

5. CONFLICT OF INTEREST
The author declares no conflict of interest.

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