Journal of Bioresource Management

Volume 7 Issue 3 Article 11

Quantitative Analysis of Phytochemical Compounds in Barks and Leaves of Okoubaka Aubrevillei Collected from Iwo, Southwestern Nigeria

Oluwatoyin Opeyemi Akintola Forestry Research Institute of Nigeria, Ibadan, Nigeria, [email protected]

Adeboyin Funmi Aderounmu Forestry Research Institute of Nigeria, Ibadan, Ibadan, Nigeria

Isaac Oluwaseyi Abiola Forestry Research Institute of Nigeria, Ibadan, Nigeria

Kolawole Emmanuel Abodurin Forestry Research Institute of Nigeria, Ibadan

Tunde Adeniran Forestry Research Institute of Nigeria, Ibadan

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Recommended Citation Akintola, O. O., Aderounmu, A. F., Abiola, I. O., Abodurin, K. E., Adeniran, T., Agboola, F., & Olokeogun, O. S. (2020). Quantitative Analysis of Phytochemical Compounds in Barks and Leaves of Okoubaka Aubrevillei Collected from Iwo, Southwestern Nigeria, Journal of Bioresource Management, 7 (3). DOI: https://doi.org/10.35691/JBM.0202.0146 ISSN: 2309-3854 online (Received: Jun 7, 2020; Accepted: Sep 6, 2020; Published: Sep 30, 2020)

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Authors Oluwatoyin Opeyemi Akintola, Adeboyin Funmi Aderounmu, Isaac Oluwaseyi Abiola, Kolawole Emmanuel Abodurin, Tunde Adeniran, Festus Agboola, and Oluwayemisi Samuel Olokeogun

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This article is available in Journal of Bioresource Management: https://corescholar.libraries.wright.edu/jbm/vol7/ iss3/11 Akintola et al. (2020). Phytochemical Compounds in Barks and Leaves of O. Aubrevillei, Southwestern Nigeria J Biores Manag. 7(3): 131-142

QUANTITATIVE ANALYSIS OF PHYTOCHEMICAL COMPOUNDS IN BARKS AND LEAVES OF OKOUBAKA AUBREVILLEI COLLECTED FROM IWO, SOUTHWESTERN NIGERIA

OLUWATOYIN OPEYEMI AKINTOLA, ADEBOYIN FUNMI ADEROUNMU, ISAAC OLUWASEYI ABIOLA, KOLAWOLE EMMANUEL ABODURIN, TUNDE ADENIRAN, FESTUS AGBOOLA AND OLUWAYEMISI SAMUEL OLOKEOGUN

Forestry Research Institute of Nigeria, Ibadan, Nigeria

*Corresponding author: [email protected] ABSTRACT Okoubaka aubrevillei is an indigenous and sacred tropical tree in Africa. It is rare with allelopathic properties and has relatively little information available in terms of what is responsible for its usage for medicinal and traditional usage. The phytochemical screening and quantitative analysis of the compounds in the barks and leaves of Okoubaka aubrevillei was determined to ascertain and establish its earlier claimed usage as traditional and modern medicine. The most important classes of secondary metabolites (phytochemical compounds) specifically alkaloids, flavonoids, tannins, saponins, anthraquinnes, mucilages, oses, holosides, coumarin and glycosides were detected in both, the leaves and barks of the tree. Quantitative determination of the phytochemical compounds found in the barks and leaves of the tree revealed that the leaves of O. aubreville tree had significantly higher values of alkaloids, flavonoids and glycosides than barks. Saponins and anthraquinnes were found to be significantly more in barks than in leaves. However, there was no significant difference found in the values of tannins, mucilages, oses and holosides and coumarin in barks and leaves of O. aubreville trees. Keywords: Phytochemical compounds, quantitative analysis, flavonoids, tannins and alkaloid. INTRODUCTION flavonoids, carbohydrates and steroids among others that can offer significant The resistance of bacteria to physiological actions in the human body antibiotics and antimicrobials over the (Choo et al., 2014; Dua et al.,2013; Farhat years has become a major challenge for the et al., 2013; Adefega and Oboh, 2012; healthcare sector (Mbaeyi-Nwaoha and Chlopicka et al., 2012). Onwuka, 2014). With the increase in Phytochemicals are naturally occurrence of lethal and widespread occurring chemical, biological and active infectious diseases, there is a need for compounds found in plants that are of urgent and continuous discovery of new benefit to human health apart from those antimicrobial compounds. Recently, the that act as macronutrients and use of plants for medicinal purposes is micronutrients (Jasiem, 2016). These gaining more attention due to the presence compounds shield plants from disease, of secondary metabolites environmental damage such as (phytochemicals). In the past, parts of contamination, stress, drought, pathogenic plants such as herbs, leaves, root, bark of attack and contribute to fragrance, color trees, vegetables and fruits etc. have been and flavor (Kotche et al., 2016). The used for cure of various diseases. various compounds (phytochemicals) Studies have shown that some especially secondary metabolites such as plants have a diverse variety of bioactive flavonoids, phenols, alkaloids, glycosides, compounds such as tannins, alkaloids, saponins, tannins, steroids, anthraquinnes,

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Akintola et al. (2020). Phytochemical Compounds in Barks and Leaves of O. Aubrevillei, Southwestern Nigeria J Biores Manag. 7(3): 131-142 mucilages, coumarin, oses and holosides with a wooden batten, and no metal found in plants have anticancer, materials such as knife, cutlass and axe antimicrobial, antiviral, anti-inflammatory, should be used on the tree (Field survey, antitumor, analgesic and many other 2019). The paucity in its native range and properties (Rahaman et al., 2017; Erum et little or no information about its scientific al.,2015; Abdelwahab et al., 2010). The authentication for the plant’s medicinal fundamental means of their usage for usage draws the attention of this study. medicine is due to their bioactive This work carried out a preliminary properties which are used as substrates for phytochemical screening and biochemical and enzymatic reactions determination of their total content in (Samell et al., 2018; Dillard and German, barks and leaves of Okoubaka aubrevillei 2000). tree collected from Amera village near Iwo Okoubaka aubrevillei, is an town in Osun state, Southwestern Nigeria. indigenous and sacred tree in Africa. It has allelopathic properties. It is a tropophilous MATERIAL AND METHODS plant tree that grows up to 40 meters high with trunk as wide as 3 meters (Borokini, Geographical Location 2014). The tree has a huge shaggy crown, cylindrical and straight bowl; and reddish- Okoubaka aubrevillei is a tropical brown coarse bark surface (Borokini, tree species majorly found within the 2014). Leaves of these trees usually do not Central and West African countries wither, though they sometimes shed leaves (Figure 1) (Borokinni, 2015; Poorter et al., into cobwebs. Samples of this plant can 2004). The location of the studied tree is in only be collected using the traditional Amere village near Iwo town, Osun State ways of the locals. For this purpose, bark Southwestern Nigeria (Figure 2). The of the tree must be harvested during the study area is geographically located on day or at night and not when the sun is set within 7º 43´ 0´´ N and 4º 13´ 0´´ E in Ola- or just rising and can only be harvested Oluwa near Iwo town, Osun State.

Figure 1. Map of distribution of Genus Okoubaka in West and Central Africa (Borokini, 2014)

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Akintola et al. (2020). Phytochemical Compounds in Barks and Leaves of O. Aubrevillei, Southwestern Nigeria J Biores Manag. 7(3): 131-142

Figure 2. Location map of the Okoubaka aubrevillei tree with the sample of the leaf and tree

Sample Collection allowed to stand for 48 hours with sporadic shaking. The suspension was The dried leaves on the ground filtered with filter paper and the filtrate floor of the trees were handpicked based was exposed for evaporation in a soxhlet on the traditional method for sample apparatus for 10 hours using absolute collection, while the harvested barks were methanol. The solvent, the crude ethanolic collected from the tree. Both the leaf and extracts of leaf and bark of the tree the bark samples of the tree were labeled recovered were kept in desiccators at 4 º C accordingly. for further analyses.

Sample Preparation Qualitative Determination of Phytochemical Compounds Barks and leaves of O. aubrevillei collected from tree worship place were The ethanolic extracts of bark and rinsed with tap water followed by distilled leaf of O. aubrevillei were tested for the water to remove the dirt on the surface. presence of alkaloids, flavonoids, tannins, They were then air dried for 2 days and saponins, anthraquinnes, mucilages, oses dried until a constant mass was obtained. and holosides, coumarin, glycosides and The dried samples of the barks and leaves reducing sugar (Oloyed, 2005; Guessan et of O. aubrevillei were ground into fine al., 2009; Roopashree et al., 2008; powder and kept in desiccators for Sofowora, 1993; Trease and Evans, 1989). extraction. The extraction was carried out The qualitative results are expressed as (+) using the method described by Anowi et for the presence and (-) for the absence of al. (2012). One kilogram of dried powder phytochemicals. of leaves and barks were soaked separately in 200 ml of analytical ethanol and

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Quantitative Phytochemical Analysis of • Determination of Tannins: This the Leaf and Bark Extracts was determined using the method of Nbaeyi-Nwaoha and Onwuka (2014). 10 The total contents of alkaloids, ml of water was added to 1 gram each of flavonoids, tannins, saponins, the samples of grounded leaf and bark of anthraquinnes, mucilages, oses and O. aubrevillei put in separate conical holosides, coumarin, glycosides and flasks, shook for 30 minutes at an interval reducing sugars in the barks and leaves of of 5 mins and filtered. A fixed quantity O. aubrevillei were analyzed using 6305 (2.5 ml) of each of the filtrates was put in UV/Visible spectrophotometer two separate conical flasks, 1 ml of Follin- (Wavelength range of 198 to 100 nm, Denis reagent and Na2CO3 were added to it Jenyway UK). The procedures used for the and mixed thoroughly. The mixture was analysis are discussed as follows: left to settle for 90 minutes at room temperature and the absorbance was • Determination of Alkaloids: This measured using spectrophotometer at 720 was determined using the method of nm for the two (leaf and bark) samples. Nbaeyi-Nwaoha and Onwuka (2014). One The procedure was repeated three times gram each of the grounded samples of leaf and values were recorded. and bark of O. aubrevillei were dissolved with 20 ml of 20% H2SO4 in ethanol (1:1) • Determination of Saponins: and filtered. 1 ml of each of the filtrates Petroleum ether (10 ml) was added to 1g (leaf and barks) was put into two different of each of the grounded samples of leaf test tubes and 5 ml of 40% H2SO4 was and bark of O. aubrevillei in separate further added and mixed thoroughly. The conical flasks using the method of Nbaeyi- mixture was covered and allowed to settle Nwaoha and Onwuka (2014). This was for 4 hours before taking the measurement. further mixed with 10 ml of petroleum The measurements were taken using ether, drained and dried. Ethanol (5 ml) spectrophotometer at 568 nm for the two was added to the dried mixture and mixed (leaf and bark) samples. The procedure thoroughly and about 2 ml of the mixture was repeated three times and values were each of the samples was put in the two recorded. different test tubes allowed to settle for 30 minutes, after which the measurements • Determination of Flavonoids: This were taken at 550 nm for the two (leaf and was determined using the method of bark) samples. The procedure was Nbaeyi-Nwaoha and Onwuka (2014). One repeated three times and the values were gram each of the grounded samples of leaf recorded. and bark of O. aubrevillei were dissolved in 200 ml of ethyl and filtered. 5 ml of • Determination of Anthraquinnes: each of the filtrates (leaf and barks) was This was determined using the method of put into two different test tubes; 5 ml of Kuster and Rocha (2014). Grounded dilute ammonia was added, mixed samples (1 g each) of leaf and bark of O. vigorously and allowed to settle for some aubrevillei were soaked with 100 ml hours. After which, the absorbance was benzene in separate conical flasks for 10 measured using spectrophotometer at 490 min and filtered. 1 ml of the filtrates was nm for the two (leaf and bark) samples mixed with 5 ml of 70 % H2SO4 in test (Nbaeyi-Nwaoha and Onwuka, 2014). The tubes and allowed to settle for some procedure was repeated three times and minutes. The absorbances of the two values were recorded. samples were measured using spectrophotometer at 284 nm. The

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Akintola et al. (2020). Phytochemical Compounds in Barks and Leaves of O. Aubrevillei, Southwestern Nigeria J Biores Manag. 7(3): 131-142 procedure was repeated three times and the values were recorded. • Determination of Glycosides: This was determined using the method of • Determination of Mucilage: This Nbaeyi-Nwaoha and Onwuka (2014). was determined using the method of Fifteen percent lead acetate (2.5 ml) was Fatemeh et al. (2017). Powder of barks and mixed thoroughly with 1 g of each of the leaves (10g each) of the studied tree was samples in test tubes and filtered. mixed with distilled water for 6 h and Chloroform (2 ml) was added to the boiled for 30 mins. The mixture was left filtrate, mixed thoroughly and allowed to for 1 hour and filtered through muslin settle. The lower portion was collected and cloth. Ethanol was added to the filtrate to evaporated to dryness. Glacial acetic acid allow mucilage precipitation. The (3 ml), 0.1ml of 5% ferric chloride and mucilage was separated and dried at 0.25 ml concentrated H2SO4 was added to temperature of 50 ºC. The dried mucilage dried lower portion, mixed vigorously and was then weighed and measured. The left for 3 hours. The absorbance was procedure was repeated three times and measured using spectrophotometer at 568 values were recorded. nm. The procedure was repeated three times and the values were recorded. • Determination of Oses and Holosides: This was determined using the Data Analyses method of Peixoto Sobrinho et al. (2008). All the tests were conducted in Leaves and barks (5 g each) was put in triplicates and the data was analyzed using separate conical flasks, mixed vigorously One-way analysis of variance (ANOVA). with 100 ml absolute ethanol, mixed and The significant differences were evaluated filtered. The filtrates were mixed gently using Tukey’s HSD test with a level of with concentrated H2SO4 (2 ml) in test significance (P<0.05). ANOVA was tubes for 5 minutes and allowed to settle performed with SPSS version 20. for some minutes. The dissolved Bromohymol (in absolute ethanol) and 2 RESULTS AND DISCUSSION drops of concentrated H2SO4 was added to the mixture and allowed to settle for 15 Phytochemical Screening minutes. The absorbances of the mixture Phytochemicals are secondary were read using spectrophotometer at 510 metabolites in plants that facilitate their nm wavelength. The procedure was ability to overcome the transitory or repeated three times and values were persistent coercion associated with their recorded. environment and they are of medical

benefit to man (Molyneus et al., 2007). • Determination of coumarins: This These secondary metabolites have been was determined using the method of used as plant derived anti-cancerous drugs. Kuster and Rocha (2014). Distilled water The medicinal plants used for (2 ml) and 0.5 ml of lead acetate solution ethnomedicine have been broadly screened were added to 0.5 ml extract of each of the for the presence of phytochemicals samples in separate test tubes and (Philipson, 2001; Edeoga et al., 2005). thoroughly mixed. Hydrochloric acid (8 Phytochemicals such as alkaloids, ml) was further added to the mixture and flavonoids, saponins, tannins etc. are left for 30 minutes at room temperature. protective compounds for treatment of The absorbance was measured using persistent diseases such as cancers, spectrophotometer at 320 nm wavelength. hypertension, diabetes etc. (Craig, 1997; The procedure was repeated three times Samell et al., 2018) and the values were recorded.

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In this study, the phytochemical screening presence of alkaloids, flavonoids, tannins, of crude ethanolic extracts of leaf and bark saponins, glycosides, anthraquinnes, samples of O. aubrevillei as indicated by mucilages, coumarins, oses and holosides. positive sign (+) (Table 1) revealed the

Table 1. Phytochemical screening of leaf and bark extracts of O. aubrevillei.

Phytochemical Compounds Barks Leaves Tannin + + Saponin + + Alkaloid + + Flavonoids + + Anthraquinnes + + Mucilages + + Oses and Holosides + + Coumarin + + Glycosides + +

They were detected in both the leaf activity (Devasayam et al., 2004; Mathew and bark extracts of O. aubrevillei. The et al., 2012; Omoregie and Osagie, 2012). phytochemical compounds detected are known to have medicinal importance. The Quantitative Determination of presence of these compounds could be Phytochemical Compounds in O. responsible for antimicrobial and aubrevillei immunity stimulating properties of O. aubrevillei making it effective as a The mean contents of each of the detoxifying, antibacterial and anti- phytochemical compounds in the ethanolic inflammatory agent (Bagot, 2015). extract of O. aubreville leaf and bark are Furthermore, the presence of the shown in Table 2. It was observed that the numerous phytochemical compounds in leaves of O. aubreville tree had the barks and leaves of the O. aubreville significantly higher values of alkaloids tree may be responsible for its local (0.64 ± 0.03), flavonoids (0.35±0.02) and (traditional) usage especially the bark as glycosides (11.32±0.001) than barks with concoction to treat various skin diseases alkaloids (0.47± 004), flavonoids (effective for bruising), as antidote for (0.24±0.01) and glycosides (8.64±0.16) food poison. values. The significantly higher values of Previous studies state that saponins (0.25± 0.01) and anthraquinnes phytochemicals act as best antioxidants (0.17±0.01) were found in barks than in and protect the cells from free radical leaves. However, there was no significant damage such as carotenoids, polyphenols difference found in the values of tannins, and reduce the risk of cancer by mucilages, oses and holosides and discouraging the production of tumour or coumarin in barks and leaves of O. hormonal stimulation and antibacterial aubrevillei trees as shown in Table 2.

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Table 2. Total Mean Contents of Phytochemical Compounds of O. aubrevillei. Phytochemical Compounds Barks (mg/100g) Leaves (mg/100g) Tannin 0.29±0.02a 0.25±0.01a Saponin 0.25± 0.0a 0.18±0.01b Alkaloid 0.47± 004b 0.64±0.03a Flavonoids 0.24±0.01b 0.35±0.02a Anthraquinnes 0.17±0.01a 0.02±0.001b Mucilage 0.02±0.001a 0.01±0.001a Oses and Holosides 0.03±0.001a 0.02±0.001a Coumarin 0.01±0.001a 0.01±0.001a Glycosides 8.64±0.16b 11.32±0.001a

Values with different letters are significantly different from each other

Alkaloids, phytochemical et al., 1961). Its effects on neutral tissues compounds found in the bark and leaves of and indirect effect on electrical activities the studied tree have the most of the heart and vascular resistance as well advantageous significance among plant as capacitance has been reported chemical constituents and have analgesic, (Chukwuma et al., 2016). Saponins, found antibacterial and antispasmodic in the bark and leaves of the tree have been characteristics among others (Enujugha described as phytochemical compounds and Agbede, 2000; Harisaranraj et al., that have the ability to coalesce with 2009; Uyo et al., 2013). These beneficial cholesterol, they impart a bitter taste and properties of the alkaloids justify the cause haemolytic activity in water solution traditional and modern medicinal usage of (Sodipo et al., 2000). They can also be the studied trees. Similarly, the presence of used for fungal infections (Sheikh et al., flavonoids in the bark and leaves of the 2013). Anthraquinnes may also contribute tree is a good contributor to its medicinal to this plant’s effective medicinal usage usage. Flavonoids are useful antioxidants due to the laxative effects of anthraquinnes that take part in fighting against liver for relieving constipation (Portalatin and tumors, toxins, allergies, inflammation, Winstead, 2012). viruses and other microorganisms Tannins found in minute quantities (Harisaranraj et al., 2009; Uyo et al., are known to be anti-nutrient in diets as 2013). It is a powerful and dominant well as having some degree of astringency compound in protecting blood vessels (Chikezie et al., 2008). This compound can particularly those that are answerable to intermingle with proteins to form insoluble distribution and transportation of nutrients complexes, thus lessening protein and oxygen to cells as well as delaying bioavailability (Enujugha and Agbede, cataract enlargement in diabetic patients 2000). Tannins also possess antiviral, (Harisaranraj et al., 2009). antibacterial, and antitumor properties Glycosides found in the bark and (Kakiuch et al., 1986; Khanbabaea and leaves have been used in the treatment of Ree, 2000). Thus, they are used for congestive heart failure due to its diverse treating diarrhea, dysentery and urinary action which strengthens the force of tract infections (Fahey, 2005; Akinpelu myocardial contraction as well as its direct and Onakaoya, 2009). Mucilage, action on the smooth muscles (Braunwald coumarins, oses and holosides found in

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Akintola et al. (2020). Phytochemical Compounds in Barks and Leaves of O. Aubrevillei, Southwestern Nigeria J Biores Manag. 7(3): 131-142 parts of the tree studied can also contribute barks of O. aubreville tree. However, there to the traditional and medicinal usage of was no significant difference found in the the tree. According to Somboro et al. values of tannins, mucilages, oses and (2011), mucilages are favourable for stool holosides and coumarin. The wide firmness and can also prevent sudden diversity and numerous secondary dehydration while coumarins as metabolites found in samples of this tree polyphenolic compounds have anti- may be responsible for its copious inflammatory, antimicrobial and medicinal and traditional usage. It is anticoagulant properties. recommended that further studies should This plant has been used in the be carried out using other solvents such as production of the herbal antimalaria drug propanolic extract of the leaves and bark (Maloff-Hb) in Nigeria (Ogunkunle et al., of the tree among others. 2014). Internationally, the bark of the tree has been extensively used in Europe and REFERENCES America for production of homeopathic drugs and in veterinary medicine (Bagot, Abdelwahab SI, Abdul AB, Elhassan MM, 2015; Borokini, 2015). Previous studies Mohan S, Al-Zubairi AS and (Borokini, 2015) have shown ability of Mariod AA (2010). Antimicrobial Okoubaka for stimulating body’s defense and free radical scavenging mechanism against poisoning and the activities of dichloromethane extract from its bark has shown to be extract of Goniothalamus effective for upset stomach, constipation, umbrosus. Int J Trop Med., 4 (1): pesticide poisoning, lethargy, depression 32-36. and all this can be attributed to the Adefega SA and Oboh G (2012). Effect of presence of the numerous and varying diets supplemented with Ethiopian quantities of the phytochemical pepper [Xylopia aethiopica (Dun.) compounds found in this study. Okoubaka A. Rich (Annonaceae)] and tree is listed as one of the top 300 herbal medicinal plants sold in United Kingdom Ashanti pepper [Piper guineense while the bark of the trees is known to be Schumach. et Thonn (Piperaceae)] used in pediatric medicine in Germany on some biochemical parameters in (Borokini, 2015; Kohlrausch, 2011). Based normal rats. Asian Pac J Trop on the numerous phytochemicals in the Biomed., 2 (2): S558-S566. bark and leaves of O. aubreville tree, the Akinpelu OA (2009). Posers on the reality tree can be also termed as a protective of Igi Nla (Nnune Ebe?). The Nig health agent. Field., 7: 92 –96. CONCLUSION Akotto FO, Alphonse AK, François MD, Josselin KK, Albert YK and The most important classes of Kamanzi K (2014). Soil landscape secondary metabolites (phytochemical and stand conditions in Cola compounds) specifically alkaloids, attiensisin Côte d'Ivoire. Int J flavonoids, tannins, saponins, Biosci., 4 (5): 102-113. anthraquinnes, mucilages, oses and http://dx.doi.org/10.12692/ijb/4.5.1 holosides, coumarin and glycosides were 02-113. detected in both the leaves and bark of the Aubréville A and Pellegrin F (1937). Deux tree. Quantitative determination of the nouveautes de la Cote d’Ivoire. phytochemical compounds revealed Bull Soc Bot Fr., 84: 390-393. significantly higher values of alkaloids, http://dx.doi.org/10.1080/0037894 flavonoids and glycosides in leaves than 1.1937.10837397

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