The phytochemical constituents and antioxidant properties of Nauclea latifolia leaf decoction were investigated.

Dried leaves were extracted in ethanol. Qualitative and quantitative phytochemical analysis was determined

spectrometrically. The antioxidant activities were examined in vitro using 2,2-diphenyl-1-picrylhydrazyl radical, total

antioxidant capacity and ferric reducing antioxidant power assays. Phytochemical screening confirmed the presence of

flavonoids, alkaloids, anthocyanins, betacyanins, phenols, saponins, terpenoids, cardiac glycosides and quinones. The total

lycopene, β-carotene, phenolics, flavonoid and alkaloid content were found to be 0.038 ± 0.01 mg CAE/g, 0.120 ± 0.04 mg

CAE/g, 58.08 ± 0.58 mg GAE/g, 10.75 ± 0.17 mg RE/g and 0.32 ± 0.08% respectively. N. latifolia ethanol leaf extract

demonstrated effective antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl with an IC50 of 2.58 ± 0.08 mg/mL

compared to 0.86 ± 0.02 mg/mL and < 0.01 ± 0.01 mg/mL for butylated hydroxytoluene and ascorbic acid respectively.

Total antioxidant capacity and ferric reducing antioxidant power of the extract were 73.81 ± 2.27 and 1314.45 ± 197.64

mg AAE/g respectively. Excellent positive correlations between the phenolic content and antioxidant activities of the

extract were observed. The leaf of N. latifolia is of therapeutic value and may be exploited for its rich antioxidant

component

Chinedu, S. N.

Emiloju, Opeyemi C.

Iheagwam, Franklyn N

Kayode, Kazeem Oyindamola

Nsedu, Emmanuel Israel

Ogunlana, Olubanke Olujoke

Covenant University Repository

Bioactive screening and in vitro antioxidant assessment of Nauclea latifolia leafdecoctionFranklyn Nonso Iheagwam, Emmanuel Israel Nsedu, Kazeem Oyindamola Kayode, Opeyemi ChristianahEmiloju, Olubanke Olujoke Ogunlana, and Shalom Nwodo ChineduCitation: AIP Conference Proceedings 1954, 030015 (2018); doi: 10.1063/1.5033395View online: https://doi.org/10.1063/1.5033395View Table of Contents: http://aip.scitation.org/toc/apc/1954/1Published by the American Institute of PhysicsBioactive Screening and In Vitro Antioxidant Assessment of Nauclea latifolia Leaf Decoction Franklyn Nonso Iheagwam1, 2, a), Emmanuel Israel Nsedu1, Kazeem Oyindamola Kayode1, Opeyemi Christianah Emiloju1, 2, Olubanke Olujoke Ogunlana1, 2 and Shalom Nwodo Chinedu1, 2 1Department of Biochemistry, College of Science and Technology, Covenant University, Canaanland, P.M.B. 1023 Ota, Ogun State, Nigeria 2Covenant University Public Health and Wellness Research Cluster (CUPHWERC), Covenant University, Canaanland, P.M.B. 1023 Ota, Ogun State, Nigeria.  a)Corresponding author: franklyn.iheagwam@covenantuniversity.edu.ng.  Abstract. The phytochemical constituents and antioxidant properties of Nauclea latifolia leaf decoction were investigated. Dried leaves were extracted in ethanol. Qualitative and quantitative phytochemical analysis was determined spectrometrically. The antioxidant activities were examined in vitro using 2,2-diphenyl-1-picrylhydrazyl radical, total antioxidant capacity and ferric reducing antioxidant power assays. Phytochemical screening confirmed the presence of flavonoids, alkaloids, anthocyanins, betacyanins, phenols, saponins, terpenoids, cardiac glycosides and quinones. The total lycopene, β-carotene, phenolics, flavonoid and alkaloid content were found to be 0.038 ± 0.01 mg CAE/g, 0.120 ± 0.04 mg CAE/g, 58.08 ± 0.58 mg GAE/g, 10.75 ± 0.17 mg RE/g and 0.32 ± 0.08% respectively. N. latifolia ethanol leaf extract demonstrated effective antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl with an IC50 of 2.58 ± 0.08 mg/mL compared to 0.86 ± 0.02 mg/mL and < 0.01 ± 0.01 mg/mL for butylated hydroxytoluene and ascorbic acid respectively. Total antioxidant capacity and ferric reducing antioxidant power of the extract were 73.81 ± 2.27 and 1314.45 ± 197.64 mg AAE/g respectively. Excellent positive correlations between the phenolic content and antioxidant activities of the extract were observed. The leaf of N. latifolia is of therapeutic value and may be exploited for its rich antioxidant components. INTRODUCTION There is an intensified research on medicinal plants due to various therapeutic potentials these herbal plants possess. The medicinal properties of these plants are believed to be conferred by phytochemicals present in the plant [2]. Phytochemicals are known as natural organic compounds/substances that are found in plants which gives them their characteristic colour, flavour, smell and texture in addition to their potential in treating, managing and preventing diseases such as cancer and cardiovascular diseases [1]. Nauclea latifolia Sm. is a small treecommonly found in tropical Africa especially Nigeria, which grows as tall as 20 feet in height, forming canopies [3]. It is commonly known as pin cushion or African peach tree; In Nigeria, the Igbos call it “Ubuluinu”, “Mbom-mbong” in Efik, “Uche” by the Igede’s,“Egbesi” among the Yoruba’s and “Tafashiya” by the Hausas [5]. N. latifolia is used extensively in traditional medicine by the locals. Its infusions and decoctions from stems and roots are used mainly against malaria by traditional healers; as a chewing stick and as a remedy against stomach ache and tuberculosis [4]. It can be prepared together with other plants and applied against diarrhoea. Various researched pharmacological effect of N. latifolia include lowering blood pressure, anticonvulsant, antipyretic, analgesic, anxiolytic and sedative properties [6]. However, there is a dearth of information on the evaluation of its bioactive chemical contents, antioxidant properties and relationship between both properties. Hence this study evaluated these parameters. Proceedings of the 2nd International Conference on Applied Sciences (ICAS-2)AIP Conf. Proc. 1954, 030015-1–030015-7; https://doi.org/10.1063/1.5033395Published by AIP Publishing. 978-0-7354-1653-6/$30.00030015-1MATERIALS AND METHODS Chemicals and Reagents All chemicals and reagents were purchased from Surechem Products and Sigma Aldrich. They were all of analytical grade. Collection of Plant Samples, Identification and Extract Preparations Healthy looking leaves of Nauclea latifolia were sourced from local farmers in Oyo State, Nigeria and identified by a botanist, Dr. J.O. Popoola from the Department of Biological Sciences, Covenant University, Ota, Nigeria. The leaves were cut into bits with knife and air dried to uniform size at room temperature for 10 days to remove all moisture. The samples were then ground into fine powder. The pulverised sample (800 g) was macerated in 80% ethanol (8 L) for 72 hrs. The filtrate obtained was concentrated using a rotary evaporator to yield a greenish-brown paste extract.  Qualitative Phytochemical Analysis  Test for tannins  To 1 mL of plant extract, 2 mL of 5% ferric chloride was added. Formation of greenish black indicated the presence of tannins. Test for saponins  To 2 mL of plant extract, 2 mL of distilled water was added and shaken in a graduated cylinder for 15 minutes lengthwise. Formation of 1 cm layer of foam indicated the presence of saponins. Test for flavonoids 5 mL of dilute ammonia solution was added to portions of the aqueous filtrate of plant extract followed by addition of concentrated sulphuric acid. The appearance of yellow colouration indicated the presence of flavonoids.  Test for alkaloids  To 2 mL of plant extract, 2 mL of concentrated hydrochloric acid was added. Then few drops of Mayer’s reagent were added. Presence of green colour indicated the presence of alkaloids. Test for anthocyanin and betacyanin To 2 mL of plant extract, 1 mL of 2 N sodium hydroxide was added and heated for 5 minutes at 100 oC. Formation of yellow colour indicated the presence of betacyanin. Test for quinones To 1 mL of extract, 1 mL of concentrated sulphuric acid was added. Formation of red colour indicated the presence of quinones. Test for glycosides To 2 mL of plant extract, 3 mL of chloroform and 10% ammonia solution was added. Pink colour formation indicated the presence of glycosides. 030015-2Test for cardiac glycosides To 0.5 mL of extract, 2 mL of glacial acetic acid and few drops of 5% ferric chloride were added. This was under layered with 1 mL of concentrated sulphuric acid. Brown ring formation at the interface indicated the presence of cardiac glycosides. Test for terpenoids To 0.5 mL of extract, 2 mL of chloroform was added and concentrated sulphuric acid was added carefully. Red-brown colour formation at the interface indicated the presence of terpenoids. Test for triterpenoids To 1.5  mL of extract, 1mL of Liberman-Buchard reagent (0.5 mL of acetic anhydride + 0.5 mL of concentrated sulphuric acid) was added. Blue-green colour formation indicated the presence of triterpenoids.  Test for phenols To 1 mL of the extract, 2 mL of distilled water followed by few drops of 10% ferric chloride was added. Formation of green colour indicated the presence of phenols.  Test for coumarins To 1 mL of extract, 1 mL of 10% sodium hydroxide was added. Formation of yellow colour indicated the presence of coumarins. Quantitative Phytochemical Analysis Total Phenolic content Total phenolic content (TPC) was determined according to the method of Sharma et al. [8]. Folin-ciocalteu reagent was added to 100 μl of sample/standard in ratio 1:10. The solution was mixed and incubated at room temperature for 1 minute followed by the addition of 1.5 mL to 20% sodium carbonate. The final mixture was shaken and incubated for 90 minutes in the dark at room temperature. The absorbance was taken at 725 nm and the phenolic content was expressed as gallic acid equivalents GAE/g of sample. Total flavonoid content Total flavonoid content (TFC) was determined according to the method described by Sharma et al. [8]. In different test tubes, 0.5 mL extract, 2 mL of distilled water, followed by 0.15 mL of sodium nitrite (5% w/v) was added. After 5 min, 0.15 mL of aluminium trichloride (10 %) was added and incubated for 6 min. After incubation 2 mL of sodium hydroxide (4% w/v) was added. After 15 min of incubation reaction mixture turns to pink and absorbance was measured against blank e.g. distilled water at 510 nm. A natural flavonoid rutin was used as standard. The TFC was expressed in RE/g of sample.  β-carotene and Lycopene The test for β-carotene and lycopene was carried out according to the method described by Sharma et al. [8]. The dried extract was vigorously shaken with 10 mL of acetone-hexane mixture (4:6) for 1 minute and filtered through Whatman N0.4 filter paper. The absorbance of filtrate was measured at 453, 505, 645 and 663 nm. The content of lycopene and β-carotene were calculated using equation (1) and (2), respectively. The values are expressed as μg/g of extract.   453505663 A0806.0A372.0A0458.0(mg/100mL)  Lycopene  (1) 030015-3  453505663 A452.0A304.0A216.0(mg/100mL) carotene  (2) Total Alkaloids According to the methods of Senguttuvan et al. [7]. 200 mL of 20% acetic acid was added to 5 g of sample and covered to stand for 4 h. This mixture containing solution was filtered and the volume was reduced to ¼ using water bath. To this sample, concentrated ammonium hydroxide was added drop wise until the precipitate was complete. The whole solution was allowed to settle and the precipitate was collected by filtration and weighed. The percentage of total alkaloids was calculated using equation (3).   100 takensample ofweight residue ofweight (%) alkaloids otalT  (3) Antioxidant Assessment 2,2-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay DPPH radical scavenging activity of NLE was carried out according to the method described by Sharma et al. [8]. 0.5 mL of DPPH was added to 0.5 mL aliquots of sample/standard in different concentrations. Control test tubes were loaded with 0.5 mL of dimethyl sulfoxide (DMSO) and 0.5 mL DPPH. After incubation at 37 °C for 30 min in dark, the absorbance was recorded at 517 nm. Ascorbic acid was used as a standard. The percentage scavenging by test sample at each concentration was calculated using the formula (4).   100)((%) DPPH ScavengedcontrolsamplecontrolAbsAbsAbs (4) Ferric reducing antioxidant power (FRAP) The reducing power of NLE was determined according to the method described by Sharma et al. [8]. Briefly, 1 mL of sample was mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide (1%). The reaction mixture was incubated at 50 oC for 20 minutes. Then 2.5 mL of trichloroacetic acid (10%) was added and centrifuged for 10 minutes. An aliquot 2.5 mL was mixed with 2.5 mL of distilled water and 0.5 mL of FeCl3 (0.1%). The absorbance of all solutions was measured at 700 nm and expressed as mg of ascorbic acid equivalent per g of powder (mg AAE/g powder). Total antioxidant capacity (TAC) TAC was determined according to the method described by Sharma et al. [8]. 0.3 mL of extract was combined with 3 mL reagent solution (0.6 M sulphuric acid, 28mm sodium phosphate and 4mM ammonium molybdate). The reaction mixture was capped and incubated at 95 oC for 90 min. After cooling to room temperature, the absorbance was measured at 695 nm against blank (methanol 0.3 mL). Ascorbic acid was taken as the standard. Statistical Analysis Results were expressed as mean ± standard deviation (SD) of replicate determinations. Statistical analysis of mean values was carried out by one-way analysis of variance (ANOVA) supplemented by Duncan multiple range test. Values were considered statistically significant at p < 0.05. 030015-4RESULTS The qualitative phytochemical analysis of N. latifolia aqueous extracts as illustrated in table 1 revealed the presence of flavonoids, alkaloids, anthocyanins, betacyanins, phenols, saponins, terpenoids, cardiac glycosides and quinones; tannins, glycosides, triterpenoids and coumarins were not detected.  TABLE 1. Qualitative phytochemical analysis of N. latifolia ethanolic leaf extract. Phytochemicals Nauclea latifolia Tannins  -ve Saponins  +ve Flavonoids  +ve Alkaloids  +ve Anthocyanin and betacyanins  +ve Quinones  +ve Glycosides  -ve Cardiac glycosides  +ve Terpenoids  +ve Triterpenoids  -ve Phenols  +ve Coumarins  -ve +ve=present; –ve=absent  When quantitatively analysed the total flavonoid, phenolic, lycopene, β-carotene, phenolics, flavonoid and alkaloid content were estimated to be 10.75 ± 0.17 mg RE/g, 58.08 ± 0.58 mg GAE/g, 0.038 ± 0.01 mg CAE/g, 0.120 ± 0.04 mg CAE/g and 0.32 ± 0.08% respectively as shown in table 2. TABLE 2. Total flavonoid, phenolic, β- carotene, lycopene and alkaloid content of N. latifolia ethanolic leaf extract Phytochemicals N. latifolia Content TFC (mg RE/g)  10.75 ± 0.17 TPC (mg GAE/g)  58.08 ± 0.58 β-carotene (mg CAE/g)  0.12 ± 0.04 Lycopene (mg CAE/g)  0.038 ± 0.01 Total Alkaloid (%)  0.32 ± 0.08 Data are mean±SD of triplicate determinations.  Figure 1 shows a dose-dependent increase in scavenging ability of DPPH radical for N. latifolia ethanolic leaf extract, butylated hydroxytoluene and ascorbic acid. N. latifolia ethanolic leaf extract scavenging ability was comparable with butylated hydroxytoluene while ascorbic acid exhibited the most scavenging ability. The IC50 of N. latifolia ethanolic leaf extract was 2.58 ± 0.08 mg/mL compared to 0.86 ± 0.02 mg/mL and < 0.01 ± 0.01 mg/mL for butylated hydroxytoluene and ascorbic acid respectively as presented in table 3. TABLE 3. IC50 of N. latifolia ethanolic leaf extract, butylated hydroxytoluene and ascorbic acid DPPH scavenging ability.  IC50 (mg/mL) Nauclea latifolia  2.58 ± 0.08a BHT  0.86 ± 0.02b AA  <0.01 ± 0.01c Data are mean ± SD of triplicate determinations. Values with different superscripts in a column are significantly different at p < 0.05. 030015-5 FIGURE 1. DPPH radical scavenging ability of butylated hydroxytoluene, ascorbic acid and N. latifolia ethanolic leaf extract  Figure 2 depicts the total antioxidant capacity and ferric reducing antioxidant power. A dose dependent increase was observed for both assay with 73.81 ± 2.27 mg AAE/g being the highest antioxidant capacity and 1314.45 ± 197.64 mg AAE/g being the highest reducing power recorded. The observed potent DPPH radical-scavenging, total antioxidant and ferric reducing capacity of the extract significantly corroborated the total phenolic content with a good correlation coefficient as illustrated in table 4. TABLE 4. Result of correlation and regression analysis Dependent Variable Pearson Correlation Linear Regression (Sig.) DPPH TAC FRAP DPPH TAC FRAP TPC 0.925* 0.977** 0.981** 0.024 0.004 0.003 **Correlation is significant at 0.01 level (2-tailed).  *Correlation is significant at 0.05 level (2-tailed) at p < 0.05.    FIGURE 2. a) Total antioxidant capacity of N. latifolia ethanolic leaf extract, b) Ferric reducing antioxidant power of N. latifolia ethanolic leaf extract 030015-6 DISCUSSION The presence of flavonoids, alkaloids, anthocyanins, betacyanins, phenols, saponins, terpenoids, cardiac glycosides and quinones in N. latifolia ethanolic leaf extract is an indication that these secondary plant metabolites have a synergistic effect on the various pharmacological properties reported by Onyesom et al. [6]. These phytochemicals play a major role in preventing disease and promoting the general health status of individuals. They are also involved in scavenging free radicals, inhibiting carcinogen-activating enzymes and increasing the synthesis and activity of enzymes that detoxify carcinogens [16]. β-carotene and lycopene have been reported to be potentially active in reducing the risk of both breast and prostate tumours [13]. Strong antioxidant activity has been attributed to be the molecular mechanism underlying the association between carotenoids and cancer prevention. Phenolic compounds are known antioxidants, due to their ability to mop up free radicals and reactive oxygen species [11]. DPPH is a stable free radical which delocalises its spare electron to give a deep violet colour. In the presence of an antioxidant, a hydrogen atom is donated followed by loss of colour [12]. The results generated from this study demonstrated that N. latifolia ethanolic leaf extract possessed good free radical scavenging activity. This potential may be attributed to the appreciable amounts of phenolic and flavonoid content [10]. Donating electron to reduce ferric ion to ferrous ion and Mo (VI) to Mo (V) is an indication of reducing power. The reducing capacity of a compound may be directly translated as an indicator of antioxidant activity [14]. The strong reducing power of N. latifolia ethanolic leaf extract might be as a result of its phenolic content, as they are good electron donors [15]. The extract’s antioxidant activity correlated positively with their phenolic contents as corroborated in a study by Awah et al. [17]. CONCLUSION This study provides evidence that N. latifolia possesses significant antioxidant activities which can be attributed to the phenolic content and other bioactive compounds present in the extract. However, further in vitro and in vivo study is required to better understand the efficacy and mechanism of action of these bioactives present in these plant extracts to provide scientific proof for clinical employment of N. latifolia secondary metabolites in modern medicine. REFERENCES 1. S. Renu, Biotechnology 4, 79–93 (2005). 2. O. P. Ukoha, L. O. Nnamdi, C. E. Cemaluk and P. E. Madus, Afr. J. Pure Appl. Chem. 5, 237–244 (2011). 3. A. O. Ayeleso, O. O. Oguntibeju and N. L. Brooks, Sci. 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Dhawale, Alexandria J. Med. 53(3), 237–243 (2017). 15. A. Pereira and M. Maraschin, J. Ethnopharmacol. 160, 149–163 (2015). 16. M. Saxena, J. Saxena, R. Nema, D. Singh and D. Gupta, J. Pharmacog. Phytochem. 1(6), 168–182 (2013). 17. F.M. Awah, P.N. Uzoegwu, P. Ifeonu, J.O. Oyugi, J. Rutherford, X. Yao, F. Fehrmann, K.R. Fowke and M.O. Eze, Food Chem. 131(4), 1279–1286 (2012). 030015-7

Bioactive Screening and In Vitro Antioxidant Assessment of

Nauclea latifolia Leaf Decoction

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Bioactive Screening and In Vitro Antioxidant Assessment of Nauclea latifolia Leaf Decoction

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