Evaluation of bioactive constituents and In vitro antioxidant potentials of the ethanolic leaf extracts of Dracaena

The current study investigated the phytochemical constituents and in vitro antioxidant activities of the ethanolic leaf extracts of Dracaena mannii , Euphorbita hirta and Senna alata . The phytochemical analyses of the leaf extracts showed varying amounts of important compounds such as tannins, phenols, alkaloids, flavoniods, protein and carbohydrates. Findings from the in-vitro free radical mopping abilities of the ethanolic leaf extracts against 1,1-diphenyl-2-picryl hydrazyl and nitric oxide radical (NO • ) as well as the reducing power and total antioxidant capacities revealed abilities to quell oxidative stress with S. alata > E. hirta > D. mannii excluding the NO • radical inhibition activity. This relationship was also reflected in the IC 50 /EC 50 values obtained, suggesting that the S. alata ethanolic leaf extract has the best antioxidant potential while E. hirta showed better activities than D. mannii . Thus, the leaves of S. alata could be further investigated for other biological potentials and possibly employed in the application of leaves against diseases that are implicated by oxidative stress


INTRODUCTION
Numerous bioactive compounds present in plants are synthesized from primary metabolites and have become the main source of herbal therapeutic products (Anigboro et al., 2014;Al-Snafi, 2017). The medicinal properties of plants is dependent on the content of the phytochemical constituents (Essiett and Okoko, 2013) and have gained increased attention among rural dwellers as well as a great part of the civilized populations, for the maintenance of health (Anigboro, 2017;Anigboro et al., 2018;Ghosh et al., 2019;Anigboro et al., 2019;Anigboro et al., 2021). It is therefore important to determine the phytochemical constituents of such medicinal plants to further enhance their therapeutic efficacy and safety (Mohammed et al., 2010).
Dracaena mannii (commonly called small-leaf dragon tree) is a small-to medium-size tree that is widespread in tropical Africa, and can record up to about 30 m and 2 m in height and diameter respectively (Damen et al., 2018).
Euphorbia hirta is a small pan tropical erect annual herb with hairy stem that grows up to 50cm, and is commonly found in North East Coast of Tamil Nadu and South Western Ghats of India as well as Nigeria (Essiet and Okoko, 2013). Senna alata (commonly called candle bush or Christmas candles) is both an ornamental and medicinal plant that is natively found in the tropics (Adedayo et al., 2001). It is locally called "Nelkhi" and "Asunwo oyibo" in eastern and western Nigeria respectively (Mutiu et al., 2015). While flowers of Senna alata and leaf of Euphorbia hirta had been reported to possess antimicrobial, antifungal, anti-inflammatory, laxative and analgesic effects (Adedayo et al., 2001;Adjeroh et al., 2015;Ilodibia et al., 2015;Al-Snafi, 2017;Damen et al., 2018;Thu et al., 2020), there is still limited information on the relative antioxidant capacities of the ethanolic leaf extracts of Dracaena mannii, Euphorbita hirta and Senna alata ( Figure 1). Hence, the present investigation explored the comparat ive phytochemical constituents and in vitro capacity of the ethanolic leaf extracts of D. mannii, E. hirta and S. alata.

Chemicals/reagents used
All the chemicals and reagents that were used in the various biochemical processes are of analytical quality.

Collection and authentication of plant materials
Fresh leaves of Dracaena mannii, Euphorbia hirta and Senna alata were collected from bushes at Oviorie-Ovu, Okpe-Olomu and Abraka in Delta State, Nigeria. Before use, the plants' specimens were identified at the Department of Plant Biology and Biotechnology (with the Voucher number; UBH-D019, UBH-E023 and UBH-S491 respectively), Universit y of Benin, Edo State, Nigeria.

Preparation of plant extract
The modified method of Anigboro et al. (2019) was used for the preparation of the plant's leaf extracts . Summarily, the fresh leaves of Dracaena mannii, Euphorbia hirta and Senna alata were rinsed with tap water to remove dirts, air-dried under shade for about two weeksand ground to coarse powder using an electric A B C blender, from which 100g of coarsely powdered leaves were soaked in 800 mL of ethanol for 48hours (with stirring at 24 hours intervals). The extract solutions were filtered firstly with a double-layer cheese cloth and thereafter with filter paper (Whatman No. 1). The filtrates obtained were concentrated in a rotary evaporator at 50 o C for two hours and then evaporated to dryness over water bath (50 o C). The yield percent of the extraction process were evaluated using equation 1 and the obtained dried extracts were stored at 4 o C in air-tight sterile containers till when needed for analysis.

Qualitative phytochemical screening
The standard protocols reported by Njoku and Obi (2009) and Borokini and Omotayo (2012) were used for the preliminary phytochemical screening of the various leaf extracts of Dracaena mannii, Euphorbia hirta and Senna alata, for the presence of phlobatannins , cardiac glycosides, flavonoids, saponins, terpenes/terpenoids, carbohydrates, tannins, phenols, steroids, proteins and alkaloids.

Estimation of phenols and tannin contents
The method of Singleton and Rossi (1965) was employed for the evaluation of the phenol and tannin contents of the ethanolic leaf extracts of Dracaena mannii, Euphorbia hirta and Senna alata. To a mixture of the extracts' solutions (1.0 mL; 0.02 g/mL) and Folin-Ciocalteau's reagent (1.0 mL; 1:10 v/v with water) which was allowed to stand for three minutes, a saturated solution of Na2CO3 (1.0 mL) and distilled water (10.0 mL) were added and kept in the dark for 90 minutes after which the optical densities (OD) were read with the Thermo-Fisher G10S (TFG) UV -Vis Spectrophotometer at 725 nm. Different concentrations (20 -100 µg/mL) of gallic acid and tannic acid were used to generate calibration curves from which the amounts of phenols and tannins were respectively deduced from the graphs [expressed as gallic acid equivalent (GAE) tannic acid (TAE) equivalents/g extract] respectively.

Estimation of total flavonoids
The standard method of Jia et al.(1999) was used for the estimation of the flavonoid contents of the extracts. A mixture of the various extract solutions (0.5 mL; 0.02 g/mL), NaNO2 (0.075 mL; 5 %) and distilled water (1.25 mL) was incubated for 5 minutes at room temperature. Thereafter, AlCl3 (0.15 mL; 10 %) was added and incubated again for 6 minutes followed by addition of NaOH (0.5 mL; 1.0 M) and dilution with distilled water (0.275 mL). The OD were read immediately at 510 nm using the TFG UV-Vis Spectrophotometer. Catechin was used at different concentrations (20 -100 µg/mL) to generate a standard calibration curve, from which the flavonoids content was deduced and expressed as equivalents of catechin (mg/g) .

Determination of carbohydrates contents
The Miller method (1959) was used for the estimation of carbohydrates contents of the various extracts. The extracts' solutions (1.5 mL; 0.02 g/mL) were mixed DNSA reagent (1.5 mL) and incubated at 90˚C over water for 10 minutes. Potassium sodium tartrate solution (0.5 mL; 40%) was added and cooled to room temperature before reading the OD at 575 nm using the TFG UV-Vis Spectrophotometer. Using a standard calibration graph of glucose concentrations (0.2 -1.0 mg/mL), the total carbohydrates content was presented as glucose equivalent/g extracts.

Total protein determination
Protein contents of sample extracts was assessed by employing the Biuret method (Gornall et al., 1949). A mixture of the extracts' samples (1.0 mL; 0.02 g/mL) and Biuret reagent (3.0 mL) was incubated for 30 minutes at room temperature, and read at 540 nm using the TFG UV-Vis Spectrophotometer.
Bovine serum albumin (BSA) (200 -1000 µg/mL) was used to obtain a standard calibration graph, from which the protein contents of the samples were deduced.

Estimation of alkaloids contents
The alkaloids contents of extract samples was evaluated using the standard procedure reported by Shamsa et al. (2008). A mixture of the extracts' samples (0.5 mL),phosphat e buffer (2.5 mL; pH 4.7; 2.0 M), bromocresol green solution (2.5 mL) and chloroform (4.5 mL) was read spectrophotometrically at 470 nm against blank. Atropine (40 -120 µg/mL) was used as a standard compound to obtain a standard calibration graph, from which the alkaloid contents were presented as atropine equivalents.

Determination of phytate contents
The standard procedure reported by Wilcox et al. (2000) was used for the estimation of the phytate contents of the extracts. The extracts' samples (1.0 mL) were vortexed thoroughly with HCl (1.0 mL; 0.4mM) followed by centrifugation at 3000rpm for 5 minutes. To the supernatants (0.1 mL), distilled water (0.9 mL) and 1.0 mL of the colour reagent (3.0 M sulfuric acid, 2.5%w/v ammonium molybdate and 10% ascorbic acid mixed in equal volume with two equal volume of distilled water) were added, followed by incubation for one hour at room temperature. The optical densities were measured against blank at 650 nm with TFG UV-Vis Spectrophotometer. Potassium dihydrogen phosphate was used as standard compound to obtained a standard calibration graph, from which the alkaloid contents were presented as its equivalent.

In vitro free radical quelling and antioxidants activities 1, 1-Diphenyl-2-picryl hydrazyl (DPPH) Radical Inhibitory Assay
The free radical inhibitory abilities of the ethanolic leaf extracts of Dracaena mannii, Euphorbia hirta and Senna alata were assessed using the standard procedure reported by Manzocco et al. (1998). A mixture of the extracts' solutions (1.0 mL; 1.0 -7.0 mg/mL) and DPPH solution (2.0 mL; 0.3 mM) was incubated in the dark for 30 minutes and read spectrophotometrically at 517 nm. The inhibition of DPPH radical was calculated in percentage using the equation (2)  Where As and Ac are the respective absorbanc e values of the sample extract and the control (which was carried out as described above but using distilled water in lieu of the extract sample). The effective concentration that results to 50% inhibition of DPPH radical (IC50) was deduced graphically.

Nitric oxide radical (no • ) inhibitory assay
The NO • inhibitory potentials of the extracts' samples was measured using the standard procedure reported by Marcocci et al. (1994). A mixture of the extracts' solutions (1.0 mL; 1.0 -7.0 mg/mL), sodium nitroprusside (2.0 mL; 10.0 mM) and phosphate buffer saline (0.5 mL; 10 mM; pH 7.4) was incubated for 150 minutes at 25°C, from which 0.50 mL was mixed with 1.0 mL of 0.33% sulfanilic acid in 20% glacial acetic acid, followed incubation at 25°C for 5 minutes. Naphthylethylenediamine dichloride (1.0 mL; 0.1 %w/v) was added before another incubation at 25°C for 30 minutes. The absorbance were then measured at 546 nm against blank using the Thermo-Fisher G10S UV-Vis Spectrophotometer and the percentage inhibition of nitric oxide radical was calculated using the equation (2). The effect ive concentration that resulted to 50% inhibition of NO • (IC50) was deduced graphically.

Assessment of total antioxidant capacity
The TAC of the ethanolic leaf extracts of Dracaena mannii, Euphorbia hirta and Senna alata were assessed using the method Prieto et al. (1999).A mixture of the extracts' samples (0.1 mL; 1.0 -7.0 mg/mL) and 1.0 mL of the working reagent (mixture of 0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) was incubated at 95°C for 90 minutes. After cooling to room temperature, the OD was measured at 695 nm against blank using the TFG UV-Vis Spectrophotometer. Gallic acid (GA) (20 -100 µg/mL) was used as standard antioxidant compound to generate a calibration graph from which were the TAC was deduced as GA equivalents.

Statistical analysis
All data obtained from the various biochemical procedures were analysed by One-way ANOVA using the SPSS-PC programme software while graphical works were done with Microsoft Excel. The values were presented as Mean ± Standard deviation while significanc e was considered at p-values of less than 0.05.

RESULTS AND DISCUSSION
Plants are known to be treatment source in both ethno-therapeutic and conventional medicine and are mostly employed as alternative therapy since they contain phytochemicals which are secondary metabolites in one or more parts of the plants and also due to several advantages ranging from cost, minimal side effects to high efficacy (Anigboro, 2018;Anigboro et al., 2018;Anigboro et al., 2019;Anigboro et al., 2020). The phytochemicals present in plants play certain physiological roles in plants and have the ability to produce definite physiological action in human body (Dzialo et al., 2016;Mahmoudi et al., 2016;Nyamai et al., 2016;Anigboro et al., 2020).
In this study, the ethanolic leaf extracts of Dracaena mannii, Euphorbita hirta and Senna alata were shown to possess phytochemicals at varying degrees both qualitatively and quantitavely. From the result obtained in this study (Table 1), saponin was found only in the D. manniiand S. alata extracts; cardiac glycosides were only found in the E. hirta and S. alataextracts; terpenes, steroids and phlobatannins were absent in all three extracts; while tannins, alkaloids, flavonoids, phenols, carbohydrates and proteins were present in all three extracts. Also, there was variation in the quantities of the commonly present bioactive compounds in these extracts (Table 2) such that: phenol and flavonoids contents in S. alata extract are significantly higher than in the E. hirta extract which in turn has higher amounts than the D. mannii extract (p<0.05). The order of tannins, alkaloids and phytate contents is such that: E. hirta>S. alata>D. mannii. Thes e phytochemical compounds have previous ly been reported to possess pharmacologic al properties. Antioxidant activity is essential in terminating free radicals generation as well as diseases associated with same (Sharma et al., 2008). The DPPH radical inhibition assays as well as RP and TAC assays in this study revealed the ethanolic leaf extracts' of S. alata > E. hirta > D. manni and may have the ability to quell oxidative stress while the NO • inhibition assay had a different pattern. This relationship was also reflected in the IC50/EC50 values that were deduced from the graphs thus, suggesting the S. alata ethanolic leaf extract as possessing the best antioxidant potential while the E. hirta is better than D. mannii (Figures 2,3,4 and 5). This antioxidative order may be attributed to the amounts of phenolic and flavonoid compounds in the leaf extracts (Table 2). Phenolic and flavonoids compounds are reportedly known to exert antioxidant property (Mohammed et al., 2010;Dzialo et al., 2016;Mahmoudi et al., 2016;Thu et al., 2020).    possess high antioxidant capacities (Mohammad et al., 2010;Dzialo et al., 2016;Anigboro et. al., 2018Anigboro et. al., , 2020Anigboro et. al., ,2021. This suggests the likely medicinal benefits of the ethanolic leaf extracts of S. alata, E. hirta, and D. Mannii reported in this study, especially against disease conditions in which oxidative stress is implicated.

CONCLUSION
In the present study, the ethanolic leaf extracts of Dracaena mannii, Euphorbita hirta and Senna alata were shown to possess phytochemicals at varying quantities. However, S. alata expressed a higher in vitro antioxidant capacity as compared with E. hirta and D. mannii. Thus, the leaves of S. alata could be further investigated for its antioxidant potentials in additional to other biological potentials and be possibly employed in the application leaves against diseases that are implicated by oxidative stress.

Conflict of interest
The authors declare that they have no known competing interests that could appear to influence the work reported in this paper.

Author contribution
The experiment was conceived and designed by AAA, OJA and NJT. OA carried out the experimental work; OA, AAA and OJA analyzed the data; OA, AAA, OJA and NJT contributed reagents, materials and provided the resources used for analysis; OJA, OA and AAA undertook the manuscript writing and proofreading; while AAA, OJA and NJT supervised the experiment s and revised the manuscript. All authors approved the final draft of the manuscript.