A periodical of the Faculty of Natural and Applied Sciences, UMYU, Katsina
ISSN: 2955 – 1145 (print); 2955 – 1153 (online)
ORIGINAL RESEARCH ARTICLE
Ndu Chukwukaike Favour1, Victoria Moltong
Yilwa1, Onwumere G. Brian1, Joseph
Appah1, Sunday Gandu Audu1,2, Enoh
Emmanuel1,2, Nwankwo Cornelius Tochukwu2,3
,
Duru Daniel4 and Pai Yunusa Yusuf2,3.
1Department of Biological Sciences, Faculty of Science, Nigerian Defence Academy, Kaduna, P.M.B. 2109, Nigeria
2Department of Biotechnology, Faculty of Science, Nigerian Defence Academy, Kaduna, P.M.B. 2109, Nigeria
3Department of Biotechnology, Faculty of Science, Mewar International University, Masaka, Nasarawa, Nigeria
4Department of Psychology, University of Ibadan, P.M.B. 5017, Ibadan, Nigeria
This study evaluated and compared the antioxidant properties of the reddish-brown and green leaves of Terminalia catappa. The reddish-brown and green leaves of T. catappa were collected from Agwan-kadara Television, Chikun LGA of Kaduna State in Nigeria. These were collected and processed separately; 270g of each of the ground samples was used to prepare the methanol and aqueous extracts, respectively. Phytochemical analysis was conducted to determine the quantities of polyphenols, flavonoids, tannins, saponins, and alkaloids in the leaf samples. The antioxidative potential of the plant was determined by analyzing the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and nitric oxide reducing activity. Methanol and aqueous extracts of the reddish-brown leaves of T. catappa yielded 31.6 g and 52.2 g of extract, respectively. The phytochemical constituents found in the methanol and aqueous extracts of reddish-brown leaves of Terminalia catappa showed a significant difference in flavonoids and polyphenols, but there was no significant difference in alkaloids (13.9±0.02mg/g), tannins (90.4±0.07 mg/g), and saponins (152.2±0.85 mg/g). The methanol and aqueous extracts of the green leaves of Terminalia catappa showed no significant difference in flavonoids (96.9±7.02mg/g), polyphenols (110.0±14.1mg/g), and alkaloids, but there was a significant difference in tannins (123.2±0.38mg/g) and saponins (143.6±1.03mg/g). The high presence of phytochemicals and antioxidant potential of Terminalia catappa justifies its use in traditional medicine because of its beneficial medicinal activity. This study also suggests both the reddish-brown and green leaves of T. catappa as effective antioxidants.
Keywords: Terminalia catappa, Phytochemicals and Antioxidants.
Terminalia catappa L. known as the Tropical almond/Indian almond, is often regarded as one of the most significant medicinal plants that can be found everywhere in the globe. Many regions inside and outside of India employ it as a traditional form of medical treatment for anti-inflammation, antimicrobial, hepatoprotective, vermifuge, antioxidant, anticancer, and antidiabetic agent (Mwangi et al., 2024; Chinaka et al., 2017). It is a member of the Combretaceae family (Combretum family); it is cultivated for its decorative value and can also grow wild because it does so well in tropical environments, has a single stem that can grow to a height of around 10 meters, after which it branches out horizontally and forms rosette-like clusters of leaves at the tips of each branch. Before they fall off, the leaves go through a transformation in which they transition from green to red, yellow, or gold and a reddish-brown copper tint.
Terminalia catappa can be beneficial in the treatment of inflammatory disorders, the healing of wounds, allergies, skin issues, asthma, ulcers, cardiovascular disease, diarrhoea, and many other conditions. It is also believed to be beneficial for reviving one's sense of vigour and vitality. It is believed that the chemical constituents of T. catappa leaves include essential chemicals such as chebulagic acid, chorilage, gentisic acid, Grenadine-B, and kaempferol; on the other hand, the seeds are abundant in arachidic acid, ascorbic acid, fibre, fat, linoleic acid, palmitic acid, and other similar substances. It has a high concentration of minerals, including calcium, potassium, phosphorus, and sodium (Chang et al., 2018). Phytochemicals such as phenolic compounds found in medicinal plants can have biological effects and are thought to be helpful to human health because they lower the chance of developing degenerative diseases by scavenging oxidative agents, preventing macromolecular oxidation and boosting the immune system (Olaoye et al., 2021).
It has been determined that the LD50 value of T. catappa is larger than 5000mg/kg bwt, which suggests that it is practically non-toxic in accordance with the classification provided by the Organization for Economic Cooperation and Development Guideline for Chemical Testing (OECD) (Ugwah-Oguejiofor et al., 2019). Previous findings record that numerous species of Terminalia, when extracted using a variety of solvents, provide LD50 values that are so low as to be considered virtually non-toxic (less than 2,000 to 5,000 mg per kg of bwt) (Arjariya et al., 2013).
A subacute study of 28-day repeat-dose treatment of T. catappa in experimental animals demonstrated that it did not elicit any clinical signs of morbidity, toxicity, or mortality in any of the treatment groups. (Dongmo et al., 2019). Alterations in organs and changes in body weight are crucial indications for access to the tested compound, the unfavourable effect of the medicinal plant, and the overall health conditions of experimental animals (Dongmo et al., 2019).
Although the antioxidant properties of T. catappa leaves have been studied, there is limited information on the comparative effects of green versus reddish-brown leaves. This study explores the potential of both leaf types as natural antioxidants, focusing on their chemical composition and medicinal value. Therefore, this research aimed to determine and compare the phytochemical and antioxidant properties of the reddish-brown and green leaves of T. catappa responsible for its medicinal properties.
Terminalia catappa leaves were collected in Agwan-kadara Television, Chikun LGA Kaduna state, Nigeria. The reddish-brownish and green leaves were collected separately in a clean bag.
Terminalia catappa reddish-brown and green leaves were rinsed, air dried, and crushed. Separately, 270g of reddish-brown leaf powder and 270g of green leaves were weighed and soaked for 48 hours at room temperature in 1.2 litres of 100 percent methanol. The mixture was filtered using Whatman filter paper. The filtrate was concentrated dry using a rotary evaporator at 40oC and the extract yield was weighed and labelled accordingly.
Terminalia catappa’s reddish-brown and green leaves were washed, air dried, and crushed. 270 g of reddish-brown leaf powder and 270 g of green leaves were weighed and soaked for 48 hours at room temperature in 3.2 liters of separately distilled water. The mixture was filtered using number 1 Whatman filter paper. The filtrate was concentrated dry in a water bath at 40oC, and the yield of the extract was weighed correspondingly.
Procedure for Estimating Polyphenols
In a clean, dry beaker, 20 mg of gallic acid was weighed, 10 mL of methanol was used to dissolve it, and the volume was increased to 20 mL using the same solvent to get the solution concentration to 1 mg/mL.
The polyphenol content of T. catappa leaf extract was determined using a modified Folin-Ciocalteu colorimetric technique. The standard was gallic acid, and the results were given as Gallic Acid Equivalent (GAE) per gram of material.
Gallic acid concentrations ranging from 0.2 to 0.7 mg/mL were produced in methanol. In a 96-well plate, aliquots of 20 µL each of the sample and standard will be mixed with 150 µL of Folin–Ciocalteu and left to stand for 3-5 minutes at room temperature. Following that, 80 l of a saturated sodium carbonate solution (7.5 percent w/v) was added to each reaction well and incubated at room temperature for one hour with intermittent shaking. At 765 nm, the absorbance was measured, and a calibration curve for the gallic acid standard was created (Kim et al., 2024)
Polyphenol Content = V x C
M
Where; C = concentration of gallic acid from calibration curve in mg/ml
V = volume of extract in mL
M = weight of extract in grammes
A stock solution of 1 mg/mL quercetin was prepared prior to the commencement of the experiment. Total flavonoid content (TFC) of T. catappa leaf extract was determined using a colorimetric assay of aluminum chloride with slight modifications. 20 µL of extract and standard solution (1 to 0.5 mg/mL quercetin) were mixed with 15 µL of sodium nitrite solution (5 percent NaNO2 w/v) in a 96-well plate and incubated for 6 minutes at room temperature. Following that, 15µL of aluminum chloride solution (10% AlCl3, w/v) was added to each reaction well and left for 6 minutes before adding 80µL of sodium hydroxide (4 percent NaOH, w/v) to each well. The absorbance at 510 nm was measured after an additional 15 minutes of incubation. The total flavonoid content (TFC) was estimated as mg of quercetin equivalent per gramme of sample using a quercetin calibration curve. (Jang et al., 2019).
Total flavonoid Content = V x C
M
Where; C = concentration of quercetin from calibration curve in mg/mL
V = volume of extract in ml
M = weight of extract in grammes
Terminalia catappa leaf extract (20mg) was diluted in 4 ml of NHCI and then filtered. One milliliter of this solution was placed to a separatory funnel and rinsed with ten milliliters of chloroform. Then 0.1N NaOH was used to modify the PH of the phosphate buffer solution to neutral. In a separating funnel, 1 milliliter of this solution was transferred, and 5 milliliters of bromcresol solution and 5 milliliters of phosphate butter were added (7.0). The complex formed was fractionated using chloroform after the mixture was violently agitated. The fractions were collected in a 10 mL graduated flask and diluted to volume with chloroform. At 470 nm, the complex's absorbance in chloroform was determined. As a control, atropine was used. (Jang et al., 2019)
To evaluate the tannins, 0.2mL of T. catappa extract was put to a volumetric flask (2 mL) containing 1.5 mL of distilled water and 0.1 mL of Folin-Ciocalteu phenol reagent, 0.2 mL of 30% Na2C03 solution, and 2 mL of distilled water. The mixture was properly combined and set aside for 30 minutes at room temperature. A series of reference standard solutions of gallic acid (20, 40, 60, 80, and 100µg/mL) were prepared in the same manner as stated previously. At 725 nm, the absorbance of the test and reference solutions was measured using a UV/VIS spectrophotometer against a blank. The tannin content was determined in milligrams of GAE per gram of extract. (Olaoye et al., 2021).
(5mg/mL) of T. catappa leaf extract was taken after diluting 5ml in 50% aqueous methanol and suitable aliquot. Following the vanilin reagent, 2.5mL of sulfuric acid (72 percent v/v) was added (0.25mL; 8 percent). It will be properly mixed and incubated for 10 minutes in a 60°C Water bath. The absorbance at 544 nm (UV visible spectrophotometer) was measured after incubation against a blank containing no extract. The standard calibration curve was made with aliquots of diosgenin (0.5mg/mL in 50% aqueous methanol). The total saponin concentration was given in milligrams of diosgenin equivalent (DE) per gram of dry weight (DW) (Olaoye et al., 2021).
The antioxidant activity of T. catappa leaf extract was evaluated using the free radical scavenging method DPPH (2,2-diphenyl-1-picrylidrazyl) according to the protocol with minor changes. The 180µL of 0.2 mm DPPH methanolic solution was prepared and mixed with 12µL of samples, ascorbic acid, and methanol (blank) for 30 minutes at 37°C in the dark on a microwell plate. The absorbance values for each combination were measured at 517nm after incubation. The proportion of radical scavenging activity (percent inhibitions) was calculated from the absorbance data using the formula below. (Hoque et al., 2023)
\[\%\ of\ Inhibition = \ \frac{A\ of\ Blank\ - A\ of\ Sample}{A\ of\ Blank}\ x\ 100\ \]
Where: A of Blank is the Absorbance of the control reaction (containing all reagents except the test sample). A of Sample is the Absorbance of the extracts/standard.
Terminalia catappa and ascorbic acid extracts were prepared using 50% methanol at a concentration of 200mg/mL. The samples are then serially diluted with the same methanol to achieve 10, 20, 30, and 40 mg/mL concentrations.
Griess' reagent was made by combining equal parts of 2% sulfonylamide, 5% phosphoric acid, and 0.1% naphthyl ethylenediamine dihydrochloride right before use.
A volume of 50µL of 10mmol/L sodium nitroprusside in 0.1mmol/L phosphate buffer pH 7.4 was added to 50µL of the extracts made in 96-well plates, which were then incubated at 25°C for 180 minutes. After that, 100µL of Griess' reagent was added. Similarly, a control sample was made with an equal volume of methanol but no extract.
The absorbance values at 542nm were obtained, and the percentage of inhibition of the extract and ascorbic acid at various concentrations was determined using the same formula as the DPPH. The falling optical density readings indicate that nitric oxide radicals have a significant scavenging activity. (Akinyede et al., 2022).
Data obtained from the study were summarized and expressed as mean ± standard error of mean. Data analysis was performed using Statistical Package for Social Science (SPSS), version 23.0. To compare the results obtained from different groups, one-way ANOVA followed by Duncan Multiple Range Test comparison tests were performed to determine statistical significance. P values less than 0.05 were considered significant.
Figure 1: Percentage yields of methanol and aqueous extracts of the Reddish-brown and green leaves of Terminalia catappa. (MEBL: Methanol Extract of the Reddish-brown leaves of T. catappa MEGL: Methanol extract of the Green leaves of T. catappa; AEBL: Aqueous extract of the Reddish-brown leaves of T. catappa; AEGL: Aqueous extract of the green leaves of T. catappa).
The results of the phytochemical screening indicated the presence of flavonoids, polyphenols, alkaloids, and tannins (Table 1). Quantitatively, no significant differences (p > 0.05) emerged in the flavonoids and polyphenols in methanol and in the aqueous extracts of the reddish-brown or green leaves of T. catappa; however, significant differences (p<0.05) in alkaloids, tannins, and saponins were observed in the extracts of T. catappa leaf samples (Table 1).
Table 1: Quantitative phytochemical constituents of the methanol and aqueous extracts of the Reddish-brown and green leaves of Terminalia catappa
| Samples | Phyto-constituents (mg/g of extract) | ||||
| Flavonoids | Polyphenols | Alkaloids | Tannins | Saponins | |
| MEBL | 96.4±9.07a | 100.0±0.00a | 14.3±0.02a | 90.4±0.07b | 152.2±1.85a |
| MEGL | 96.9±7.02a | 110.0±14.1a | 14.2±0.03a | 123.2±0.38a | 143.6±1.03b |
| AEBL | 102.4±11.8a | 105.0±7.07a | 13.9±0.02b | 74.9±0.73c | 110.2±1.06c |
| AEGL | 97.5±7.62a | 110.0±14.1a | 14.0±0.02a | 56.9±0.07d | 66.6±0.20d |
Values are given as mean±standard deviation; in each column, values with different superscripts have statistical significant difference (p < 0.05)
KEY: MEBL: Methanol Extract of the Reddish-brown leaves of T. catappa; MEGL: Methanol extract of the Green leaves of T. catappa; AEBL: Aqueous extract of the Reddish-brown leaves of T. catappa; AEGL: Aqueous extract of the green leaves of T. catappa.
Radical Scavenging Action of DPPH: Radical scavenging activity standards were much higher than that of T. catappa leaf extracts, with a mean percentage inhibition of 2.68\(\pm\)0.36. No significant difference was seen among all the extracts' radical scavenging activity (Table 2). The methanol extract of T. catappa's reddish-brown leaves had the highest DPPH radical scavenging activity, with a mean value of 0.55\(\pm\)0.31 percent, and the aqueous extract of T. catappa's green leaves had the lowest, with a mean inhibition of 0.24\(\pm\)0.26 percent. T. catappa's methanol extract of green leaves and aqueous extract of reddish-brown leaves had mean values of 0.48\(\pm\)0.45 and 0.43\(\pm\)0.46 percent, respectively (Table 2).
Assay for Nitric Oxide Reduction: Except for the aqueous Reddish-brown leaf extract, all three solvent extracts of T. catappa displayed concentration-dependent nitric oxide lowering action, with increasing activity linked with graded extract concentrations (Table 3). At the highest concentration (40mg/mL) the methanol and aqueous green leave extract value was 99.74 and 98.28 percent, respectively, while methanol and aqueous reddish-brown leave extract were 36.34 and 52.28 percent, respectively. The results also showed that methanol and aqueous green leaf extracts had considerably higher (p<0.05) nitric oxide lowering activities than methanol and aqueous reddish-brown leaf extracts at all doses examined (Table 3).
Table 2: DPPH radical scavenging activities of the methanol and aqueous extracts of the Reddish-brown and green leaves of T. catappa
| Samples | % inhibition |
| DPPH | 2.68±0.36a |
| MEBL | 0.55±0.31b |
| MEGL | 0.48±0.45b |
| AEBL | 0.43±0.46b |
| AEGL | 0.24±0.26b |
Values are given as mean±standard deviation; in each column, values with different superscripts have statistical significant difference (p < 0.05)
KEYS: DPPH: 2, 2-diphenyl-1-picrylhydrazyl; MEBL: Methanol Extract of the Reddish-brown leaves of T. catappa; MEGL: Methanol extract of the Green leaves of T. catappa; AEBL: Aqueous extract of the Reddish-brown leaves of T. catappa; AEGL: Aqueous extract of the green leaves of T. catappa;
Table 3: Nitric oxide reducing activity of the methanol and aqueous extracts of the Reddish-brown and green leaves of T. catappa
| Conc. (mg/mL) | MEBL | MEGL | AEBL | AEGL |
| 10 | 0.106d | 43.23b | 12.43c | 40.74b |
| 20 | 16.22c | 98.25a | 10.19d | 98.33a |
| 30 | 23.59b | 98.36a | 38.02b | 98.07a |
| 40 | 36.34a | 99.74a | 52.28a | 98.28a |
Values are given as percentage inhibition. Values with different superscripts in each column have statistically significant differences (p < 0.05).
Key: MEBL: Methanol Extract of the Reddish-brown leaves of T. catappa; MEGL: Methanol extract of the Green leaves of T. catappa; AEBL: Aqueous extract of the Reddish-brown leaves of T. catappa; AEGL: Aqueous extract of the green leaves of T. catappa
The plant leaves subjected to different extractants gave different yields, which could be attributable to differences in compound composition and polarity of the extraction solvent (Kuppusamy et al., 2015). The presence of phytochemical constituents such as flavonoids, polyphenols, tannins, saponins and alkaloids in the leaves of T. catappa have also been documented by Kankia, (2014). High content of these bioactive compounds, except for alkaloids, were recorded. Past research has strongly encouraged long-term consumption of diets rich in plant polyphenols because it could help in the prevention of several degenerative diseases.
Many studies have recorded that tannins are polyphenolic compounds that exhibit antioxidant, tissue repair, and antimicrobial properties (Vasconselos et al., 2010). Flavonoids are made up of 4000 compounds out of 8000 known polyphenolic compounds that are water soluble and anti-allergen, anti-oxidative, anti-microbial, and anti-inflammatory. Alkaloids from Lotus plumules have been recorded to have hepato-protective efficiency (Liu et al., 2019). Saponins have been seen from past research to control dietary hypercholesterolemia, suppress colon cancer cell proliferation, prevent peroxidation of lipids, and protect the liver by accelerating the secretion of thyroid hormones (Sayama et al., 2012).
Free radical scavenging activity of the plant is similar to the reducing ability which is attributable to different quantities of the plant’s phytochemicals constituents (Akinyede et al., 2022). Generally, the antioxidant activities of plant extracts are attributed to the constituents of total phenolic, total flavonoid, and total antioxidant capacity. The analysis of the antioxidant activity showed that the DPPH scavenging activities of the methanol extract of T. catappa leaves were higher than that of the aqueous extract, and this agrees with earlier publication reported by Ebrahimzadeh et al. (2009) for the free radical scavenging activity of Hydroxyanmus squareous, and by Omenna, (2015) on the antioxidant activity of almond leaves of T. catappa prove the efficacy of the solvent. Muhie and Endalew (2023), reported similar results on in vitro antioxidant and free-radical scavenging activities of polar leaf extracts of Vernonia amygdalina. In their study, the methanolic extract of the plant had the highest scavenging activities among other extracts used.
Sodium nitroprusside reacts with oxygen to produce nitric oxide, and nitrite scavenges free radicals through diazotisation with a sulphanilamide acid coupled reaction, giving rise to a pink colour (Akinyede et al., 2022). The Nitric Oxide assays’ antioxidant activities involved the donation of protons to the nitrite radicals that resulted decrease in absorbance. In this study, the Nitric Scavenging activity of the green leaves of T. catappa was higher than that of the reddish-brown leaves, and this agrees with Olaoye et al. (2021) in their study analyses of antioxidants of Moringa oleifera leaf extracts from southwestern states in Nigeria. Their results revealed a concentration-dependent free radical scavenging activity of the plant extracts. Following the decrease in absorbance observed, points out the extent of nitrite radical scavenging potentials, which could be attributed to components such as flavonoids, as reported in Akinyede et al. (2022).
The green and reddish-brown leaf extracts of T. catappa contain high Polyphenols, Flavonoids, Tannins, Saponins and low Alkaloids. The DPPH radical scavenging activity ability of the Green and Reddish-brown leaves extract of T. catappa are not significantly different, and the Green leaves of T. catappa demonstrated higher Nitric oxide scavenging activity than the Reddish- brown leaves. This suggests that both the Green and Reddish-brown leaf extracts of T. catappa are both effective oxidants since the scavenging activity and Nitric Oxide assay of T. catappa leaf extracts (Aqueous and Methanol) revealed no significant difference.
The authors declare no conflict of interest.
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