Oxidative stress is a condition characterized by an imbalance between the production of reactive oxygen species (ROS) and the body’s ability to detoxify these reactive intermediates or repair the resulting damage. Oxidative stress leads to the many problems in humans, and they are connected with pathophysiology problems of many diseases [1,2], which includes inflammation [3], cancer, atherosclerosis, and aging [4]. Orange fleshed sweet potatoes, a variant distinguished by its vibrant orange color are valued for their high beta-carotene content, a precursor of vitamin A, which is essential for maintaining healthy vision, immune function, and skin integrity. In addition to beta-carotene, the tubers and leaves of this plant are rich in vitamins C, E, iron, potassium, and dietary fiber, making them a highly nutritious food source bb [5]. Orange fleshed sweet potato leaves have special attributes such as adaptability in wider topography, ability to grow in subsidiary circumstances, good productivity in short durations, and balanced nutritional composition [6].The leaves are an underutilized part of the plant despite their rich nutrient profile. They contain significant levels of polyphenols, including chlorogenic acid and caffeoylquinic acid, which contribute to their antioxidant activity. These compounds have been associated with various health benefits, including anti-inflammatory, anti-carcinogenic, cardio-protective and antioxidant effects [7]. While previous studies have primarily focused on the tubers of the orange-fleshed sweet potato, the leaves, which are often considered a byproduct, are an underutilized source of nutrients and antioxidants. The potential health benefits of incorporating orange-fleshed sweet potato leaves (OFSP leaves) into the diet, particularly their role in modulating antioxidant enzyme activity, remain largely unexplored.Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) are critical components of the body’s defense system against oxidative stress. Enhancing the levels of these enzymes through dietary interventions could be a viable strategy to improve overall health and reduce the risk of chronic diseases related to oxidative stress, such as cardiovascular diseases, diabetes, and neurodegenerative disorders [8].

This study aims to fill this gap by investigating the effects of orange fleshed sweet potato leaves supplemented diet on the antioxidant enzyme levels in rats compared with rats fed with pumpkin leaves. Pumpkin is used as positive control for this experiment. The natural phenolic compounds that are present in plants are responsible for antioxidant activity confirmed in numerous in vivo and in vitro studies. Phenolic compounds also have other important biological activities, which makes them applicable as alternatives to synthetic additives [9]. Antioxidant can be classified as either “primary antioxidants” or “secondary antioxidants.” Primary antioxidants actively inhibit oxidation reactions, whereas secondary antioxidants act in an indirect way; for example, they react with pro-oxidants or are able to scavenge oxygen [33,10]. Polyphenols are the major plant antioxidants with various structural and functional characteristics and biological properties [11], found in plant foods such as fruits, cereals, seeds, berries, and plant-based products such as wine, tea, and vegetable oils [12].

Figure: 1: Chemical Structures of (a) Polyphenolic compounds (b) Terpenes and (c) β-Glucan.

One of the numerous Source: (Single et al., 2019; 13]

Phenolic Acids

Phenolic acids are the derivatives of benzoic acids and cinnamic acids, and salicylic acid, gentisic acid, p-Hydoxybenzoic acid, protocatechuic acid, vanillic acid, syringic acid, gallic acid, p-coumaric acid, ferulic acid, caffeic acid, and sinapic acid are among the most common phenolic compounds present in the food plants and common phenolic acid compounds [14].

Flavonoids

Flavonoids consist of two outer aromatic rings with three carbon rings. These flavonoids include flavone, flavanol, flavanone, flavanonol, flavonone, flavononol, flavanol (catechin), isoflavone, and anthocyanidin [15]. The derivatives of the flavonoids have inhibitory properties against acetylcholinesterase [16].

Stilbenes

Stilbenoides/stilbenes are another phenolic compound commonly found in berries, grapevines, and peanuts. Stilbenoids are hydroxylated stilbene derivatives (i.e., resveratrol). The most common stilbenes are piceid, resveratrol, piceatannol, and pterostilbene. [17]. However, only resveratrol has antioxidant potential against proteins and lipids [18]. Endogenous compounds such as superoxide dismutase (SOD), catalase, and glutathione neutralize oxidative stress induced by UV radiation. Stilbenes, such as resveratrol, can increase the activity of antioxidant enzymes (glutathione S-transferase) and increase the SOD level. In addition, pterostilbenes reduce oxidative damage by activating the endogenous antioxidant enzymes [19].

Lignans

Lignans are precursors to phytoestrogens and synthesized from phenylalanine with dimerization of substituted cinnamic alcohols. Sesame seeds and flax seeds are the main sources of lignans. Secoisolariciresinol, matairesinol, pinoresinol, and lariciresinol are the common lignans from flax seeds and sesamin, sesamoiln, sesamolinol, and sesaminol are the common lignans from sesame seeds [20]. Sesamin and sesamolin possess antioxidant, neuroprotective, and anticancer activities, but sesamol, their decomposition product during the roasting process of sesame seeds, is the major antioxidant of the sesame seeds [21].

Terpenes and Terpenoids

Terpenes and terpenoids are also good antioxidants from plant sources. They are the largest secondary metabolites of plants. Terpenes and terpenoids contain a hydrocarbon skeleton with five carbons (isoprene), and two or more isoprene molecules polymerize and form various terpenes. Most of them are non-polar compounds [22]. Plant oils such as pine oil, vegetables such as carrots, and some fruits such as lemon and orange are rich sources of terpenes and terpenoids. These compounds can further classify into monoterpenes (C-10), sesquiterpenes (C-15), diterpenes (C-20), triterpenes (C-30), tetraterpenes (C-40), or carotenoids, polyterpenes, norisopernoids, and sesquatreterpenes. These compounds have antioxidant and antimicrobial activities, contributing odor and flavor and other health-promoting properties such as relieving stress and depression, reducing depression and migraines, and antiaging and anticancer properties [23].

Tannins

Tannins are another group of phenolic antioxidants in plants and can be divided into two main sub-classes: condensed tannins and hydrolyzable tannins [24]. The condensed tannins are biopolymers based on flavan-3-ols, and gallic and ellagic acid derivatives (gallotannins and ellagitannins) are the main components with antioxidant properties [26] Gallotannins are natural polymers formed by the esterification of D-glucose and gallic acid hydroxyl groups. Proanthocyanidins can donate hydrogen atoms/electrons and act as an antioxidant compound. Proanthocyanidins are abundant in green tea and bearberry [27]. Tannin extracts from red beans, adzuki beans, lentils, fava beans, and broad beans showed better antioxidant activity than the flavonoids and phenolic acids separated from the same plant materials [28].

β-Glucan

Beta glucans are soluble fibers found naturally in various food sources, including oats, barley, and sorghum. They have been associated with several health benefits, including lowering cholesterol levels, improving blood sugar management, and boosting immune health [29].

Plant Collection and Experimental Design

Orange fleshed Sweet potato leaves were obtained from Nigeria’s National Root Crops Research Institute (NRCRI)-Umudike, Abia State. The plant was identified and authenticated at the Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Benin, Nigeria. The herbarium specimen was deposited with voucher number: UBH-1493. After harvesting of the leaves, the leaves were separated from the vine, washed under running water and dried at room temperature. The leaves were then grinded using electric grinder into fine powder. [34].

Figure: 2: Orange fleshed sweet potato leaves.

Source: National Root Crops Research Institute (NRCRI)-Umudike, Abia State. Nigeria.

Thirty (30) albino rats were purchased from the Department of Anatomy (Animal house) University of Benin, Benin City. The rats were randomly divided into five (5) test groups (n=6) of animals each and transferred to standard steel cages [30]. The rats were acclimatized for two weeks. Group 1, serving as the control group, was fed a formulated diet consisting of maize, corn flour, fish meal, groundnut meal, bone meal, and a vitamin premix. Group 2 received the same formulated diet with the addition of 25% orange-fleshed sweet potato (OFSP) leaves. Group 3 diet was enhanced with 50% OFSP leaves, while Group 4 was provided with a diet containing 100% OFSP leaves. Lastly, Group 5, the positive control group, was fed a diet containing 100% ugwu leaves (Telfairia occidentalis) [11]. Blood samples were collected by Cardiac puncture intro plane bottles [31].

Figure: 3: Blood sample, dissection, collection and preservation.

Proximate Composition of Pumpkin Leaves (Telfairia Occidentalis)

Nutritional (carbohydrate, ash, protein, crude fat, crude fibre and moisture) analysis was carried out to ascertain the nutrient compositions present in pumpkin leaf sample. These were done using the standard methods described by Association of Official Analytical Chemist (AOAC, 2005). Telfairia occidentalis leave was used as positive control due to its high antioxidant properties revealed from scientific researches. [32].

Table 1: The table below shows the rats feed composition for the different groups.

Where g = Gram

Biochemical Analysis

Assay of Antioxidant Enzymes

Antioxidant enzymes assayed in this study include; Catalase (CAT), Superoxide Dismutase (SOD), Gluthatione Peroxidase (GPX) and GluthationeReductase (GR).

Assay of Catalase Enzyme

The kinetic process of Cohen was utilized to evaluate the activity of the serum enzyme [35].

Principle of Asssay:

The enzyme catalase is present practically in all plants and organisms that are subjected to oxygen, where it catalyzes the dissolution of hydrogen peroxide into oxygen and water.

2H₂ O² → H₂O + O₂

Similarly in the assay serum, Hydrogen peroxide is broken down into water and oxygen by catalase, of which reacts and catalyzes tzhe whole process. Since water and oxygen are unable to absorb at the same wavelength, the absorbance of hydrogen peroxide at 480 nm is directly measured to determine the reaction rate. The rate at which reactions happen is linearly (proportionally) increased in the presence of catalase [36].

Assay of Glutathione Peroxidase (Gpx)

GPx assay was done according to the kinetic method described by Flohe and Gunzler, 1984.

Principle of Assay:

The biological function of glutathione peroxidase is to convert free hydrogen peroxide to water and to metabolize lipid hydroxyl-peroxides into their respective alcohols. Glutathione donates the hydrogen atoms used in the reduction reaction, while it will be oxidized.

2GSH + H₂O₂ → GPx GSSG + 2H₂O

 2GSH + ROOH GPx → GSSG + ROHH₂O

GPx catalyses the reaction of pyrogallol with hydrogen peroxide to form purpurogallin (purple to black colored) whose absorbance is read at 420nm.

2Pyrogallol + 3H₂O₂ → Purpurogallen + 5H₂O + CO₂

Assay of Glutathione Reductase (GR)

Assay of GR was done according to the method described by Ell man, 1959.

Principle of Assay

Glutathione reductase (GR) reduces oxidized glutathione (GSSG) to the reduced sulfhydryl form GSH which is an important cellular antioxidant. A high GSH/GSSG ratio is important for protection against oxidative stress. Thus, measurement of GR activity is used as indicator for oxidative stress.Dithionitro benzoic acid; 5, 51-Dithiobis (2-nitrobenzoicacid) (DTNB) reacts with the GSH generated from the reduction of GSSG by the GR in a sample to form a yellow product3-thio-6-nitrobenzoate (TNB2-). The rate of change in the optical density, measured at 412 nm, is directly proportional to GR activity in the sample.

Assay of Superoxide Dismutase (SOD)

Activity of serum Superoxide dismutase enzyme was assayed according to the kinetic method of Misra and Fridovich, 1972.

Principle of Assay:

Superoxide dismutases are class of enzymes that catalyse the dismutation of superoxide into oxygen and hydrogen peroxide. As such they are an important antioxidant defense in nearly all cells exposed to oxygen.

2O₂^- + 2H + SOD → H₂O₂ + O₂

Adrenaline auto-oxidises rapidly in aqueous solution to adrenochrome, whose concentration can be determined at 420nm. The auto-oxidation of adrenaline depends on the presence of superoxide anions. The enzyme, SOD inhibits the auto-oxidation of adrenaline by catalysing the breakdown of superoxide anions. The degree of inhibition is thus a reflection of the activity of SOD, and is determined at one unit of the enzyme activity.

Data Analysis

Data were subjected to statistical analysis using the IBM SPSS statistics software (Statistical Package for Social Science) (Version 25 and relevant statistical values were obtained. One-way analysis of variance (ANOVA) was carried out and data were presented as mean ± SEM. LSD post-hoc test was used. Values of P<0.05 were considered significant.

Table 2: Antioxidant enzyme activities in experimental diet of rats supplemented with 0%, 25% 50%, 100% (OFSP) and 100%Pumpkin leaves.

AOE - antioxidant enzymes; SOD - superoxide dismutase - (x10-2U/mL protein);CAT – catalase (x10-3K/S/mL protein);GPx- glutathione peroxidase (U/mL protein);GPx - glutathione reductase; (U/mL protein)

Superoxide Dismutase (SOD) activity significantly varied among the groups (p = 0.000). Group 1 (Control) showed a baseline SOD activity of 2.28 ± 0.20 U/mg protein. The inclusion of 25% OFSP leaves in Group 2 resulted in a decrease in SOD activity (1.783 ± 0.30 U/mg protein). However, a significant increase was observed in Groups 3 and 4, with SOD activities of 3.887 ± 0.60 U/mg protein and 4.103 ± 0.30 U/mg protein, respectively, suggesting that higher concentrations of OFSP leaves can enhance SOD activity. The 100% ugwu leaves group (Group 5) showed slightly lower SOD activity (3.488 ± 0.03 U/mg protein) compared to the 100% OFSP leaves group, indicating that while ugwu leaves also have a positive effect, OFSP leaves may be more effective in increasing SOD activity [37-39].

There was a significant difference in catalase (CAT) activity among the groups (p < 0.05). The control group (Group 1) exhibited a CAT activity of 100.03 ± 2.0 U/mg protein. Similar to SOD, Group 2 (25% OFSP leaves) showed no significant increase (100.37 ± 8.00 U/mg proteins). However, a marked increase in CAT activity was observed in Group 3 (50% OFSP leaves) at 121.25 ± 1.00 U/mg protein, which was the highest among all groups. Group 4 (100% OFSP leaves) and Group 5 (100% ugwu leaves) demonstrated slightly lower CAT activities (113.52 ± 2.00 and 111.33 ± 0.20 U/mg protein, respectively) compared to Group 3, suggesting an optimal concentration for OFSP leaves in enhancing CAT activity.

Glutathione Peroxidase (GPx) activity showed significant differences among the groups (p < 0.05). The control group (Group 1) had a GPx activity of 2.03 ± 0.03 U/mg protein. Groups 2 (25% OFSP leaves) and 5 (100% ugwu leaves) showed slightly reduced GPx activities (1.97 ± 0.07 U/mg protein and 2.27 ± 0.08 U/mg protein, respectively). However, the highest GPx activities were observed in Groups 3 (50% OFSP leaves) and 4 (100% OFSP leaves) with values of 2.30 ± 0.05 and 2.34 ± 0.02 U/mg protein, respectively, indicating that higher percentages of OFSP leaves can enhance GPx activity.

Glutathione Reductase (GRx) activity also showed a significant difference (p < 0.05). The control group (Group 1) had a GRx activity of 2.088 ± 0.02 U/mg protein. Group 2 (25% OFSP leaves) showed a slight increase in GRx activity (2.182 ± 0.01 U/mg protein), whereas Groups 3 (50% OFSP leaves), 4 (100% OFSP leaves), and 5 (100% ugwu leaves) had lower GRx activities (2.138 ± 0.007, 2.117 ± 0.005, and 2.112 ± 0.007 U/mg protein, respectively). This indicates that a moderate inclusion of OFSP leaves may enhance GRx activity, but higher concentrations do not further increase this effect.

The study demonstrated that the inclusion of OFSP leaves in the diet of experimental rats significantly affects the activities of key antioxidant enzymes, with the effects varying depending on the concentration of the leaves. Specifically, higher concentrations (50% and 100%) of OFSP leaves resulted in significant increases in SOD, CAT, and GPx activities, indicating enhanced antioxidant capacity. The positive effects of OFSP leaves were more pronounced than those observed with 100% ugwu leaves, suggesting that OFSP leaves might offer superior antioxidant benefits. These findings suggest that OFSP leaves could be a valuable dietary supplement to enhance antioxidant defense mechanisms, potentially offering protective benefits against oxidative stress. Further studies are needed to explore the underlying mechanisms and the potential health benefits in different populations.

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