Inhibition of advanced glycation end product and lipid peroxidation by extract and fractions of Newbouldia laevis

 Corresponding author: Mbagwu Ikechukwu Sonne. Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Inhibition of advanced glycation end product and lipid peroxidation by extract and fractions of Newbouldia laevis Daniel Lotanna Ajaghaku 1, Ikechukwu Sonne Mbagwu 2, *, and Amara Anwuchaepe Ajaghaku 3, 4 1 Department of Pharmacology, Faculty of Pharmaceutical Sciences, Enugu State University of Science and Technology, Enugu State, Nigeria. 2 Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University Agulu, Anambra State, Nigeria. 3 Department of Pharmacognosy and Traditional Medicine, Faculty of Pharmaceutical Sciences, David Umahi Federal University of Health Sciences, Uburu, Ebonyi State, Nigeria. 4 International Institute for Pharmaceutical Research and Innovation, David Umahi Federal University of Health Sciences, Uburu, Ebonyi State, Nigeria. World Journal of Advanced Research and Reviews, 2025, 26(02), 2514-2530 Publication history: Received on 02 April 2025; revised on 10 May 2025; accepted on 12 May 2025 Article DOI: https://doi.org/10.30574/wjarr.2025.26.2.1836 Abstract Newbouldia laevis is generally known for its significant antidiabetic potentials. Advanced glycation end-products (AGEs) and lipid peroxidation contribute significantly to the pathogenesis of diabetic complications through oxidative stress and protein damage. This study evaluated the inhibitory effects of extract and fractions of Newbouldia laevis leaves on AGE formation and lipid peroxidation. The leaves were extracted using 70% aqueous ethanol and partitioned into nhexane, ethyl acetate, butanol, and water fractions. In vitro assays revealed the ethyl acetate fraction with the highest total phenolic content and exhibited the strongest ferric reducing antioxidant power and inhibition of lipid peroxidation. The formation of AGEs and glycation-induced protein carbonylation were significantly suppressed in a concentrationdependent manner, with the ethyl acetate fraction demonstrating the most potent activity in weeks 2 – 4 at 0.4 – 1.6 mg/ml concentrations, comparable to aminoguanidine, a standard AGE inhibitor. The butanol and water fractions also showed notable inhibitory effects, whereas the n-hexane fraction exhibited the least activity. The inhibitory potential of the fractions correlated with their phenolic content, suggesting a phenol-mediated mechanism. These findings support the therapeutic potential of N. laevis, particularly its phenol-rich fractions, in mitigating oxidative stress and protein glycation associated with diabetes mellitus and its complications. Keywords: Newbouldia Laevis; Advanced Glycation End-Products (Ages); Lipid Peroxidation; Diabetic Complications; Phenolic Compounds; Antioxidant Activity 1. Introduction Diabetes mellitus is a metabolic condition marked by hyperglycemia [1]. Long term exposure to high glucose concentration in the blood is associated with numerous complications that lead to impairment of kidney, eyes, nerves and blood vessels structures and functions. These complications represent major cause of morbidity and mortality associated with diabetes [2]. One of the major pathways involved in the development and progression of different diabetic complications is protein glycation reaction leading to the formation of advanced glycation end products [3]. Some of the diabetic complications associated with this process include but not limited to nephropathy, retinopathy and neuropathy [4]. Other consequences of advanced glycation end products (AGEs) include cardiovascular complications, neurodegenerative derangement, bone disorders and erectile dysfunction to mention but a few [5]. World Journal of Advanced Research and Reviews, 2025, 26(02), 2514-2530 2515 Advanced glycation end-products are the final products of the non-enzymatic reaction between reducing sugar and amino groups in proteins, lipoproteins and nucleic acids [6]. During sustained hyperglycemia, glucose creates covalent adducts with amino acid residues in proteins, which initiates this process. This process of protein glycation induces conformational changes that affects the structural and functional activities of proteins including their ability to act as receptors for ligand/drug interaction as well as their metabolic functions [7]. The pathological implications of AGEs are ascribed to their ability to produce reactive oxygen and nitrogen species as well as oxidative stress and inflammatory conditions that lead to structural and functional protein alteration, cellular dysfunction and other tissue and organ damages [8]. The free radicals generated through various glycation steps and other hyperglycaemic mediated processes also produce lipid peroxidation with its associated reactive carbonyl products [9]. The lipid peroxidation reactive products act as both precursor of AGEs formation and as direct protein damage initiator thereby amplifying the hyperglycaemia mediated protein damage [5]. The inhibition of advanced glycation end products ad lipid peroxidation therefore represents an important pharmacological target for the prevention or mitigation of life threatening complications of diabetes. For many decades, natural products have been used as a source of therapeutic agents because of the existence of various bioactive compounds and they are believed to be non-toxic to a larger extent [10]. Newbouldia laevis commonly referred to as the African Border Tree or Boundary Tree can be seen growing in tropical rain-forest regions and has been used for many years in the practice of traditional medicine. This plant is also rich in many phytochemical constituents and secondary metabolites including Alkaloids, Flavonoids, Saponins, Tannins, Glycosides Phenols and the vitamins A, B1, B2, B6, B12, C, D, E and K which are ethno-pharmacologically significant [11]. N. laevis also exhibit anti-inflammation, antihypertensive, anticoagulant, antibacterial, and antioxidant properties which are comprehensively reported in the current literature [12]. Newbouldia laevis has been shown in recent research to have antidiabetic potential, with hypoglycemia effects similar to those of glibenclamide [13, 14]. In type 2 diabetes animals, the butanol fraction improved insulin secretion, lipid profile, and glucose/fat tolerance while significantly lowering blood glucose, body weight, and insulin resistance [15]. Additionally, the plant has also been reported to exert it’s antidiabetic effects by lowering oxidative stress and decreasing the absorption of glucose, which affects insulin sensitivity and pancreatic βcell activity [16]. In addition, Newboulasides A and B, two novel caffeic acid glycosides discovered from N. laevis, demonstrated potent α-amylase inhibition [17], indicating that enzyme inhibition is a crucial mechanism. These findings support its long-standing use in the treatment of diabetes. This study seeks to investigate further therapeutic potentials of N. laevis in the prevention and management of diabetes complications. 2. Materials and Methods 2.1. Plant material Newbouldia laevis leaves were collected in Oba, Enugu State, Nigeria, and confirmed by a trained taxonomist, Mr. Felix Nwafor from the Department of Pharmacognosy and Environmental Medcine, University of Nigeria Nsukka, Enugu State, Nigeria. The plant material was air dried and crushed to powder. The voucher specimen was placed in the herbarium of the Faculty of Pharmaceutical Science at Nnamdi Azikiwe University's Agulu Campus (PCG 474/A/035). 2.2. Extraction A 2.5 kg quantity of pulverized leaves of N. laevis was cold macerated in 10 L of aqueous ethanol (70%) for 72 h with intermittent shaking. The resulting solution was filtered, and the filtrate was pre-concentrated in vacuo using a rotary evaporator at 40°C and thereafter, dried to a constant weight using an open water bath at the same 40°C to obtain the ethanol extract. 2.3. Fractionation (Liquid-liquid Chromatography) The ethanol extract (112 g) was dissolved in distilled water and subjected to liquid–liquid partition successively with 2.5 L of n-hexane, ethyl acetate, and then butanol using separating funnel to obtain the n-hexane, ethyl acetate and butanol soluble fractions, respectively. The leftover portion after partitioning was used as the water fraction. The fractions were pre-concentrated using rotary evaporator at 40oC and dried using water bath at 40oC. The water fraction was freeze dried. World Journal of Advanced Research and Reviews, 2025, 26(02), 2514-2530 2516 2.4. In vitro analysis 2.4.1. Total phenolic content of the extract and fractions by Folin ciocalteu’s assay The total phenolic content of the fractions were determined using the method described by Kim et al. [18]. One milliliter of the extracts (100 µg/ml) was mixed with 0.2 ml of Folin-Ciocalteu’s phenol reagent. After 5 minutes, 1 ml of 7.6% Na2CO3 solution was added to the mixture followed by the addition of 2 ml of distilled water. The mixture (in duplicate) was incubated at 40 °C for 30 minutes, after which the absorbance were read at 760 nm using UV-VIS spectrophotometer against blank (containing every other component of the mixture except sample). The total phenolic content was estimated from the calibrated curve which was made by preparing gallic acid solution and expressed as milligrams of gallic acid equivalent (GAE) per gram of the extracts. 2.4.2. Ferric reducing antioxidant power assay The FRAP assay was conducted according to the method reported by Benzie and Strain, [19]. FRAP reagent was freshly made by mixing three solutions a, b and c, 300 mM sodium acetate buffer, pH = 3.6 (solution a), 10 mM 2,4,6-tris(2pyridyl)-s-triazine (TPTZ) solution in 40 mM HCl solution (b) and 20 mM ferric chloride (FeCl3) solution (c) in proportions of 10:1:1 (v/v/v). The reaction was completed by keeping the reagent in darkness for 30 minutes. In the test, 0.1 mL of fractions and positive control ascorbic acid (100, 200, 400, 800, and 1600 µg/mL) and FeSO4 (200, 400, 600, 800, and 1000 µM) were mixed with 2.9 mL of FRAP reagent individually. An equal amount of 5% Tween 80 (0.1 mL) was used as a blank (control). All samples were prepared in triplicate and vortexed for 1 minute before being incubated in the dark at 37 °C for 30 minutes. Each sample's rise in absorbance was evaluated using a UV-Visible spectrophotometer at 593 nm. The results were compared with ascorbic acid as positive control and FeSO4 was used for calibration. FRAP activity was estimated using ferrous equivalent (FE) in µM. 2.4.3. Linoleic acid peroxidation assay The procedure was performed according to modified method of Choi et al. [20]. The fractions were mixed with 550 μL linoleic acid solution (0.28 mg linoleic acid and 0.28 mg Tween-20 in 100 μM phosphate buffer,pH 7.4) at various concentrations (100 – 1600 μg/mL), 500 μL of phosphate buffer (100 μM, pH 7.4) and 150 μl of ascorbic acid (10 μM). Same concentrations of Trolox where used as reference standard while the blank contained the vehicle (5% Tween 80) in place of sample/standard. The linoleic acid peroxidation was initiated by the addition of 0.1 mL FeSO4 (10 μM) and incubated at 37°C for 60 min. The reaction mixture was cooled and 1.5 mL of trichloroacetic acid (10% in 0.5% HCl) added. Then, 3 mL TBA (1%, in 50 mM NaOH) was added. The reaction mixture and TBA solution were heated in the water bath at 90oC for 60 min. After cooling, 2 mL portions were taken from each sample and vortexed with 2 mL butanol and centrifuged at 1000 x g for 30 min. The upper layer of the resulting solution was separated for spectrophotometric measurement. The absorbance of solution was read at 532 nm and calculated the percentage of linoleic acid peroxidation inhibition in the following equation: Linoleic acid peroxidation inhibition (%) = ((Acontrol-Asample)/ Acontrol) x100 2.4.4. In vitro glycation of bovine serum albumin The glycated BSA formation was determined according to a previously described method (Adisakwattana et al. [21] with slight modification. Bovine serum albumin (BSA) was used as the model protein in a concentration of 40 mg/ml corresponding to physiological albumin concentration in human blood; similarly, glucose (0.5 M) was used as the glycating agent. Briefly, BSA (40 mg/ml) was incubated with 0.5 M glucose in 0.1 M phosphate buffer saline (PBS) (pH 7.4), containing 0.02 % sodium azide in the dark at 37 °C for 1, 2, 3, and 4 weeks. The solution containing N. laevis fractions (0.1–1.6 mg/ml) dissolved in PBS was added to the mixtures, before incubation. All incubations were performed under sterile conditions. A small drop of chloroform was added to the solution and the corks moistened with toluene to inhibit bacterial growth. Fluorescent intensity at an excitation wavelength of 355 nm and an emission wavelength of 460 nm was used to measure glycated BSA formation. Aminoguanidine (AG, 1 mg) was used as a positive control for the study. AGE inhibition (%) = [1 − 𝐹𝑠−𝐹𝑠𝑏 𝐹𝑐−𝐹𝑐𝑏] 𝑥 100 Where Fs – Fsb is the difference between the fluorescent intensity of sample + BSA incubated with or without glucose while Fc - Fcb is the difference between the fluorescent intensity of BSA incubated with or without glucose. World Journal of Advanced Research and Reviews, 2025, 26(02), 2514-2530 2517 2.4.5. Determination of protein carbonyl content Carbonyl content is regarded a glycation indicator and is utilized as a measure to evaluate protein oxidation as well as a confirmatory assay for AGE production. The level of carbonyl group in glycated BSA was slightly modified according to Levine’s method [22]. Concisely, 100 μl of glycated BSA was mixed with 400 μl of 10 mM 2,4-dinitrophenylhydrazine (DNPH) in 2.5 M HCl. After incubation for 60 min at room temperature, glycated BSA was then precipitated using 500 μl of 20 % (w/v) trichloroacetic acid (TCA), left on ice for 5 min, and centrifuged at 10,000 g for 10 min at 4 °C. The protein pellet was washed 3 times by 500 μl of 1:1 (v/v) ethanol: ethyl acetate solution. The final protein pellet was resuspended in 250 μL of 6 M guanidine hydrochloride. The absorbance was read at 370 nm. The protein carbonyl group of each sample was calculated by using absorption coefficient (ɛ=22,000 M−1.cm−1). The protein carbonyl content was expressed as nmol carbonyl/mg protein. 2.5. Statistical analysis Data were expressed as means ± standard error of mean (S.E.M), N=3. Data were analyzed using One way ANOVA followed by Tukey’s HSD post hoc test. P-value 3. Results 3.1. Total phenolic content, ferric reducing antioxidant power and lipid peroxidation The intermediary polar solvent (ethyl acetate) partitioned majority of the phenolic compounds making it the phenol rich fraction (Table 1). Lowest content was found in the non-polar solvent fraction (n-hexane) while water fraction contained about double fold lower the quantity recorded in ethyl acetate fraction. The fractions showed graded concentration dependent reduction of ferric ion to ferrous ion (Figure 1). Using regression equation generated from the ferrous calibration curve (Y = 0.0009x – 0.1324), the fractions showed similar trend of effect based on their phenolic content. Ethyl acetate fraction showed the strongest ferric reducing power while n-hexane fraction the least. These reducing powers shown by the fractions were however lower when compared to corresponding concentrations of the reference standard (ascorbic acid). For the inhibition of lipid peroxidation, n-hexane fraction produced linear concentration-effect curve compared to other fractions that produced hyperbolic curve shape just like the reference standard (trolox) (Figure 2). The order of inhibition of lipid peroxidation by the fractions followed the same trend with phenolic content. Ethyl acetate fraction inhibited 50% of lipid peroxidation at 0.349 mg/ml which is the closest compared to trolox - the reference standard (0.15 mg/ml). n-hexane required upto 6768 mg/ml concentration to produce same inhibition of 50% lipid peroxidation. Table 1 Total phenolic content of the fractions of N. laevis extract Fractions of N. laevis Extract Total Phenolic content (mgGAE/g) N-hexane 46.87 + 1.10 Ethyl acetate 389.91 + 4.74 Butanol 236.85 + 2.56 Water 107.79 + 4.02 Where GAE = Gallic acid equivalent World Journal of Advanced Research and Reviews, 2025, 26(02), 2514-2530 2518 Figure 1 Ferric reducing antioxidant power effect of the fraction World Journal of Advanced Research and Reviews, 2025, 26(02), 2514-2530 2519 Figure 2 Inhibitory effect of the fraction on lipid peroxidation 3.2. Effect of treatment on advanced glycation end (AGE) product formation The formation of AGE in bovine serum albumin (BSA) was measured by the fluorescence intensity. As shown in figure 3, the fluorescent intensity increased with time during 4 weeks of experiment in the vehicle control group (BSA-F). The butanol and water fractions showed concentration dependent inhibition of AGE formation. The fluorescent intensity recorded by the graded concentrations of the fractions were significantly (P<0.05) lower than that of the vehicle control at all points of measurement (figure 3). The effect recorded by these fractions were significantly (P<0.05) lower than that shown by the reference standard (Aminoguanidine). The n-hexane fraction showed lower ability to inhibit AGE formation compared to other fractions. Significant (P<0.05) inhibition of AGE formation where shown in week 1 at the initiation of AGE formation (figure 4). With time, higher concentrations of n-hexane fraction were required to produce significant (P<0.05) inhibition of AGE formation. Ethyl acetate fraction recorded the highest effect of inhibition of AGE formation compared to other fractions as shown by significantly (P<0.05) lower fluorescent intensity compared to the vehicle control. At week 1, all the tested concentrations except 0.1 mg/kg showed similar strength of inhibition of AGE formation compared to the reference standard with no significant (P>0.05) difference when these concentrations were compared between groups. In weeks 2 - 4, 0.4 – 1.6 mg/ml concentrations of ethyl acetate fraction produced similar effect when compared to reference standard. The inhibition of AGE produced by the fractions followed the same trend in all the weeks of the experiment with ethyl acetate fraction having the highest inhibition while n-hexane the least (figure 5). The half-maximal inhibitory concentration of ethyl acetate fraction at the end of the experiment (week 4) was 0.058 mg/ml while that of n-hexane was 1575 mg/ml. Similarly, butanol and water fractions required higher concentrations to inhibit 50% of AGE formation compared to ethyl acetate fraction (figure 6). However, these polar fractions (butanol and water fractions) produced better than the polar fraction (n-hexane fraction). World Journal of Advanced Research and Reviews, 2025, 26(02), 2514-2530 2520 Where: * = P<0.05 compared to BSA-F (vehicle control); # = P<0.05 compared to Aminoguanidine (standard reference drug). Where BF = butanol fraction and WF = water fraction Figure 3 Effect of butanol and water fractions on Advanced glycation end product formation World Journal of Advanced Research and Reviews, 2025, 26(02), 2514-2530 2521 Figure 4 Effect of Hexane and Ethyl acetate fractions on Advanced glycation end product formation Where: * = P<0.05 compared to BSA-F (vehicle control); # = P<0.05 compared to Aminoguanidine (standard reference drug). 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