Glycemic Responses, Enzyme Activity, and Sub-Acute Toxicity Evaluation of Unripe Plantain Peel Extract in Rats

Abstract

Plantain (Musa paradisiaca L.) is a tropical monocotyledonous, succulent plant of the Musaceae family commonly grown for food in the tropical regions of the African, Asian, and South American continents, where its parts are also sought for ethnomedicinal purposes in the treatment of burns, inflammation, and diabetes, among others. In the present preliminary exploratory study, the ethanol extract of the underutilized Musa paradisiaca peel (MPE) was evaluated for its in vitro inhibitory effects on α-amylase and α-glucosidase, as well as its in vivo hypoglycemic activity and potential biochemical toxicity. MPE (100, 200, 400 mg/kg) was orally administered to normal experimental rats for 30 days, following which the lipid profile, antioxidant status, and serum/tissue indices of hepatic, renal, and cardiac functions were evaluated. MPE produced significant inhibition (p < 0.05) of α-amylase (37%) and α-glucosidase (46%) at 120 µg/mL in vitro. The effect was lower than that of acarbose (IC₅₀ = 44.4 ± 1.14 and 15.60 ± 0.01 µg/mL, respectively). A modest blood glucose-lowering effect of MPE was observed at the highest tested dose (400 mg/kg) following subacute oral administration. During this treatment period, no biochemical alterations of toxicological importance were caused by MPE, as the organ–body weight ratio and serum/tissue indicators of organ function/damage were not adversely altered. In conclusion, MPE demonstrated inhibitory activity against both α-amylase and α-glucosidase, which may contribute to its potential hypoglycemic effects. Additionally, the findings indicate that the peel extract is non-toxic in rats following sub-acute administration at doses up to 400 mg/kg body weight. Further studies involving diabetic models and chronic exposure will substantiate and extend these preliminary observations.

1. Introduction

Plants provide a rich repertoire of phytochemicals. These compounds are responsible for many biological effects in humans and other higher animals [1]. Many potential medicinal plants are yet to be realized or given due attention, thus making their therapeutic value underestimated and underexploited. There are now global efforts geared towards developing these categories of plants (and plant parts) as sources of viable phytocomponents with nutritional and therapeutic values [2]. For such ethnobotanicals, preliminary studies are essential not only to characterize their phytochemical constituents but also to evaluate and identify potential toxic or adverse effects that may compromise their therapeutic value. Further, toxicity investigations could also reveal the therapeutic margins of such ethnobotanical preparations with a view to allowing safer and viable alternatives [3].

Plantain (M. paradisiaca) is a tropical monocotyledonous, perennial, and succulent plant belonging to the Musaceae family [4,5]. Along with banana (M. sinensis), its very close cousin with marked morphological similarities, plantain has been around with humans for ages. Plantain is cultivated across tropical and subtropical regions, and West Africa is a major production hub ([6]). With an aerial pseudostem, rhizome, huge leaves, and elongated starchy fruits, the plant can reach heights of 3 to 15 m. The cultivars are classified as French or Horn types, and plantain exhibits polyploid genomes (AAB, ABB, BBB) [7,8]. Plantain is rich in potassium, and the peel contains substantially more potassium (729.41 ± 2.51 mg/100 g) than the flesh (284.65 ± 3.42 mg/100 g). This makes it a potential source of the mineral essential for maintaining energy balance and proper muscle function for those in humid tropical regions [4,9]. While the fruit of plantain serves as a staple in some cultures, the flowers, leaves, stems, and roots are commonly sought ethnomedicinals [8].

In the traditional system of medicine, M. paradisiaca is employed in the management of burns, inflammation, bronchitis, diabetes, hypertension, and diarrhea [10]. Various plant parts are rich in phytochemicals, including polyphenols, flavonoids, saponins, triterpenes, and phytosterols [4]. Preclinical studies show that plantain extracts exhibit a broad spectrum of bioactivities, like antioxidant, antimicrobial, antidiabetic, wound-healing, and anticancer effects [11]. The antidiabetic property of the flowers was confirmed in a streptozotocin-induced diabetic rat [5]. The plantain fruit peel is often discarded as waste and is highly underutilized. Work by Parmar and Kar [12] showed that the water extract of plantain peel affects glucose metabolism, exhibits antithyroidal activity, and inhibits H₂O₂-induced membrane peroxidation in rats’ tissues in vitro.

Even though the literature is replete with information on the folkloric uses of M. paradisiaca roots and flowers as well as the scientific validation of some of these claims, little or no study has been done to ascertain the chemical composition and safety of the unripe fruit peel following subacute administration in a rodent model. Also, there is a paucity of information on its hypoglycemic potential or effects on carbohydrate-metabolizing enzymes like α-amylase and α-glucosidase in vitro. An attempt has, therefore, been made in the present investigation to explore these possibilities and ascertain how well the peel is tolerated following a subacute exposure in rats.

2. Materials and Methods

2.1. Chemicals

Thiobarbituric acid (TBA), 5,5′-Dithiobis (2-nitrobenzoic acid) (DTNB), hydrogen peroxide, and malonaldehyde bis-(dimethyl acetal) (MDA) were procured from Sigma Chem., Co. (London, UK). Lactate dehydrogenase (LDH), bilirubin, creatinine, uric acid, urea, creatine kinase MB (CK-MB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), cholesterol, triglyceride, high-density lipoprotein (HDL-c), and low-density lipoprotein (LDL-c/VLDL-c) assay kits were obtained from MTD Diagnostics (Maddaloni, Italy) and Fortress Diagnostics (Antrim BT41 1QS, Antrim, UK). All other chemicals were of analytical grade.

2.2. Equipment

Gas chromatograph (BUCK M910; BUCK Scientific Instruments LLC, Ansonia, CT, USA), microplate reader (SpectraMax Plus 384; Molecular Devices, San Jose, CA, USA), laboratory centrifuge (Hettich Rotina 380R; Tuttlingen, Germany), tissue homogenizer (Polytron PT 1200E; Kinematica AG, Lucerne, Switzerland), water bath (Thermo Fisher Scientific; Waltham, MA, USA), boiling water bath (Thermo Fisher Scientific; Waltham, MA, USA), adjustable micropipettes (Eppendorf; Hamburg, Germany), and analytical balance (Ohaus Pioneer PA214C; Parsippany, NJ, USA).

2.3. Acquisition of Plant Material and Extract Preparation

Fresh unripe bunches of plantain (M. paradisiaca) were obtained from a local farm in Ilara-Mokin, Nigeria (7.3497° N, 5.1067° E), West Africa, between July and August 2021. After botanical identification and authentication at the Department of Crop, Soil and Pest (CSP) of the Federal University of Technology, Akure, Ondo State, Nigeria, the peels were carefully separated from thoroughly washed fruits, air-dried and stored at room temperature until needed. The dried peels were then ground to fine powder (0.45 mm to 0.75 mm particle size) using an electric blender. The powdered plant material (500 g) was macerated in 1200 mL of locally distilled absolute ethanol for 72 h. The resulting suspension was filtered, and a rotary evaporator was used to remove the entire ethanol from the filtrate to obtain the M. paradisiaca unripe peel extract (MPE).

2.4. Phytochemical Screening

Screening of MPE for its phytochemical constituents was carried out using standard methods [13,14,15]. The screening involves detection of flavonoids, phenolics, alkaloids, terpenoids, coumarins, saponins, steroids, tannins, anthraquinones, and cardiac glycosides.

2.5. Quantification of Phytochemicals Using Gas Chromatography-Flame Ionization Detection (GC-FID)

Phytochemicals in MPE were quantified using gas chromatography equipment (BUCK M910, BUCK Scientific Instruments LLC, Ansonia, CT, USA) equipped with a flame ionization detector and an SE 30 glass capillary column (L × I.D. 30 m × 0.25 mm). The extract was first ground into powder, dissolved in water for 48 h, and then filtered. The filtrate was reconcentrated to a solid, after which a 10 g sample was extracted with ethanol and concentrated to 1 mL. n-Hexadecane (≥99%, Sigma-Aldrich, St. Louis, MO, USA) was used as the internal standard (10 µg/mL in ethanol). A 1 µL aliquot of the sample was injected in splitless mode at 280 °C while nitrogen served as the carrier gas (40 mL/min, 5.0 Pa). The programming of the oven temperature was from 200 °C (initial) to 330 °C at 3 °C/min, while the detector temperature was maintained at 320 °C. Using external calibration curves constructed for each reference standard (caffeic acid, syringin, myricetin, luteolin, quercetin, kaempferol, β-sitosterol, 4-hydroxybenzoic acid, apigenin, sitoindoside I, sitoindoside II, and capsaicin), quantification was performed in the range of 0.1–100 µg/mL. Linearity was established from calibration curves (R² > 0.995). The limits of detection (LOD) and quantification (LOQ) were determined based on signal-to-noise ratios of 3 and 10, respectively. To conduct recovery experiments, known amounts of standards were spiked into pre-analyzed extracts, and the rates of recovery ranged between 92.3% and 106.4%. Retention times for standards were used to identify compounds, which were further confirmed by comparison with the literature. Quantitative results were expressed in mg/g of extract using peak area ratios relative to the respective internal standard [16]. All calibration curves exhibited excellent linearity (R² ≥ 0.995). The limits of detection (LOD) and quantification (LOQ) ranged from 0.10–0.25 µg mL⁻¹ and 0.32–0.76 µg mL⁻¹, respectively. Mean recoveries (92–106%) and precision values (%RSD < 5%) confirmed the reliability and reproducibility of the GC–FID method.

2.6. Evaluation of In Vitro Antioxidant Contents and Potentials

2.6.1. Quantification of Total Phenol Content

The total phenolic content of MPE was determined using the modified Folin–Ciocalteu method described by [17]. Briefly, a mixture containing deionised water (0.5 mL), Folin–Ciocalteu reagent (125 µL) was added to 125 µL of MPE dissolved in distilled water (0.25, 0.5 and 1.0 mg/mL) and allowed to stand for 6 min before adding 1.25 mL of 7% (w/v) Na₂CO₃ solution. The reaction mixture was then allowed to stand for an additional 90 min before taking the absorbance at 760 nm against the distilled water blank. The amount of total phenolics was expressed as gallic acid equivalents (GAE, mg gallic acid/g sample) through the calibration curve of gallic acid.

2.6.2. Quantification of Total Flavonoid Content

The total flavonoid content was determined spectrophotometrically as described by [18]. Briefly, 5% NaNO₂ solution (75 µL), freshly prepared 10% AlCl₃ (0.150 mL), and 1 M NaOH solutions were added to 250 µL of MPE dissolved in distilled water (0.25, 0.5, and 1.0 mg/mL), and the final volume was adjusted to 2.5 mL with deionized water. The mixture was allowed to stand for 5 min, following which the absorbance was measured at 510 nm against the blank. The amount of total flavonoids was expressed as quercetin equivalents (QE, mg quercetin/g sample) through the calibration curve of quercetin.

2.6.3. Evaluation of Total Antioxidant Capacity

The total antioxidant capacity was measured by the ferric reducing ability of plasma (FRAP) assay described by [19]. A working FRAP reagent was prepared by mixing together acetate buffer (300 mM, pH 3.6), 2,4,6-tripyridyl-s-triazine, TPTZ (10 mM in 40 mM HCl), and FeCl₃·6H₂O (20 mM) in the ratio of 10:1:1. About 0.5 mL of MPE (0.25, 0.5, and 1.0 mg/mL) or the reference compound, gallic acid (2, 4, 8, and 10 µg/mL final concentrations), was mixed with 3 mL of the working reagent and vortexed, following which the absorbance was measured at 593 nm. The calibration curve of known Fe²⁺ concentration was prepared using a preparation of methanol solutions of FeSO₄·7H₂O ranging from 50 to 1000 μM. The concentration of antioxidant having a Ferric-TPTZ reducing ability equivalent to that of 1 M FeSO₄·7H₂O was used to define the parameter equivalent concentration.

2.7. In Vitro Assessment of Carbohydrate-Digestive Enzyme Inhibition

2.7.1. Assay of α-Amylase Inhibition Potential

The α-amylase inhibitory activity of M. paradisiaca extract (MPE) was determined as earlier described [20]. Briefly, 100 µL of MPE (0, 20, 50, 75, 100, 120 µg) and 100 µL of reaction buffer (0.02 mol/L sodium phosphate buffer, pH 6.9 with 0.006 mol/L NaCl) containing swine pancreatic α-amylase (EC 3.2.1.1) (0.5 mg/mL) was incubated for 10 min at room temperature (25 °C). Thereafter, 100 µL of 1% starch solution in reaction buffer was added to the reacting mixture, which was further incubated for 10 min at room temperature and stopped with 200 µL of dinitrosalicylic acid color reagent. After incubation in a boiling water bath for 5 min, the reaction mixture was cooled to room temperature, diluted with distilled water (2 mL), and the absorbance was measured at 540 nm using a microplate reader (SpectraMax Plus 384, Molecular Devices). For assay validation, positive controls (acarbose, 0–120 µg/mL) and negative controls (reaction buffer without extract) were included in each plate. Blanks without enzyme were also run to account for background absorbance. Percentage inhibition of α-amylase activity by the extracts was calculated as follows: % inhibition = [(AbsControl − AbsSamples)/AbsControl] × 100.

2.7.2. Assay of α-Glucosidase Inhibition Potential

The ability of MPE to inhibit α-glucosidase activity was determined in accordance with the procedure described by [21]. A mixture containing MPE (100 uL) at different concentrations (0, 20, 50, 75, 100, 120 µg) and 100 µL of α-glucosidase solution was incubated at 25 ᵒC for 10 min. Thereafter, 50 µL of p-nitrophenyl-α-D-glucopyranoside solution (5 mmol/L in 0.1 mol/L phosphate buffer, pH 6.9) was added. The reacting mixture was further incubated at room temperature for 5 min. Finally, the absorbance was taken at 540 nm using a microplate reader. Appropriate positive and negative controls were included as described earlier. The α-glucosidase inhibitory activity was expressed as percentage inhibition as follows: % Inhibition of α-glucosidase activity = [(AbsControl − AbsSamples)/AbsControl] × 100

2.8. Animals

Male albino rats (Wistar strain) weighing 180–220 g, obtained from a private breeder and housed in the rodent colony of the Department of Biochemistry, Federal University of Oye, Oye-Ekiti, Nigeria, were used for this study. The rats, which were kept in wire mesh cages and maintained under a controlled light cycle (12 h light/12 h dark), were fed with commercial rat chow (Chikum Feeds, Ibadan, Nigeria) ad libitum and liberally supplied with water. All laboratory protocols and animal experiments were conducted following approval of the departmental committee on laboratory and animal ethics of the Federal University of Technology, Akure (FUTA), with approval number FUTA/ETH/21/05.

2.9. Experimental Design

Age-matched rats were randomly assigned to four groups (n = 6) and treated as follows:

• Group I: Control; vehicle (distilled water, 1 mL/kg)
• Group II: 100 mg/kg Musa paradisiaca ethanol extract (MPE)
• Group III: 200 mg/kg MPE
• Group IV: 400 mg/kg MPE

Distilled water and MPE (100, 200, or 400 mg/kg) were administered by oral gavage to healthy rats once daily for thirty (30) consecutive days. The group size (n = 6) was based on a previous rodent study with similar biochemical endpoints, which achieved adequate power (≥80%) to detect moderate effects (Cohen’s d ≈ 1.0) at α = 0.05. The primary outcome was the effect of the extract on hepatic (ALT, AST) and renal (creatinine, urea) indices. The dose selection was guided by our previous preliminary investigation and a cardioprotective study in which aqueous extract of the plantain peel was administered at comparable doses as preventive treatments [22]. This design also adhered to ethical considerations aimed at minimizing animal use in accordance with the 3Rs principle. Animals were sacrificed under mild ether anesthesia 24 h after the last administration. Blood was collected by cardiac puncture for serum preparation, and major tissues (liver, kidney, heart) were dissected out for biochemical evaluation, which was conducted in a blinded manner to reduce bias.

2.10. Serum and Tissue Homogenates Preparation

Blood collected through cardiopuncture from animals was allowed to clot and thereafter centrifuged at 3000 rpm for 15 min. The supernatant (serum) was carefully separated and stored at −20 °C for biochemical analysis. Organ tissues (liver, kidney, heart) were rinsed in KCl solution (1.5%) and homogenized in aqueous Tris–HCl buffer (50 mM, pH 7.4). Thereafter, tissue homogenates were centrifuged at 10,000 g for 20 min at 4 °C to obtain the supernatant, which was stored below −4 °C until needed for biochemical analyses.

2.11. Assessment of Serum Lipid Profile

Serum lipid profiles (total cholesterol TC, triglyceride TG, high-density lipoprotein HDL-c, low-density lipoprotein LDL-c, and very low-density lipoprotein VLDL-c) were determined by commercial assay kits following the manufacturer’s instructions.

2.12. Assessment of Tissue Function Indices

The levels or activities of biochemical indices and markers in the liver (albumin, total/direct bilirubin, AST, and ALT), kidney (creatinine, uric acid, and urea), and heart (CK-MB and LDH) homogenates and in the serum were estimated using assay kits obtained from Fortress Diagnostics (Antrim, UK) and MTD Diagnostics (Maddaloni, Italy) according to the instructions from the manufacturer.

2.13. Assessment of Tissue Antioxidant Status

In order to assess the antioxidant status, levels of reduced glutathione (GSH) and thiobarbituric acid reactive substances (TBARS), as well as the activities of endogenous antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the tissues, were estimated as previously documented [15,23].

2.13.1. Quantification of Tissue Membrane Lipid Peroxidation

Lipid peroxidation was determined by measuring the formation of thiobarbituric acid reactive substances (TBARS) [23].

2.13.2. Assay of Reduced Glutathione (GSH)

Tissue levels of GSH were estimated in tissue homogenates as previously described [23]. Briefly, sulphosalicylic acid (5%, 150 µL) was added to the supernatant (100 µL) and gently mixed. This allowed for the precipitation of protein after 5 min, and the filtrate was collected using Whatman no 2 filter paper (GE Healthcare, Maidstone, UK). The filtrate (50 µL) was added to 200 µL of 0.1 M phosphate buffer (pH 7.4), followed by Ellman’s reagent (25 µL). The blank was prepared with 200 µL buffer, 50 µL of diluted precipitating solution (three parts to two parts of distilled water), and 25 µL of Ellman’s reagent. The absorbance was measured at 412 nm using a microplate reader (SpectraMax Plus 384, Molecular Devices). The GSH estimate was obtained from a GSH standard curve.

2.13.3. Assay of Superoxide Dismutase (SOD) Activity

The method described by Komolafe, Akinmoladun and Olaleye [15] was deployed to assay for the activity of superoxide dismutase in tissue homogenates.

2.13.4. Assay of Glutathione Peroxidase Activity (GPx)

The assay of glutathione peroxidase in the tissue homogenates proceeded as previously described [15].

2.14. Statistical Analysis

Data are expressed as mean ± SEM. Statistical evaluation was done using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. The threshold for statistical significance was set at p < 0.05. In all cases, levels of significance are reported uniformly as follows: p < 0.05, p < 0.01, and p < 0.001. GraphPad Prism (ver. 8.0a) was used for statistical analysis, graphing, and IC₅₀ determination. IC₅₀ values were calculated by plotting percentage inhibition versus extract concentration and fitting the data to a nonlinear regression curve.

3. Results

3.1. Extract Yield, Phytochemicals and Antioxidant Potential of MPE

The ethanolic extraction of Musa paradisiaca peels resulted in a yield of 12.7 ± 0.5% (w/w) across three independent batches, indicating the reproducibility and consistency of the extraction process. Preliminary screening revealed the presence of alkaloids, tannins, phlobotannins, phenolics, flavonoids, steroids, cardiac glycosides, and terpenoids but no anthraquinones and saponins in MPE. GC-FID analysis detected and quantified (mg/g) the flavonoids (myricetin (0.65), luteolin (1.33), apigenin (0.95), kaempferol (1.50), quercetin (1.75), and leucocyanidin (0.48)), phenolic acids (4-hydroxybenzoic acid (1.56) and caffeic acid (2.67)), glycosides (b-sitosterol (0.96), syringin (0.36), sitoindoside I (0.36), and sitoindoside II (0.67)), and alkaloid (capsaicin (0.49)), as well as unclassified cyclomusatenone (0.76) and cyclomusatenol (1.69) in MPE (

Table 1. GC-FID quantification of phenolics and other phytochemicals in M. paradisiaca unripe peel extract.

Component	Retention	Area	Height	Concentration (mg/g)
Caffeic Acid	2.8000	3585.21	176.139	2.6734
Lanosterol	3.7160	534.241	28.8810	0.3984
Syringin	4.4000	489.100	23.5380	0.3647
Myricetin	4.8830	865.163	40.0710	0.6451
Sitoindoside I	5.3330	443.163	34.5420	0.3305
Sitoindoside II	5.5830	893.413	68.1380	0.6661
Cyclomusatenol	6.7660	2267.58	105.220	1.6909
Cyclomusatenone	7.2500	1022.77	89.5790	0.7627
Luteolin	7.4830	1786.61	160.532	1.3322
Quercetin	7.9500	2346.70	76.5430	1.7499
Kaempferol	9.3000	2016.48	160.053	1.5036
Capsaicin	9.6000	651.599	49.9540	0.4859
B-Sitosterol	10.316	1283.35	51.6850	0.9570
P-Hydroxybenzoic Acid	11.016	2089.50	123.220	1.5581
Leucocyanidin	11.566	640.560	31.9610	0.4776
Apigenin	12.216	1266.82	49.1180	0.9446

; Figure S1). The GC–FID method showed excellent linearity, sensitivity, and precision (Table S1). The structures of some of these phytochemicals are presented in Figure 1. The total phenolic and flavonoid contents of the extract were 101.40 ± 3.69 mg gallic acid equivalent/g extract and 88.62 ± 3.64 mg quercetin equivalent/g extract, respectively. Furthermore, the total antioxidant capacity was found to be 35.24 ± 0.64 mg gallic acid equivalent/g extract.

3.2. Inhibitory Effects of MPE on α-Amylase and α-Glucosidase

As shown in Figure 2, MPE caused dose-dependent in vitro inhibition (p < 0.05) of α-amylase (37%) and α-glucosidase (46%) at 120 µg/mL. The inhibitory effects of MPE were significantly lower (p < 0.05) compared with those produced by the reference drug, acarbose, on both α-amylase (IC₅₀ = 44.4 ± 1.14 µg/mL) and α-glucosidase (IC₅₀ = 15.60 ± 0.01 µg/mL). The IC₅₀ values (µg/mL) for MPE were estimated from the dose–response curve and found to be 184 ± 2.8 for α-amylase and 136 ± 1.2 for α-glucosidase. These values exceeded the highest concentration tested in the assay (120 µg/mL).

3.3. Fasting Blood Glucose Levels and Organ-to-Body-Weight Ratio

A significant reduction (p < 0.05) in fasting blood glucose level was observed at the highest MPE dose (400 mg/kg; 10% decrease) compared with the untreated control, whereas the lower doses (100 and 200 mg/kg) produced only slight, non-significant decreases (Figure 3A). However, the liver/body weight (Figure 3B), kidney/body weight (Figure 3C), and heart/body weight (Figure 3D) ratios of experimental animals did not change after thirty days of oral MPE administration.

3.4. Serum Lipid Profile of Rats

Altogether, the serum lipid profile of rats was not altered by MPE treatment (Figure 4A–E). Except for the reduction in high-density lipoprotein cholesterol (HDL-c) in the group administered 400 mg/kg MPE, the changes in serum total cholesterol, triglyceride, low-density lipoprotein (LDL-c), and very-low-density lipoprotein cholesterol (VLDL-c) as a result of MPE treatment were not statistically significant (p > 0.05).

3.5. Hepatic Function Indices

The biochemical indices of hepatic functions were evaluated in the serum and liver of rats following subacute administration of M. paradisiaca unripe peel extract (MPE) (

Table 2. Effect of M. paradisiaca unripe peel extracts (MPE) on serum markers of hepatic function.

	Albumin		ALT		AST		Total Bilirubin		Direct Bilirubin
Group	Serum	Liver	Serum	Liver	Serum	Liver	Serum	Liver	Serum	Liver
	(g/dL)	(g/dL)	(U/L)	(U/mg Protein)	(U/L)	(U/mg Protein)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)
Control	3.23 ± 0.02	0.048 ± 0.002	30.36 ± 4.02	3.00 ± 0.28	65.63 ± 3.15	3.03 ± 0.30	3.60 ± 0.27	5.78 ± 0.29	2.23 ± 0.14	3.57 ± 0.28
MPE 100 mg/kg	3.38 ± 0.05	0.050 ± 0.003	16.16 ± 2.58 **	2.82 ± 0.29	71.02 ± 3.90	3.06 ± 0.25	4.07 ± 0.31	5.31 ± 0.26	3.21 ± 0.43	3.23 ± 0.20
MPE 200 mg/kg	3.35 ± 0.10	0.064 ± 0.004 **	14.54 ± 2.56 **	3.52 ± 0.15	46.05 ± 6.88 *	3.71 ± 0.23	3.78 ± 0.20	5.29 ± 0.26	2.78 ± 0.21	2.85 ± 0.07
MPE 400 mg/kg	3.33 ± 0.13	0.050 ± 0.003	17.95 ± 1.57 **	3.51 ± 0.51	54.87 ± 1.88	3.47 ± 0.39	4.03 ± 0.17	5.24 ± 0.10	3.73 ± 0.65	2.88 ± 0.26

Values are expressed as mean ± SEM (n = 6). Control: distilled water; 0 mg/kg MPE. MPE: M. paradisiaca unripe peel extract. * p < 0.05; ** p < 0.01 vs. control.

). MPE did not affect serum albumin levels but caused elevated (by 34%) hepatic albumin at 200 mg/kg dosages. The reduction in serum ALT activity of rats was statistically significant (p < 0.01) following treatment with MPE at 100 (44%), 200 (50%), and 400 (36%) mg/kg dosages. Serum AST activity was also significantly reduced by treatment with 200 mg/kg MPE (p < 0.05; 29%). However, changes in hepatic levels of both marker enzymes were not significant (p > 0.05) for all treatment groups compared with control. Also, liver function indices, typified by total and direct bilirubin, were not affected by MPE treatment.

3.6. Renal and Cardiac Function Indices

The levels of uric acid and urea were, respectively, reduced in the serum following treatment with MPE at 200 (64% and 39%) and 400 (72% and 45%) when compared with normal rats (

Table 3. Effect of M. paradisiaca unripe peel extracts (MPE) on serum markers of renal and cardiac functions.

	Uric Acid		Urea		Creatinine		LDH		CK-MB
Group	Serum	Kidney	Serum	Kidney	Serum	Kidney	Serum	Heart	Serum	Heart
	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(µmol/L)	(µmol/L)	(U/L)	(U/mg Protein)	(U/L)	(U/mg Protein)
Control	6.53 ± 0.86	16.71 ± 0.69	46.30 ± 4.51	19.27 ± 1.54	109.0 ± 4.63	43.00 ± 3.19	126.9 ± 6.28	10.48 ± 1.22	309.2 ± 13.4	0.73 ± 0.12
MPE 100 mg/kg	6.33± 1.08	20.28± 0.40 *	28.57 ± 4.25 **	18.43 ± 1.02	106.5 ± 10.0	50.78 ± 5.23	134.5± 7.60	16.98 ± 4.80	311.0 ± 13.28	1.39 ± 0.53
MPE 200 mg/kg	2.08 ± 0.24 **	18.02 ± 1.59	26.91 ± 1.14 **	19.00 ± 1.11	96.20 ± 5.88	60.94 ± 10.5	89.46 ± 5.32 **	15.20 ± 5.67	215.8 ± 13.28 ***	0.80 ± 0.11
MPE 400 mg/kg	1.94 ± 0.43 **	15.63 ± 0.62	24.03 ± 2.49 ***	17.99 ± 1.21	97.48 ± 5.13	29.92 ± 3.60	99.03 ± 7.32 **	13.96 ± 2.14	231.5 ± 8.80 ***	1.96 ± 0.73

Values are expressed as mean ± SEM (n = 6). Control: distilled water; 0 mg/kg MPE. MPE: M. paradisiaca unripe peel extract. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control.

). However, levels of the biochemical indices of renal function were not altered in the renal tissue at those dosages. Similarly, the level of creatinine in the serum and kidney was not changed by oral MPE treatment for 30 days. Cardiac status and function were assessed by measuring the activities of creatine kinase-MB (CK-MB), the isoenzyme specific to the heart, and lactate dehydrogenase (LDH) in the serum and heart of MPE-treated rats. Treatment caused significantly decreased serum CK-MB (p < 0.001) and LDH (p < 0.01) in rats at 200 (30% and 28%) and 400 mg/kg (24% and 21%) dosages, respectively, compared with the control (Table 3). The levels of marker enzymes were not, however, affected by MPE treatment.

3.7. Tissues Oxidative Stress Profile

The oxidative stress profile in the tissues of rats was assessed by the level/activities of antioxidant molecules/enzymes (GSH, GPx, and SOD) and the extent of biological membrane peroxidation (Figure 5). MPE treatment increased GSH levels in hepatic tissue at 200 mg/kg (43%) and 400 mg/kg (27%) dosages, whereas renal and cardiac levels were not affected. MPE also caused a significant increased (p < 0.001) in activity of the antioxidant enzyme SOD in the liver (200 mg/kg, 60%; 400 mg/kg, 52%), but the increase in the heart (4-fold) was only significant (p < 0.05) in 100 mg/kg-treated rats. Similarly, the activity of GPx was increased in the liver (198%) and heart (66%) of rats subjected to the highest dosage (400 mg/kg) of MPE.

4. Discussion

4.1. Phytochemical Profile of Plantain Peel Extract

The highly diverse plant secondary metabolites are the components of medicinal plants that are relevant to phytomedicine. GC-FID analysis revealed a plethora of phytochemicals, including polyphenols, alkaloids, and glycosides, in the crude ethanol extract of unripe M. paradisiaca peel [24], in line with the preliminary phytochemical screening. Biologically active polyphenol phytochemicals, including flavonoids and phenolic acids, possess powerful antioxidant activities to which the therapeutic actions of some medicinal plants are ascribed [25]. In the present study, plantain peel was found to contain a considerably high amount of phenolic components, in line with the work of Boua, et al. [26] who reported higher phenolic contents in the peel compared with the pulp. The phenolic and flavonoid contents were found to be comparable to or higher than those of the plantain pulp and flour [27,28], ripened banana peel [29] or whole banana [30] extracts. Plant extracts usually show a strong correlation between phenolic contents and total antioxidant capacity in vitro, owing to the antioxidant potential of polyphenols and related phytochemicals [25,31]. These bioactive, functional components are often retained in food byproducts like the plantain peel and can be incorporated into human diets or formulations to promote antioxidant defense and metabolic health [32].

4.2. In Vitro Antioxidant and Glycemic Regulatory Activities

In the present study, the FRAP assay revealed higher antioxidant potential in plantain peel than banana flour [33]. Going by folkloric claims on the use of some parts of the plantain plant in the management of blood glucose, the effect of unripe plantain peel extract on the activity of α-amylase and α-glucosidase, two enzymes relevant to diabetes pathophysiology, was evaluated in vitro. α-amylase in the saliva and pancreatic juice facilitates glucose absorption by breaking down large insoluble starch molecules into simpler, absorbable ones, while α-glucosidase in the mucosal brush border of the small intestine facilitates the digestion of dietary disaccharides into the corresponding monosaccharides to encourage absorption [34]. Substances capable of inhibiting the two enzymes could thus be effective at lowering postprandial hyperglycemia since glucose uptake in the small intestine is delayed/inhibited [35]. The inhibition of α-glucosidase and α-amylase in vitro by the peel extract may contribute to its potential for modulating glucose metabolism [36,37], especially since this was accompanied by a modest reduction in glucose levels in rats. Biologically active compounds in the peel, especially phenolic compounds (including apigenin and quercetin), which are known to be strong inhibitors of α-amylase and α-glucosidase, are likely contributors to the observed effects on the carbohydrate-metabolizing enzymes [38]. Phenolics have been touted as potential alternatives to synthetic inhibitors, which possess harsher side effects like flatulence and abdominal distention [38,39,40]. Although exploratory, the demonstrated enzyme-inhibitory activity of the extract suggests possible dietary applicability in moderating postprandial glucose spikes if consumed as part of a meal or developed into functional food formulations.

4.3. In Vivo Toxicological Evaluation

Conducting a toxicity evaluation of highly unexplored plant products, such as the unripe plantain peel, is appropriate. Such an evaluation would ascertain a safe dose margin, lethal clinical signs, or selective toxicity to specific tissues and allow informed decisions on use [41]. From a translational perspective, it provides an essential foundation for considering plantain peel as a source of food-derived bioactive candidates for long-term dietary use or formulation into functional foods [42].

4.3.1. Effects on Body and Organ Weights

Weights and morphometric parameters of the major organs (liver, heart, and kidney) could serve as sensitive indicators of general health status or assist in the detection of possible organ defects or diseases [43]. Unripe plantain peel extract did not affect the organ–body weight ratios of experimental animals, possibly suggesting that normal metabolism and growth might not be affected at the evaluated dosages and within the treatment duration [44].

4.3.2. Impact on Lipid Profile and Cardiac Biomarkers

Lipid profile indices are valuable tools for toxicological evaluation of the cardiovascular system and overall health [15,45]. For instance, atherosclerotic progression is underpinned by elevated LDL and lower concentrations of functional HDL [45]. Except for the reduction in serum HDL at 400 mg/kg b.w., the ethanol extract from plantain peels did not alter the lipid profile in rats after 30 days of oral administration. The modest reduction in HDL-c observed at the highest evaluated dosage of 400 mg/kg suggests a possible dose-dependent response and should be interpreted with caution in light of the overall cardiometabolic safety. These biphasic responses to plant bioactives are thought to reflect complex effects on liver lipid metabolism that involve pathways like HMG-CoA reductase and AMPK signaling [46,47]. As observed in the present case, reduced doses may provide metabolic advantages, while increased exposure could disrupt lipid homeostasis. It is expedient for future research to assess lipid endpoints over a broader dose range and include longer follow-up to define the nutritional risk–benefit balance better.

The activities of CK-MB and LDH were assessed in the serum and cardiac tissues of rats to correlate events at the cellular level. The two enzymes are normally present in the cardiac tissue but are released from the cells into the blood upon serious injury. Both have been used to quantify the extent and degree of injury to the myocardium [15,48]. In the present study, CK-MB and LDH activities in cardiac tissues were maintained, whereas blood levels of the enzymes decreased in a dose-dependent manner after MPE administration. Since elevated serum levels of these enzymes normally indicate cardiac injury, the observed decrease implies that the extract did not cause myocardial damage but may rather exert a protective effect on cardiac tissues [15,48]. Taken together, the peel might not be toxic to the heart or predispose to cardiovascular-related disorders [15,49].

4.3.3. Hepatic and Renal Function Indices

The important role played by the kidney and liver in drug metabolism makes them vital for the assessment of drug toxicity [50,51]. While the liver is involved in the metabolism and detoxification of harmful xenobiotics, the kidney is involved in the removal of those waste products. In the present study, biochemical indices related to the functions of the two organs were evaluated. ALT and AST are related to the cellular integrity of the hepatocytes [52], while albumin, globulin, and bilirubin could be related to both hepatocellular and secretory functions of the liver [44]. Serum activities of AST and ALT were decreased but largely remained within physiological range [53], whereas other parameters were not affected by MPE treatment. The decrease in serum ALT and AST without corresponding tissue changes in the marker enzymes should be interpreted conservatively. This may reflect disparities between circulating enzyme activity and hepatic enzyme content since serum levels are influenced by enzyme release and clearance rather than tissue concentration [54].

Creatinine, uric acid, and urea are important biochemical indices for the assessment of renal functions [55]. A serum increase in creatinine without a concomitant muscle mass increase could indicate compromised nephron function and kidney damage [56]. In cases of renal injury or disease, the kidney may retain urea, which it normally transports and excretes. In the same vein, accumulation of uric acid, a by-product of purine nucleotide metabolism, in the blood could result from a compromised ability of the kidney to clear waste products [55,56]. In line with the submissions of [44], subacute administration of MPE did not cause significant alterations in the evaluated renal parameters, as serum levels of kidney function markers remained unchanged and no evidence of organ retention was observed, even at higher dosages.

4.3.4. Oxidative Stress and Antioxidant Status

Toxic responses could alter the critical redox balances and result in oxidative stress, OS (detectable by an increase in thiobarbituric acid reactive species, TBARS), and its attendant pathological consequences. The maintenance of redox homeostasis is the function of both enzymatic (like SOD, GPX, and GST) and non-enzymatic (like GSH) antioxidants [15,57], which are the antidotes to the potential damages caused by OS-causing free radicals [15]. In the present study, MPE maintains lipid peroxidation at baseline levels in tissues because the TBARS contents, produced as a result of various free-radical-driven propagations of oxidative insult to polyunsaturated fatty acids (PUFAs), remained at baseline levels [57]. Concomitantly, GSH contents and SOD and GPx activities were not depleted (but rather increased in some organs like the liver and heart) in the tissues. Taken together, it could be submitted that the subacute treatment did not cause oxidative toxicity but rather boosted the antioxidant profile in hepatic and extrahepatic tissues [15]. The observed boosts in antioxidant indices (GSH, SOD, GPx) are consistent with the presence of polyphenolic compounds such as gallic acid, rutin, and myricetin in Musa paradisiaca peel [25]. The findings here are associative to a large extent, and studies with purified compounds and pathway-specific assays are needed to confirm the underlying mechanisms.

4.3.5. Limitation and Translational Relevance

Although the present study shows the safety and modest hypoglycemic potential of unripe plantain peel extract in healthy rats, additional research in diabetic models is needed. The carbohydrate metabolizing enzyme-inhibitory and blood glucose-lowering effects of MPE indicate potential relevance for human dietary interventions. There is a need for future work to explore physiologically relevant intake levels and dietary incorporation to better inform translational applications in human dietetics and functional nutrition. The results in this study provide a useful basis for future studies aimed at translating the findings into clinically relevant contexts.

5. Conclusions

MPE demonstrates inhibitory activity against α-amylase and α-glucosidase, which may contribute, at least in part, to its observed effect on blood glucose. Subacute oral administration of MPE up to 400 mg/kg b.w. appears safe in rats, since it does not induce any significant biochemical alterations. As an initial exploratory study, these results provide foundational evidence rather than definitive proof of efficacy. Further studies integrating histopathology, hematology, and chronic toxicity are desirable to thoroughly ascertain the safety and therapeutic potential of the peel in animal models and humans.