In Vitro α-Amylase and α-Glucosidase Inhibitory Potential of Green Banana Powder Extracts

This study investigated the proximate composition and inhibitory potential of hot water and ethanolic extracts of the pulp, peel and whole fruit of green banana (Musa sapientum) on α-amylase and α-glucosidase. Bioactive compounds were identified using GC-MS analysis. In addition, the cytotoxic effect on human gingival fibroblast (hGF) was evaluated using the sulphorhodamine B (SRB) assay. The results showed that the peel of green banana had the highest amount of ash (10.05%), fat (2.83%), protein (3.64%) and total dietary fibre (36.62%). The carbohydrate content of the whole fruit (81.79%) and pulp (81.50%) was higher than that of the peel (71.90%). The moisture content of the pulp (13.08%) was higher than that of the peel (11.58%) and whole fruit (11.30%). The ethanolic green banana peel extract showed a good inhibitory effect of α-amylase and α-glucosidase with the concentration necessary for 50% inhibition (IC50) of 0.512 and 0.100 mg·mL−1, respectively. The α-glucosidase inhibitory effect of the ethanolic green banana peel extract and the hot water green banana peel extract was not significantly different from that of acarbose (IC50 0.108 mg·mL−1). GC-MS analysis of the ethanolic green banana peel extract revealed fatty acids and fatty acid ester (9-octadecenamide (Z), octadecanamide and other compounds). The ethanolic peel extract exhibits a significant noncytotoxicity effect on hGF cells at concentrations ranging from 0.0001 to 1.0 mg·mL−1.


Introduction
According to the International Diabetes Federation (IDF), there are more than 530 million diabetic patients globally.Tis number is increasing rapidly since diabetes mellitus (DM) is one of the most prevalent metabolic disorders worldwide.Te condition is closely linked to chronic hyperglycaemia, and it is anticipated that the number of cases will exceed 640 million by 2030, a number that could be more than 780 million by 2045 [1].DM usually involves inadequate secretion of insulin caused by damage to the β cells that produce insulin in the pancreas.Hyperglycaemia follows, which in turn can cause severe problems in the long term, including organ failure in some cases, typically affecting the nerves, kidneys, eyes and heart [2].Te condition has been linked to lifestyle factors such as obesity and a lack of exercise, as well as oxidative stress.DM can be addressed initially through exercise programmes and a healthy eating plan, while subsequent treatments include insulin or drugs designed to reduce the blood sugar levels of patients [3].
One of the therapeutic approaches in diabetes treatment is based on the inhibition of α-amylase and α-glucosidase activity, since these enzymes are responsible for digesting and absorbing carbohydrates.Type 2 diabetes can be treated by the use of miglitol, acarbose and voglibose, which are α-glucosidase inhibitors.Te problem, however, is that hypoglycaemic drugs can lead to adverse side efects such as diarrhoea, fatulence, liver disorders and abdominal cramping [4].As a consequence, natural sources of hypoglycaemic agents have become an important area of research; the aim is to avoid the undesirable side efects of the drugs currently used to treat diabetes as well as to reduce the costs involved.
One of the most widely grown fruits in the world is the banana, which can be found in more than a thousand diferent varieties.While production is high, more than 30% of the total crop yield typically goes to waste because people have a strong preference for only fully ripened bananas [5,6].Consequently, the commercial processing of bananas has been developed and adjusted to minimise this waste, and further research into this issue is ongoing.Waste can be utilised in the form of green banana powder (GBP), which can be obtained from bananas in Stages 1 and 2 on the Von Loesecke maturation scale [7].Tese bananas are green, or green with a hint of yellow.GBP is becoming increasingly popular for human consumption since it ofers notable benefts in terms of nutritional value and physiological support.It is an excellent source of vitamin C, vitamin B6 and provitamin A and is also high in minerals such as zinc, magnesium, potassium and phosphorus [5,8].In addition, it contains resistant starch, commonly used in the food sector since it ofers health benefts through the promotion of microbiota in the human gut [9].As a result, it is used as an ingredient in gluten-and wheat-free food products [10].
As it is rich in numerous bioactive components, GBP is a promising and attractive target for antidiabetes studies.GBP exhibits multifaceted potential by preventing oxidative damage to the liver and kidneys, enhancing the biochemical parameters in type 1 diabetic rats [11], reducing body weight and improving insulin sensitivity in obese type 2 diabetics [12].Furthermore, it shows promise in ameliorating type 2 diabetes and associated cardiovascular risks [13].However, the nutritional composition of GBP depends on the soil, climate, banana variety, mutation stage, local production and other factors [5].Te study of various parts of plants has contributed signifcantly to the discovery of numerous biologically active drugs [14].Various components of bananas, including the peel, pulp and whole fruit, exhibit dissimilar percentages of phenolic compounds and favour components [15].It is widely recognised that various solvents have the capacity to extract distinct phytochemical groups depending on their polarity, consequently leading to variations in the biological activity of the extracts [16].Hence, the objective of the present study was to investigate the proximate composition and inhibitory potential of hot water and ethanolic extracts of the pulp, peel and whole fruit of green banana (Musa sapientum) on α-amylase and α-glucosidase.In addition, the bioactive compounds can be identifed using gas chromatograph interfaced with a mass spectrometer (GC-MS) analysis.

Plant Material.
Green bananas (Musa sapientum; Musa ABB cv.Kluai "Namwa") at Maturation Stage 1 were collected in May 2022 from the Chu-Jit banana cultivating farm, Nong Yai To Subarea, Chai Badan Area, Lopburi Province, Tailand.Bananas were identifed by the Forest Herbarium, Department of National Park, Wildlife and Plant Conservation, Bangkok, Tailand.

Preparation of GBP.
Green bananas were washed with water and then separated into three portions, including the pulp, peel and whole fruit.Te samples were cut into small pieces and dried immediately at 50 °C for 24 h using a hot-air oven.Subsequently, the dried samples were ground to a fne powder using a mechanical blender.

Determination of α-Amylase Inhibition.
Te α-amylase inhibition was determined by assay, as previously reported [19].Porcine pancreatic α-amylase was prepared using 0.5 mg•mL −1 in a 20 mM sodium phosphate bufer (pH 6.9).Te preincubation mixture consisted of 50 μL of α-amylase and 50 μL of GBP extract.Te mixture was incubated at 25 °C for 10 min.Tereafter, 50 μL of 0.5% starch solution in 20 mM sodium phosphate bufer (pH 6.9) was added and incubated at 25 °C for 10 min.Te reaction was stopped by adding 50 μL of 96 mM 3,5dinitrosalicylic acid and incubating it in a boiling water bath for 10 min.Te absorbance of the solution was then measured spectrophotometrically at 540 nm (VICTOR Nivo, PerkinElmer, Massachusetts, USA).Subsequently, a control was prepared using the same procedure except that the extract was replaced with a bufer.Acarbose was used as a positive control.Te extent of inhibition by the addition of samples was expressed as the concentration necessary for 50% inhibition (IC 50 ).Te percentages of α-amylase inhibition were expressed as 2 Te Scientifc World Journal 2.6.Determination of α-Glucosidase Inhibition.Te α-glucosidase inhibition was determined by assay, as previously reported [19,20].α-Glucosidase from a Saccharomyces cerevisiae solution was prepared using 1 U•mL −1 in a 20 mM sodium phosphate bufer (pH 6.9).Te preincubation mixture consisted of 50 μL of α-glucosidase and 25 μL of GBP extract.Te mixture was incubated at 25 °C for 10 min.Tereafter, 25 μL of 5 mM pnitrophenyl-α-D-glucopyranoside solution in 20 mM sodium phosphate bufer (pH 6.9) was added and incubated at 25 °C for 5 min.Te absorbance was measured at 405 nm using a spectrophotometer (VICTOR Nivo, PerkinElmer, Massachusetts, USA).Subsequently, a control was prepared using the same procedure except that the extract was replaced with a bufer.Acarbose was used as a positive control.Te extent of inhibition by the addition of samples was expressed as the concentration necessary for IC 50 .Te percentages of α-glucosidase inhibition were expressed as

GC-MS Analysis.
In this study, gas chromatography and mass spectrometry were used to identify the phytochemical compounds present in the ethanol extract of green banana peel.GC-MS analysis was carried out on a GC Shimadzu QP2020 and GC-MS equipped with a SH-Rxi-5Sil MS capillary column (30 m × 0.25 mm ID × 0.25 μm flm thickness, Shimadzu, Japan).Helium (99.99%) was the carrier gas at a constant fow rate of 3 mL•min −1 .Te oven temperature programme was 50 °C (isothermal for 2 min), with an increase of 10 °C•min −1 to 280 °C for 3 min.Te total GC-MS running time was 28 min.Te relative percentage of each component was evaluated by comparing its average peak area to the total area.

Determination of Cytotoxic Activity.
For the sulphorhodamine B (SRB) assay, human gingival fbroblast (hGF) cells were seeded onto 96-well plates and treated with diferent concentrations of the GBP peel extract at 0.0001, 0.001, 0.01, 0.1 and 1.0 mg•mL −1 for 48 h.Cells were fxed by adding 100 μL of cold 10% trichloroacetic acid for 1 h at 4 °C.Tereafter, the supernatant was removed, and cells were rinsed four times with slow-running tap water.Te plate was allowed to air-dry and stained with 100 μL of 0.057% SRB solution at room temperature for 30 min.Subsequently, the wells were washed four times with 1% (v/v) acetic acid to remove unbound dye.Te bound SRB was dissolved by 100 μL of 10 μM Tris, pH 10.5, for 10 min.Te absorbance was measured at 510 nm (VICTOR Nivo, PerkinElmer, Massachusetts, USA).Cells treated with a culture medium served as the negative control, while those treated with sodium lauryl sulphate (SLS) were the positive control [21].

Statistical Analysis.
Comparison of the means of three replicates was carried out for mean ± standard deviation to establish whether there were diferences between the activities of samples, and the variance analysis was applied to the result.Values of p ≤ 0.05 were considered signifcantly diferent (α = 0.05).

Results and Discussion
3.1.Proximate Composition.Te ash, moisture, carbohydrate, fat, protein and fbre contents in diferent parts of GBP in g/100 g dry weight are shown in Table 1.GBP peel had the highest amount of ash (10.05%), fat (2.83%), protein (3.64%) and total dietary fbre (36.62%).Te carbohydrate content of GBP pulp (81.50%) and whole fruit (81.79%) did not reveal a signifcant diference, though it was higher than that in the peel (71.90%) with a signifcant diference.Te moisture content of the peel (11.58%) and whole fruit (11.30%) was less than that of the pulp (13.08%) with a statistically signifcant diference.Te results revealed that GBP is a good source of healthy minerals, nutrients and dietary fbre.Ash content in foods refers to the amount of minerals in food [18].Tis indicates that GBP peel is rich in minerals.Fat, protein and carbohydrate contents provide energy for the body.Te energy value of GBP was ranked in the order of whole fruit (345.01 kcal) > pulp (339.12 kcal) > peel (327.63 kcal).Variances in protein and fat contents may be due to geographical location and varietal diferences [5,18].Total dietary fbre has various health benefts, including the prevention of heart disease, hypertension, diabetes, obesity and gastrointestinal diseases [22].Te total dietary fbre content in GBP peel was found to be approximately three times higher than that in the pulp and whole fruit.Moisture content in food products infuences their shelf life.Te moisture in four should be at an acceptable limit of not more than 10% for long-term storage [23].Te moisture content of GBP pulp, peel and whole fruit was found to be from 11.30 to 13.08%, which is not suitable for storage stability and longer shelf life.2. Te results revealed that the α-amylase inhibition of GBP extracts increases slightly as the concentration increases.Acarbose had the highest inhibitory α-amylase activity at all concentrations.At 2.0 mg•mL −1 , the pulp, peel and whole fruit of hot water extract did not show a signifcant diference in the α-amylase inhibition (Table 2).However, there were signifcant diferences in the inhibition rates of ethanol extracts (Table 3).Te α-amylase activity of the GBP extracts is summarised in Table 4.All GBP extracts had a higher value of IC 50 than acarbose as the positive control.IC 50 values of extracts for α-amylase activity ranged from 0.512 to 1.615 mg•mL −1 , while the IC 50 value of acarbose was 0.219 mg•mL −1 .Previous reports have shown that severe inhibition of pancreatic α-amylase may lead to aberrant bacterial fermentation of undigested starch in the Te Scientifc World Journal colon.Tus, modest α-amylase inhibitory action appears benefcial [24].

Inhibition of α-Glucosidase.
Te results of α-glucosidase inhibition for hot water and ethanolic extracts of diferent parts of GBP showed that 0.05 to 0.5 mg•mL −1 concentrations of pulp, peel and whole fruit extracts were signifcantly diferent among the α-glucosidase inhibition values for all extracts (Tables 5 and 6).All extracts showed the concentration-dependent inhibition of enzyme activity.At 1 mg•mL −1 of peel with hot water (81.84 mg•mL −1 ) and ethanolic (87.48 mg•mL −1 ) extracts, there was no signifcant diference in the α-glucosidase inhibition when compared to acarbose (83.94 mg•mL −1 ).All peel extracts showed the most increasing rate of inhibition ranging from 29.50 to 87.48 mg•mL −1 with signifcant diferences.Meanwhile, the hot water extract of whole fruit showed the highest inhibition than others but was not signifcantly diferent from acarbose at a concentration of 0.05 mg•mL −1 .However, pulp with hot water and ethanolic extracts showed the lowest α-glucosidase inhibition with a signifcant diference.When increasing the concentration of extracts, the results revealed that the percentage of α-glucosidase inhibition increased in all extracts.Inhibition of α-glucosidase activity was calculated with the IC 50 values (Table 4).Most extracts had a higher value of IC 50 than acarbose.In addition, the IC 50 values of GBP peel with hot water and ethanolic extracts were determined to be 0.164 and 0.100 mg•mL −1 , respectively.Te inhibitory efect of the ethanolic peel extract was not signifcantly diferent from that of acarbose (IC 50 0.108 mg•mL −1 ).From these results, the ethanolic peel extract exhibited the potential inhibition of α-glucosidase activity.Moreover, the α-glucosidase inhibition of the hot water peel extract (0.164 mg•mL −1 ) suggested that there were no signifcant diferences between ethanolic peel, hot water peel extracts and acarbose.Natural inhibitors derived from Note: Each value is presented as mean ± SD (n � 3).Mean with diferent letters (a-c) shows statistically signifcant diferences (p ≤ 0.05).

GBP extract concentration (mg•mL
4 Te Scientifc World Journal dietary plants are benefcial because they have a reduced inhibitory activity against α-amylase and a higher inhibitory activity against α-glucosidase [25].Moreover, they can be utilised for the safe and efcient treatment of postprandial hyperglycaemia.

GC-MS Analysis.
Te ethanol extract of peel was subjected to GC-MS analysis to identify its phytochemical constituents.GC-MS analysis showed the presence of fourteen compounds in the extract (Table 7).Te chromatographic analysis indicated the presence of numerous components in the ethanol peel extract that might be involved in biological processes.Te maximum peak was shown by 9-octadecenamide (Z), followed by octadecanamide, hexadecanamide and 2,4-di-tert-butylphenol.Several of the compounds contained in the peel extract have already been demonstrated to possess antioxidant, antibacterial, hypocholesterolemic and antidiabetic characteristics [26,27].Te compound 1-undecanol has previously been researched for its bactericidal and membrane-damaging properties [28].Several publications have underlined the antioxidant and anti-infammatory properties of 2,4-di-tertbutylphenol.Most signifcantly, the phenol displayed a wide range of toxicity in all tested species, including the producers, comprising cytotoxicity in human cells and animals, insecticidal and nematicidal activity, antibacterial activity and phytotoxicity [29].1-Hexadecanol is a 16-C fatty alcohol that can protect the skin against irritations triggered by insect bites, rashes and stings.It has been found that 1hexadecanol inhibits the development of Mycoplasma gallisepticum and Mycoplasma pneumoniae [30], while 1nonadecene has antitubercular, anticancer, antioxidant and antibacterial properties [28,31].n-Hexadecanoic acid demonstrates potential pharmacological activities including antioxidant, anti-infammatory, anticancer, antitumour, hypocholesterolemic, nematicide and pesticide [32][33][34][35].Decanoic acid has been demonstrated to possess substantial antibacterial and antifungal properties, and its esters have been employed in the medicinal, nutritional and dietetic felds [36].Butyrate and its derivatives have the potential to serve as efective antibacterial and immunomodulatory therapeutics in the treatment of bacterial infections [37].
Hexadecanamide is a saturated fatty acid amide produced from palmitic acid.It has been investigated for potential therapeutic applications, such as its capacity to modulate infammation, decrease oxidative stress and enhance cognitive performance [38].Moreover, 9-octadecenamide (Z) is abundant in natural volatile oil with strong antioxidant, antibacterial, anti-infammatory, antifungal and hypolipidaemic properties [39,40].
3.5.Cytotoxic Activity.hGFs are accountable for a substantial portion of the extracellular matrix production in connective tissue and signifcantly impact wound healing [21].GBP could be considered a functional food and has fewer side efects in comparison with conventional drugs, ofering a safer alternative for the management of diabetes [5,22].Terefore, it is essential to verify the safety and efectiveness of GBP before their utilisation.In this respect, the cytotoxic potential of the GBP peel extract was assessed on hGF cells in comparison with SLS as a positive control.Te results are presented in Table 8. hGF cells were treated with diferent concentrations of ethanolic peel extract (0.0001 to 1 mg•mL −1 ) to determine cell survival.Te results showed that cell survival was 50% at 0.086 mg•mL −1 when cells were treated with SLS.However, when increasing the concentration of the peel extract to 1.0 mg/mL, hGF cells survived (87.62 mg•mL −1 ).Te results indicated that the ethanolic GBP peel extract exerts cytotoxic efects on hGF cells with lower potency compared to SLS.Te Scientifc World Journal

Conclusion
GBP has the potential to serve as a signifcant component in health food products.GBP peel is a good source of minerals and fbre.Further, it efectively inhibits carbohydrate-hydrolysing enzymes.Te ethanolic peel extract displayed the highest potency in inhibiting both α-amylase and α-glucosidase activities, suggesting that the solvent has the capability to extract the active constituents present in GBP.GC-MS analysis indicated the presence of numerous components in the ethanolic peel extract that might be involved in biological processes.GBP peel appears to be a promising candidate that might be benefcial for various applications in plant-based superfoods, nutraceuticals and alternative medicine.Note: Each value is presented as mean ± SD (n � 4).Mean within column with diferent letters (a-b) shows statistically signifcant diferences (p ≤ 0.05). 6 Te Scientifc World Journal

Table 1 :
Proximate composition of GBP.

Table 7 :
Chemical profle of the ethanolic GBP peel extract by GC-MS.

Table 8 :
Percentage of survival of human gingival fbroblast cells treated with sulphorhodamine B (SRB).