INTRODUCTIONThe World Health Organization reported that by2035 the number of people with diabetes, a major causeof mortality worldwide, will account for 471 million [1].Cheap snacks and products with high energy content arerisk factors in diabetes development [2, 3]. One of themost popular high energy snacks is ice cream, whichmainly contains milk or cream and sugar. Ice cream isa homogenized mixture of milk, flavorings, colorings,and stabilizers frozen at the temperature that is lowerthan the freezing point to avoid the formation of large icecrystals.There are many varieties of ice cream, but generallyice cream contains 10% of milk fat, less than 10%of non-milk fat (caseins, whey proteins, lactose),13–20% of sweeteners, 0.1–0.7% of stabilizers andemulsifiers, and about 64% of water [4]. Ice creamhas become a popular product due to its coolingproperties and the enormous amount of energy itsupplies. However, a high amount of carbohydratesfats in ice cream can increase bad cholesteroldeposition around the belly and have become oneof the leading causes of obesity and such diseasesas diabetes, atherosclerosis, and hypertension [5].All these diseases caused by ice cream consumptionhave been found to result from excess energy deposition,which is a central factor to hyperglycemia. Despite ahigh demand for ice cream, there has been little effort toimprove its nutritional and medicinal properties. Hence,there is a need to develop functional ice cream withoutthe mentioned disadvantages which would treat a widearray of metabolic diseases.Herbs are widely available, effective, safe, andacceptable raw materials which can be used asfunctional plants in the food industry [6]. Various typesof plants that have been used in the treatment of heart related diseases have shown promising therapeuticpotential. Soursop (Annona muricata L.) is a tropicalplant popular in ethnomedicine due to its antioxidantproperties [7]. Soursop is rich in phytochemicals suchas flavonoids, phenolic acids, phytosterols, saponins,and cardiac glycosides [7, 8]. Moringa oleifera L.(Moringaceae family) is a fast growing plant ofeconomic and medical importance widely distributedin Africa, America, and Asia [9–11]. Some of thephytochemicals present in moringa leaf, which havemedicinal potential, are mainly natural antioxidantssuch as flavonoids, carotenoids, vitamins, and phenolicacids [12–17].Therefore, this study aimed to produce soursopbasedice cream enriched with moringa leaf powder andthen assess its antioxidant properties, glycemic index,amylose and amylopectin contents, as well as α-amylaseand α-glucosidase inhibitory properties.STUDY OBJECTS AND METHODSSoursop (Anona muricata L.) and moringa (Moringaoleifera L.) leaves were collected from the botanicalgarden at the Federal University of Technology, Akure.The moringa leaves were washed, air dried, and finelypowdered using a stainless steel blender. The powderedsamples were kept dry at room temperature for furtheranalysis.The soursop was peeled and seeds were separatedfrom the pulp. Whipping cream (600 g) was stirred for15 min using a mixer. Thereafter, 600 g of the soursoppulp was mixed together with the whipping creamfor another 15 min. The mixture was divided intothree parts and frozen (Fig. 1). This produced threeexperimental samples of soursop-based ice cream: withno moringa, with 0.1 g of moringa, and with 1 g ofmoringa. Ice cream purchased at a local store served ascontrol.Sensory analyses were conducted in well illuminatedodorless laboratory booths. Water was provided formouth rinsing in between successive evaluation. Sampleattributes (color, texture, taste, aroma, etc.) were ratedfrom 1 to 7, where 1 = very poor and 7 = excellent.Panelists made their responses on score sheets whichwere designed in line with the test procedures [18].The total phenol content was determined accordingto the method reported by Singleton et al. and calculatedas gallic acid equivalent (GAE) [19].The total flavonoid content was determinedusing a slightly modified method reported by Medaet al. [20]. The absorbance of the reaction mixturewas subsequently measured at 415 nm, and the totalflavonoid content was subsequently calculated.DPPH free radical scavenging ability was evaluatedas described by Gyamfi et al. [21]. Ice cream samples(0.05 mL) were incubated in the dark for 30 min with1 mL of 0.4 mM DPPH after thorough mixing. Theabsorbance was measured at 516 nm, and the radicalscavenging ability was subsequently calculated aspercentage of the control.ABTS radical scavenging ability was determinedaccording to the method described by Re et al. [22].The radicals were generated by adding 7 mmol/L ABTSaqueous solution to a reaction mixture containing2.45 mmol/L K2S2O8, keeping in the dark for 16 h,and adjusting the absorbance to 0.700 with ethanol at734 nm. 0.2 mL of appropriate dilution of the ice cream samples was added to 2.0 mL of ABTS solutionand absorbance was measured at 734 nm after 15 min.The radical scavenging ability and Trolox equivalentantioxidant capacity were subsequently calculated.Ferric reducing antioxidant property of the sampleswas determined by assessing its ability to reduce FeCl3solution as described by Pulido et al. [23]. The reducingproperty was subsequently calculated using ascorbicacid equivalent.Hydroxyl radical scavenging ability was determinedusing the method of Halliwell and Gutteridge [24]. Thereaction mixture contained 1–100 μL of the ice creamsamples, 400 μL of 0.1 M phosphate buffer, 120 μL of 20mM deoxyribose, 40 μL of 20 mM hydrogen, and 40 μLof 500 M FeSO4. The mixture was incubated at 37°C for30 min. Thereafter, 0.5 mL of 2.8% TCA (trichloroaceticacid) and 0.4 mL of 0.6% TBA (thiobarbituric acid)solution were added. The tubes were subsequentlyincubated in boiling water for 20 min. The absorbancewas measured at 532 nm using a spectrophotometer.α-amylase activity assay. The reaction mixturecontained the sample dilution (500 μL) and 0.5 mg/mLα-amylase in 500 μL of 0.02 M sodium phosphate buffer(pH 6.9 with 0.006 M NaCl). The mixture was incubatedfor 10 min at 25°C. 500 μL of a 1% starch solution in0.02 M sodium phosphate buffer (pH 6.9 with0.006 M NaCl) was then added to the reactionmixture and incubated for another 10 min at 25°C.Dinitrosalicylic acid (DNSA) was used to stopthe reaction before incubating for 5 min at roomtemperature. Absorbance was measured at 540 nm, andthe percentage enzyme inhibitory was calculated [25].The α-glucosidase inhibitory activity wasdetermined by the method of Apostolidis et al. [26].The reaction mixture contained 100 μL of α-glucosidasesolution (EC 3.2.1.20; 1.0 U/mL) in 0.1 M phosphatebuffer (pH 6.9). Ice cream samples (50 μL each) wereput in the mixture and incubated at 25°C for 10 min.50 μL of 5 mM pnitrophenyl-α-D-glucopyranosidesolution was added, and the reaction mixture wasincubated for 5 min at 25°C. The absorbance was read at405 nm.Glycemic index and starch hydrolysis rate in vitrowere determined according to the method of Goniet al. [27]. Each ice cream sample (50 mg) was incubatedwith pepsin (1 mg) in 10 mL of HCl-KCl buffer(pH 1.5) at 40°C for 60 min. 2.5 mL of phosphate buffer(pH 6.9) and 5 mL of α-amylase solution were added tothe reaction mixture. The mixture was incubated at 37°Cin a shaking water bath. To activate the enzyme, wewere taking 0.1 mL of the mixtures every 30 min duringthree hours and boiled. The residual starch was digestedto glucose by the addition of 3 mL of α-glucosidase andincubated at 60°C for 45 min. The glucose concentrationwas assayed by the addition of 200 mL of DNSA. Afterstopping the reaction by boiling, 5 mL of distilled waterwas added and absorbance read at 540 nm.To determine amylose-amylopectin content, 1 mL of95% ethanol and 9 mL of 1 N NaOH were added to involumetric flasks containing 100 mg of each ice creamsample. Thereafter, the reaction mixture was heated inboiling water for 10 min. 1 mL of 1 N acetic acid and2 mL of iodine solution were added to 5 mL portion ofthe solution. After thorough shaking, the absorbance wasmeasured at 620 nm. Amylopectin content was derivedfrom the difference between the starch and amylosecontents [28, 29].Statistical analysis. The results were expressedas mean ± standard deviation (SD). One-way analysisof variance (ANOVA) was used to analyze the resultsfollowed by Turkey’s post hoc test, with levels ofsignificance accepted at P < 0.05.RESULTS AND DISCUSSIONThe results of the sensory evaluation of the control(commercial ice cream) and experimental (soursopbasedice cream enriched with moringa leaf powder)samples are presented in Table 1. The control icecream had higher overall acceptability compared to thesoursop-based ice cream samples. The experimentalsamples had no significant differences in their overallacceptability.Aroma, taste, color, flavor, texture, and generalacceptability of food have a significant effect on itssensory quality, which is one of the major criteriain food selection by consumers [30]. The overallacceptability and aroma of the soursop-based ice creamwas not significantly different. However, moringaleaf powder reduced such attributes as texture, taste,and color. The ice cream samples with moringademonstrated reduced acceptability, which could be dueto a peculiar taste of moringa leaf powder (no sugar inthe formulation).The soursop-based ice cream had a high amount ofphenolic and flavonoid content compared to the controlTable 1 Sensory attributes of soursop-based ice cream enriched with moringa leaf powderIce cream Texture Taste Color Aroma Overall acceptabilityCommercial ice cream (control) 6.09 ± 0.04a 6.11 ± 0.03a 6.12 ± 0.07a 6.21 ± 0.04a 6.25 ± 0.05aSS 5.37 ± 0.08b 5.89 ± 0.04b 5.92 ± 0.11a 5.91 ± 0.04b 5.82 ± 0.03bSS + MLP (0.1 g) 5.11 ± 0.03c 5.71 ± 0.03c 5.71 ± 0.06b 5.82 ± 0.03b 5.78 ± 0.05bSS + MLP (1g) 4.95 ± 0.04d 5.28 ± 0.04d 5.05 ± 0.07c 5.79 ± 0.06b 5.79 ± 0.04bSS – soursopMLP – moringa leaf powder sample (Table 2). Our results consistent with the databy Tungmunnithum et al. who studied phenolics andflavonoids in medical plants [31]. The authors foundthat these compounds are responsible for the biologicalactivity of the plants. Phenolic compounds, especiallyflavonoids, are remarkable antioxidants which havebeen widely researched for their medicinal propertiesagainst various diseases. Phenolic compounds are goodiron chelators which scavenge free radicals, preventingoxidative stress [32]. In this study, the sample withmoringa leaf powder (1 g) showed the highest totalphenol and flavonoid content compared to the othersamples.Figure 2 demonstrates that the ice cream withmoringa leaf powder (1 g) had the highest DPPHscavenging ability at all the concentrations (100–400 mg/mL) among all the samples. Also, this ice creamsample showed the highest ABTS scavenging abilitycompared to the other samples (Fig. 3). The control icecream sample had the lowest both DHHP and ABSTscavenging activities.The highest ferric reducing antioxidant propertiesand hydroxyl radical scavenging ability belonged tothe experimental sample with moringa leaf powderin the amount of 1 g (Figs. 4 and 5). Among the othersamples, these parameters decreased from ice creamwith moringa leaf powder (0.1 g) to the control sample(without soursop and moringa powder).Reducing property of the samples was assessedbased on their ability to reduce Fe3+ to Fe2+. The resultsrevealed that the control ice cream had significantlylower reducing property compared to the soursop-basedsamples. Similarly, the ice cream with 1 g of moringaexhibited the highest hydroxyl radical scavenging abilitycompared to the other soursop-based samples, while thehydroxyl radical scavenging ability of the control icecream was comparably low.The antioxidant properties of the sour-sop based icecream samples was directly proportional to increasingmoringa leaf powder proportion (Figs. 2–5). Therefore,the antioxidant properties can be linked to phenoliccompounds that majorly present in the moringa andthe soursop. Furthermore, the ability of the samples toscavenge DPPH radical could be due to the presence ofmultiple hydroxyl groups in phenolic compounds, whichare able to donate their protons to finally break the chainreaction of the free radicals [32].ABTS is a water soluble free radical initiator that isoxidized to form a stable green radical ABTS+ in thepresence of reactive oxygen [33]. All the soursop-basedice cream samples exhibited a remarkable ABTS radicalscavenging ability, with the highest radical scavengingability in the sample containing 1 g of morings leafpowder. This could also be explained by synergisticeffects of phenolic compounds present in moringa andsoursop [34, 35]. These results prove that moringa andsoursop increased the antioxidant properties of theice cream samples due to phenolic compounds in theircompositions. The correlation between antioxidantproperties and phenolic content has been established formany food products [36].The effect of moringa and soursop on the α-amylaseand α-glucosidase inhibitory activity of the ice creamsamples are presented in Figs. 6 and 7. The samplewith 1 g of moringa leaf powder showed the strongestinhibition of α-amylase activity at the concentrationstested (50–200 mg/mL) and the highest α-glucosidaseinhibitory ability compared to the other soursop-basedsamples. The control sample demonstrated the lowestα-amylase and α-glucosidase inhibitory activities.In vitro estimated glycemic indices of the samplesare presented in Fig. 8. The results revealed that thecontrol ice cream had the highest glycemic index (61.24)compared to the other samples (27.14–28.61). Figures 6and 7 revealed that the sour-sop based ice cream samplesinhibited carbohydrate hydrolyzing enzymes.The control ice cream had the lowest amylose content(14.32%) compared to the soursop-based ice cream(32.35–35.34%) (Table 3). There was no significantdifference in the amylopectin content of the sampleswith soursop and moringa leaf powder (64.66–67.65%),while the control ice cream had the highest amylopectincontent (85.68%).A therapeutic and practical way to controlpostprandial rise of glucose level in blood is the controlof carbohydrate hydrolyzing enzymes [37]. Starchis converted to disaccharides and oligosaccharidesby pancreatic α-amylase, before further conversionto glucose is catalyzed by intestinal α-glucosidase[38, 39]. Therefore, inhibition of both α-amylaseand α-glucosidase activities would result in a reductionof glucose absorbed into the blood. The ability ofthe sour-sop based ice creams to inhibit the enzymescould be of therapeutic benefit in the management ofhyperglycemia.Interestingly, this tendency for enzyme inhibitionby the samples was similar to the tendency for totalphenolic and flavonoid contents [40]. In addition, thesynergistic contribution of phenolic compounds insoursop and moringa leaves can make ice cream a potentinhibitor of α-amylase and α-glucosidase activities.Our previous studies showed the presence of phenoliccompounds, such as gallic acid, elagic acid, rutin,quercetin, kaempferol, epicatechin and chlorogenic acid,in soursop and moringa leaves [34, 35, 41].The soursop-based ice cream samples had lowglycemic indices (Fig. 8) which can be attributed to anumber of factors. First, phenolic compounds in soursopand moringa leaves are potent inhibitors of α-amylaseand α-glucosidase activities, which results in a slowerbreakdown of starch into glucose [42]. This is furtherevidenced by the fact that moringa powder increasedphenolic content and reduced glycemic indices. Second,an amylose and amylopectin ratio in food productshave a significant effect on postprandial glucoseresponse [43]. Starchy products with a high amylopectinto amylose ratio often digest faster and are absorbedquicker than those with a low amylose to amylopectinratio and, consequently, produce a high postprandialglucose and insulin response [34]. The control ice cream used in this study possessed a low amylose content andhigh amylopectin content and, thus, the highest glycemicindex compared to the experimental ice cream samples,which had a low amylopectin content and a highamylose content.CONCLUSIONMoringa leaf powder added into soursop-based icecream improved the antioxidant properties of the finalproduct, reduced its glycemic index, and enhancedinhibition of carbohydrate hydrolyzing enzymes.Soursop-based ice cream with moringa leaf powdercan be used to control postprandial hyperglycemia andoxidative stress. The results revealed that moringaenrichedsoursop-based ice cream could be analternative to the sugar-sweetened ice-cream. However,further in vivo experiments and clinical trials arerecommended.CONFLICT OF INTERESTThe author declares no conflict of interest regardingthe publication of this article.