In Vitro Experimental Assessment of Ethanolic Extract of Moringa oleifera Leaves as an α-Amylase and α-Lipase Inhibitor

Methods Phytochemical screening, antioxidant activity, α-amylase, and α-lipase inhibitory assessment were carried out on Moringa oleifera extract. Results The result of the phytochemical screening revealed the presence of total phenolic, flavonoid, tannin, and alkaloid contents of values 0.070 ± 0.005 mg gallic acid equivalent/g, 0.180 ± 0.020 mg rutin equivalent/g, 0.042 ± 0.001 mg tannic equivalent/g, and 12.17 ± 0.001%, respectively, while the total protein analysis was 0.475 ± 0.001 mg bovine serum albumin equivalent/g. Ferric reducing antioxidant power (FRAP) and total antioxidant capacity (TAC) values were 0.534 ± 0.001 mg gallic acid equivalent/g and 0.022 ± 0.00008 mg rutin equivalent/g, respectively. Diphenyl-2-picrylhydrazyl (DPPH), ABTS (2,2′-azino-bis (ethylbenzothiazoline-6-sulfonic acid)), and nitric oxide (NO) assays showed the extract to have a strong free radical scavenging activity. The 50% inhibitory concentration (IC50) values of the lipase and amylase activities of the extract are 1.0877 mg/mL and 0.1802 mg/mL, respectively. Conclusion However, α-lipase and α-amylase inhibiting activity of M. oleifera could be related to the phytochemicals in the extract. This research validates the ethnobotanical use of M. oleifera leaves as an effective plant-based therapeutic agent for diabetes and obesity.


Introduction
Diabetes mellitus is a disorder that occurs when there is little or not enough insulin production from the pancreas. It is often regarded as having excessive blood glucose in the blood (hyperglycaemia). According to the International Diabetes Federation, a total of 387 million people were diagnosed with diabetes worldwide in 2014, with the fgure expected to rise to 592 million by 2035 [1]. Te core remedy for managing diabetes is to lower hyperglycaemia and reduce intestinal glucose absorption through the inhibition of carbohydrate metabolizing enzymes (e.g., α-amylase) [2]. Te toxicity levels and the high cost of pharmaceutical drugs designed for carbohydrate metabolizing enzyme inhibition have been a major concern [3].
Obesity, a metabolic disorder, occurs as a result of excessive accumulation of fat [4][5][6]. It is also regarded as an imbalance in energy intake and expenditure [7,8]. Further complications of obesity can result in type 2 diabetes. Pancreatic α-lipase expression is vital in developing obesity towards an advanced state [9]. Phytochemicals such as quercetin, favonoids, and polyphenols are known to inhibit pancreatic lipase. Tere is a need to adopt the use of herbs because they are readily available, minimize the side efects caused by synthetic drugs such as orlistat, and also reduce the cost of drug purchases.
Alternative medicines have been adopted for healing several ailments, whereas pharmacological treatment has provided no sustained weight loss with few or no side efects [10,11]. Terefore, a cost-efective management approach is needed to be adopted to ensure minimal side efects in diabetes and obesity treatment. Te therapeutic importance of medicinal plants has accelerated researchers' interest in the discovery of the possible activities of plants to prevent and protect against chronic diseases [11]. Phenols, a bioactive compound found in plants were reported to inhibit α-amylase and α-lipase; thus, providing it an excellent approach for type 2 diabetes and obesity management [12][13][14][15][16]. Phenolic compounds act by scavenging reactive oxygen created during metabolic reactions in humans. Tus, they are known to fght against cancer, obesity, and diabetes [17]. However, various prospective studies have shown that some compounds such as catechin, isocatechin, favonoids, favones, isofavone, and anthocyanin, have exhibit antiobesity and antidiabetic properties when comparied to β-carotene, vitamin E, and vitamin E [18] to have exhibit antiobesity and antidiabetic properties.
Moringa oleifera, an Indian tree grown in various areas of Mexico, belongs to the Moringaceae family, ranging between 5-10 m high. Te fowers, seeds, pods, and leaves of the Moringa tree have several medicinal benefts used for therapeutic purposes. For instance, the fowers have been studied to have anti-infammatory activities, the seeds exhibit antihypertensive and liver-protective properties, and the leaves reported to have antimicrobial and hypoglycaemic activities [20]. It comprises three phytocompounds: rutin, quercetin-3-glycoside, and kaempferol, which have been evaluated as a possible mechanism of glucose concentration reduction after ingestion [21][22][23]. Te leaves are mostly taken for self-medication by diabetic and hypertensive patients [24,25]. Te leaves of this plant are mostly used because of their medicinal characteristics, alongside with their hypocholesterolemic and hypoglycemic efects [26,27]. M. oleifera leaves are also rich in ascorbic acid and aid in insulin secretion [22]. Reports of in vitro and in vivo studies of diferent extracts of M. oleifera have shown them to have antidiabetic and antiobesity properties, but a detailed explanation relating the phytochemicals present in the ethanolic extract of M. oleifera to its carbohydrate and lipid metabolizing enzyme inhibiting ability is yet to be properly unveiled. Tus, this investigation was aimed at elucidating the mechanism of action and thereby validating the use of M. oleifera ethanolic extract for the prevention and treatment of diabetes and obesity.

M. oleifera Leaves' Extract Preparation.
Te selected plants' fresh leaves were air-dried, and an electric blender was used to pulverize them to obtain a fne powder. Te powdered form of M. oleifera (400 g) was steeped in 90% ethanol in the ratio 1 : 5 (v/v) for three days (72 hours) and then fltered. Te obtained fltrates were concentrated using a rotatory evaporator at 50°C to obtain the solvent-soluble fractions [27].

Total Alkaloid Content Determination.
A solution of 200 cm 3 of acetic acid (10%) diluted in ethanol was added to the sample (in powdered form, 2.5 g). Te mixture was placed in a water bath to concentrate the extract to a quarter (1/4 th ) of the original volume after 4 hours. Afterwards, it was fltered and concentrated with 15 drops of ammonium hydroxide until the end of the precipitation. Te mixture was subjected to fltration, dried, and weighed after it was left to settle for some hours [22]. Experimental analysis was carried out in triplicate (n � 3).

Total Phenolic Content
Determination. Folin-Ciocalteu (FC) reagent (1 : 10 v/v) of 2.5 mL was mixed with 0.5 mL of extract (1 mg/mL). After 5 minutes of incubation at 25°C, sodium carbonate in the amount of 2 mL (7.5%) was added. Te mixture was measured at 765 nm after incubation at 40°C for 30 minutes. Te standard used was gallic acid (0.02-0.1 mg/mL), and the extract's phenol was calculated as mg of gallic acid equivalent (mg GAE/g extract) using the standard curve [27]. Te experiment was carried out in triplicate (n = 3).

Total Flavonoid Content Determination.
Two millimetres of distilled water (dH 2 O 2 ) was added to 0.15 mL of sample (1 mg/mL), and in addition, 0.15 mL of sodium nitrite (5%) was added. Te mixture was subjected to incubation at 25°C for 6 minutes. Aluminium chloride (10%) of 0.15 mL was mixed and further incubated for 5 minutes. To make up to 5 mL, a millimetre of NaOH (4%) and 1.2 mL dH 2 O 2 were added. Afterwards, the absorbance of the mixture was measured at 420 nm. Te standard used was rutin (0.02-0.1 mg/mL) [28]. A favonoid in the extract was observed by the presence of pink colouration. Te content of favonoids was calculated as mg RE/g extract as rutin equivalent using the standard curve. Experimental analysis was carried out in triplicate (n � 3).

Total Tannin Content
Determination. An aliquot sample of 0.5 mL (1 mg/mL), dH 2 O 2 of 3.75 mL, and 0.25 mL of FC reagent (1 : 10 v/v) were mixed together. Lastly, 0.5 mL (35% sodium carbonate) was added to the mixture, and the absorbance was measured. Tannic acid within the concentration range of 0.1-0.00625 mg/mL was used as a reference standard. Te extract's total tannin content was calculated using the standard curve and reported as tannic acid equivalent mg TAE/g extract [27]. Te experimental analysis was carried out in triplicate (n = 3).

Total Saponin Content
Determination. An extract of 0.05 mL and 0.25 mL of dH 2 O 2 were mixed together. Tereafter, vanillin reagents of 0.25 mL (800 mg in 10 mL ethanol) and 2.5 mL of sulphuric acid (72%) were added and further incubated at 60°C for 10 minutes. Te absorbance was then read at 544 nm after it was cooled. Te standard used was diosgenin, and values were calculated as equivalents of diosgenin (mg DE/g extract) derived from a standard curve [29]. Experimental analysis was done in triplicate (n � 3).

Total Protein Content Determination.
Alkaline copper sulphate (AlKCuSO 4 ) of 2 mL was added to varying concentrations of 0.2 mL of sample and then proceeded for 10 minutes of incubation. A volume of 0.2 mL of FC reagent was mixed and further incubated for 30 minutes. Te absorbance of the mixture was measured at 660 nm. BSA (bovine serum albumin) was used as a standard with a varying concentration of 0.05-1 mg/mL. Te values were calculated using a standard curve and represented as BSA equivalents (mg BSAE/g extract) [30]. Te experiment analysis was carried out in triplicate (n � 3).

Diphenyl-2-Picrylhydrazyl (DPPH).
Te DPPH assay was carried out following the method of Iheagwam [27]. A standard/sample of varying concentrations of 0.5 mL was mixed together with 0.5 mL of DPPH (0.1 mM). In the dark, the mixture was set for incubation for 30 minutes. Te absorption rate was measured at 517 nm against a blank (methanol), and a DPPH solution was used as a control. Ascorbic acid and silymarin were used as standards (0.00125-0.000019) mg/mL. Radical scavenging activity with a higher value is indicated by a lower absorbance value: % inhibition of DPPH scavenging activity � Ac − As Ac 100000, where Ac and As denote control and plant extract absorbance, respectively.

FRAP Activity (Ferric Reducing Antioxidant Power).
A sample of 1 mL (0.00625-1) mg/mL was mixed together with 1% potassium ferricyanide [K 3 Fe (CN) 6 ] in 2.5 mL, and the mixture was set for incubation at 50°C for 20 minutes. Te same volume of trichloroacetic acid was added; 2.5 mL was taken from the mixture, and 2.5 mL of dH 2 O 2 was added. Te absorbance of the mixture was read at 700 nm after 0.5 mL was added to the mixture. Te result was compared with ascorbic acid, and values were presented as ascorbic acid equivalents (mg AE/g extract), which were calculated using a standard curve [27]. Te experimental analysis was carried out in triplicate (n � 3).

2,2-Azino-bis(3-ethylbenzothiazoline)-6-sulfonic Acid
(ABTS) Activity. Potassium persulfate (2.45 mmol/L) and 2,2azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) (7 mmol/L) were mixed to obtain an ABTS radical solution (ABTS | ) in the ratio 1 :1 and were incubated for 12-16 hours in the dark. One mL of the radical solution was diluted with the required amount of ethanol/methanol (1 : 89 v/v) to be read using the spectrophotometer to obtain 0.700 ± 0.200 values immediately before use at 745 nm. After that, 0.1 mL of various concentrations of extract (0.0375-1) mg/mL were added to 3.9 mL of ABTS solution and then incubated for 6 minutes. Te absorbance of the solution was read at 745 nm against a blank (methanol); ABTS was used as control and butylated hydroxytoluene (0.0375-1.2 mg/mL) as standard [31]: % inhibition of ABTS scavenging activity � Ac − As Ac 100, where Ac and As denote control and plant extract absorbance, respectively.

Nitric Oxide (NO
where Ac and As denote control and plant extract absorbance, respectively.

Total Antioxidant Capacity (TAC).
One (1) mL of phosphomolybdate reagent (4 mM ammonium molybdate, 28 mM sodium phosphate, and 0.6 M sulphuric acid) was added to a 0.1 mL sample. Te absorbance of the mixture was measured at 695 nm after the mixture was set to incubate for 1 hour and 30 minutes. Distilled water was replaced with the sample/standard, and the control used was gallic acid. Te extract's phenol content was calculated as mg of gallic acid equivalent (mg GAE/g extract) as derived from the standard curve [27]. Te experimental analysis was carried out in triplicate (n � 3).

Enzyme Inhibition
where Ac and As denote control and plant extract absorbance, respectively.

Lipase Inhibitory Assessment.
In a 96-well plate, 164 mL of assay bufer and 6 mL of pancreatic lipase solution were mixed. Also, 20 mL of extract/orlistat was pipetted and set for incubation for 10 minutes at 37°C. Tereafter, 10 mL of the substrate was added and set for incubation again at 37°C for 15 minutes. Te absorbance of the mixture was read at 405 nm [34]: %inhibition of alpha lipase � Ac − As Ac 100, where Ac and As denote control and plant extract absorbance, respectively.

Antioxidant Analysis.
To investigate the antioxidant ability of the extract from M. oleifera leaves, antioxidant assays such as DPPH, nitric oxide, FRAP, TAC, and ABTS were carried out. Te results of FRAP and TAC showed that 0.022°±°0.00008 mg RE/g and 0.534°±°0.001 mg GAE/ g, respectively, as shown in Table 2. Te inhibitory percentage of ABTS at the highest concentration (1.2 mg/mL) is 10.67%, as shown in Figure 1. Moreover, the 50% inhibitory concentrations (IC 50 ) values for DPPH, nitric oxide, and ABTS values of M. oleifera shown in Table 3 are 0.0002 mg/mL, 0.6577 mg/mL, and 8.3036 mg/mL, respectively.

Percentage Inhibitory Efect of Diferent Concentrations of M. oleifera Extract on α-Amylase Enzyme Activity.
M. oleifera extract inhibits the activity of the α-amylase enzyme in a concentration-dependent manner. Te percentage inhibitory values of the extract were evaluated to be 34%, 19%, 33%, 22.58%, and 23.79% at 0.2 mg/mL, 0.4 mg/mL, 0.6 mg/ mL, 0.8 mg/mL, and 1 mg/mL concentrations, respectively. Te highest percentage inhibitory values of acarbose were estimated at 80.72% at 0.8 mg/mL concentration, as shown in   Table 4.

Percentage Inhibitory Efect of Diferent Concentrations of M. oleifera Extract on α-Lipase Enzyme Activity.
M. oleifera extracts had a concentration-dependent inhibitory efect on the α-lipase enzyme. Te highest percentage inhibitory value was 93.84% at 4.68 μg/mL concentration, and the lowest percentage inhibitory value was 79% at a concentration of 300 μg/mL. However, orlistat has the highest percentage inhibitory value of 95.76% at 75 μg/mL concentration, as shown in Figure 3. Te IC 50 values of pancreatic lipase inhibitory activity of the extract were 1.0877 mg/mL, and orlistat (the standard drug) was 0.8170 mg/mL, as shown in Table 4.

Discussion
In vitro studies in all felds of biology are aimed at elucidating the mechanisms by which biological substances perform their roles within a cell [35]. Te uses of medicinal plants in delivering cost-efective therapy for a variety of ailments are due to the existence of secondary metabolites [36]. Plants contain bioactive compounds, which have been shown in studies to have a variety of therapeutic properties [37].
M. oleifera has proven to have various antidiabetic, antiobesity, antioxidant, and anti-infammatory efects. It was reported to contain a huge amount of proteins, oils, potassium, calcium, carbohydrates, amino acids, and phenolic compounds (such as rutin, kaempferol, p-coumaric acid, and quercetin). Te antidiabetic and antiobesity pharmacological properties of Moringa oleifera are a result of its high constituents of favonoid, glucoside, and glucosinolate [38].
Te amphipathic nature of ethanol allows for the breakdown of both polar and nonpolar elements in plants [39]. It was also shown to be the best choice for extracting active principles. Te ethanolic extract of M. oleifera showed phytochemical constituents such as phenol, favonoid, saponin, and tannin, which could be related to the presence of high phytochemical content in this study. Furthermore, the onset of diabetes can be delayed, as revealed in experimental studies with M. oleifera leaves extract with high phenolic content [40,41], which is also in agreement with the high phenolic content revealed in this research. Te therapeutic potential of phytocompounds in M. oleifera has been reported in vitro and in vivo studies for reducing the risk of chronic diseases [42].
Natural antioxidants, which can be found in a variety of foods and medicinal plants such as fruits, vegetables, beverages, herbs, and spices, play a major role in the human diet, particularly in preventing cellular damage (oxidative stress) [43]. Tannins and saponins have been discovered to have strong antioxidant properties [44], which could be responsible for the radical scavenging ability of M. oleifera ethanolic extract to scavenge nitric oxide, diphenyl-2-picrylhydrazyl (DPPH), and 2,2-azino-bis(3-ethylbenzotiazoline)-6-sulfonic acid (ABTS) activity radicals in this present study. Moreover, the phytochemicals present in M. oleifera leaves have been proven to have antioxidant phytochemicals present in them.     Alpha amylase and pancreatic lipase inhibition are one therapeutic method to reduce hyperglycaemia and obesity [45]. In vivo investigation, M. oleifera was proven to have antihyperglycaemic properties via the release of insulin such as sulfonylureas and meglitinides to inhibit ATP-sensitive potassium channels in the residual beta cell channels [46]. High inhibitory efects were shown on α-amylase by M. oleifera more than the reference drug (acarbose) as a result of the saponin, phenolic, and favonoid content. Tis is related to the result of the α-amylase inhibitory assessment revealed in this study.
M. oleifera ethanolic leaf extract efectively inhibited pancreatic lipase using orlistat as the standard. In vitro, in vivo, and clinical studies have shown that M. oleifera has antiobesity properties. Bioactive compounds such as polyphenols and quercetin have antiobesity activities and are great pancreatic lipase inhibitors [47]. Tis result validates the pancreatic lipase inhibitory property of M. oleifera ethanolic leaves.

Conclusion
M. oleifera leaf extract has been validated to be an excellent α-amylase and α-lipase inhibitor with properties suitable for the prevention and treatment of diabetes mellitus and obesity when consumed. Te bioactive compounds such as favonoid, saponin, tannin, etc. present in M. oleifera ethanolic leaves extract as reported in this present and other previous studies were shown to possess antioxidant, antidiabetic, and antiobesity activity, which could be responsible for its potent radical scavenging ability and efective inhibition of α-amylase and pancreatic lipase. Tese natural bioactive compounds and M. oleifera powdered leaves can likewise be included in foods and used as drugs for weight loss management. Moreover, a natural deep eutectic solvent may be adopted in extract preparation for better extraction, low toxicity, and probably to achieve unique chemical compounds. Clinical trial studies can be explored further to maximise the gains in the use of M. oleifera leaves in humans.

Data Availability
All the data supporting this study are available upon request.

Conflicts of Interest
Te authors declare that there are no conficts of interest.   Biochemistry Research International