Introduction

Diabetes mellitus (DM) is a serious health challenge that arises from insulin deficiency or shortage; it is denoted by excessive blood glucose. Excessive blood glucose increases the chance of complications such as nephropathy, neuropathy, retinopathy, and foot ulcers (Ismail et al. 2007; Stehouwer et al. 2008). The levity of this ailment has skyrocketed over the last 30 years and projected to afflict about 439 million individuals come 2030 (Shaw et al. 2010). Regulation of elevated postprandial (PP) sugar has been demonstrated to be paramount for the prevention of DM complications. Delaying the absorption of plasma sugar is one of the curative methods to lower PP blood sugar through inhibition of polysaccharide-catabolizing enzymes (α amylase and α glucosidase) in the digestive tracts. Reports have also confirmed excessive blood glucose and hydroxyl radicals to be involved in the ontogeny and succession of DM through glycosylation of sugar and free radical damage to β-cells. With the involvement of free radicals in the ontogenical and physiological complications of DM (Lipinski 2001), antioxidant-rich agents have been used over the years as good and viable intervention for the management of diabetes. In view of these, concerted efforts have been continuously geared towards developing new plant-based therapeutic agents focusing on the inhibition of polysaccharide-catabolizing enzymes (PCE) (McCue et al. 2005; Kim et al. 2005). This is because many inhibitors of PCE have been isolated from an array of plants to serve as complementary therapy with significant efficacy and minimal adverse effects than the existing conventional therapy (Matsuda et al. 2002; Matsui et al. 2006). Myrothamnus flabellifolius (MF) is one of the botanicals with anti-diabetic potential.

Myrothamnus flabellifolius Welw. (Myrothamnaceae), commonly known as resurrection bush, bush tea, and wonderbos, is one of the known resurrection plants (Child 1960) that has biogeographically spread throughout southern Africa such as Namibia, Botswana, Zimbabwe, and South Africa (Kruger 1998). This unique feature is reflected in the English (resurrection bush) and Zulu (uvukwabafile) vernacular names and is used as a symbol of hope in traditional African psychological treatment against severe depression. The leaves and twigs of the plant have been ethnomedicinally valued against a number of debilitating disorders (Hutchings et al. 1996; Van Wyk et al. 1997). Polyphenolic compounds such as 3,4,5-tri-O-galloylquinic acid and galloylquinic acid have been isolated from MF (Moore et al. 2005; Russo et al. 2010). The former is a good radical scavenger, while the latter is known for its wound healing properties (Baratto et al. 2003; Moore et al. 2007).

Due to the safety, efficacy, availability, and affordability of folkloric medicine, WHO recently recommended traditional plant treatment for diabetes (Baishakhi and Analava 2013). According to Motlhanka (2008), the water extract of MF has found ethnomedicinal use as anti-diabetic preparation in eastern Botswana and among the Zulus of South Africa. In view of the foregoing and couple with no previous scientific reports on the anti-diabetic potential of MF, this study was designed to unravel the radical scavenging (antioxidant) and anti-diabetic potential of MF extracts. The tentative mechanism(s) underlying the mode of inhibition of the implicated enzymes was also investigated. The study is expected to validate the usage of this plant in folkloric medicine and also as part of effort geared towards finding a lasting solution to the menace of diabetics in Southern Africa through alternative or complementary medicine.

Materials and methods

Chemicals and reagents are as follows: chemicals used were 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ascorbic acid, rutin, gallic acid, nitroblue tetrazolium (NBT), dinitrosalicylic acid, quercetin, acarbose, starch, ferrozine, NADH, phenazine methosulfate (PMS), 1,1-diphenyl-2-picryl hydrazyl (DPPH), para-nitrophenol, naphthylethylenediamine dihydrochloride, tris-hydrochloric acid buffer, NADH, hydrogen peroxide, ferrous sulfate, thiobarbituric acid, potassium ferrocyanide, ferric chloride, sulfanilic acid reagent, glacial acetic acid, potassium persulfate, deoxyribose, hydrogen peroxide, thiobarbituric acid, p-nitrophenyl-α-d-glucopyranoside (pNPG), sodium carbonate, and sodium acetate which are products of Sigma-Aldrich (St. Louis, MO, USA). While the water used was glass-distilled, and the other reagents were of analytical grades from reputable companies in the world.

Sample collection and extract preparation

Freshly collected leaves of MF were thoroughly rinsed under running tap, air-dried to constant weight, and subsequently powdered. Exactly 20 g each of the powdered sample was respectively suspended in 200 mL each of ethanol, water (sterile distilled), and hydroethanol (50:50), with continuous agitation for 48 h on a Labcon shaker. Afterwards, the infusion obtained in each case was filtered (Whatman No. 1 filter paper) prior to concentration to dryness under reduced vacuum maintained at 40 °C.

Chemical group profiling of the extracts

Using standard methods (Sofowara 2006; Prashant et al. 2011), the extracts were tested for the presence of different chemical groups like saponin, alkaloids, sterols, flavonoids, anthraquinones, glycosides, triterpenes, phenols and phytosterols.

Quantitative phytochemical analysis

Total phenolic content

In this assay, 1 mL of the extract (I mg/mL) was mixed with Folin-Ciocalteu reagent (5 mL) and 75 g/1000 mL of sodium carbonate (4 mL) in a tube. For color development, the resulting mixture was vortexed (15 s) and subsequently incubated (40 °C, 30 min). Afterwards, the absorbance of the mixture was spectrophotometrically taken (Biochrom WPA Biowave II, Cambridge) at 765 nm. The determinations were triplicated in each case. Using gallic acid as standard, the phenolic content of the extract was estimated from its calibration curve and expressed as %w/w gallic acid equivalent (Wolfe et al. 2003).

Total flavonoid content

A previously adapted protocol (Ordon-ez et al. 2006) was used in this estimation. Briefly, an equal portion (0.5 mL each) of alcoholic aluminum solution (2%) and the extract (1 mg/mL) were mixed in a test tube prior to incubation (25 °C, 1 h). All determinations were carried out in triplicates. Subsequently, the absorbance was taken at 420 nm and the flavonoid content was expressed as quercetin equivalent using its (quercetin) standard calibration curve.

Total flavonol content

For this estimation, the rutin calibration curve was prepared by mixing 2000 μL of different concentrations (200–1000 μg/mL) of rutin with 2000 μL (20 g/L) aluminum chloride and 6000 μL (50 g/L) sodium acetate in test tubes. Following incubation (20 °C, 2.5 h), the absorbance was read at 440 nm. The determinations were carried out in triplicates, and the protocol was also performed with 2000 μL of the plant extract (100/1000 μg/mL) instead of rutin solution. The total flavonol in the extract was thereafter estimated from a rutin standard curve (Kumaran and Karunakaran 2007).

In vitro antioxidant assays

DPPH radical scavenging assay

The adapted method of Turkoglu et al. (2007) was employed in this assay. In brief, different concentrations (200–1000 μg/mL) of the extracts and methanolic ascorbic acid solutions were mixed with methanolic solution of DPPH (0.2 M). Following 30 min of incubation (25 °C), the absorbance of the resulting solutions were colorimetrically determined against blank at 517 nm.

Reducing power assay

This was evaluated as previously reported (Oyaizu 1986). Different concentrations (200–1000 μg/mL) of the extracts and ascorbic acid were made in distilled water (1 mL) and mixed with 2.5 mL each of 0.2 M phosphate buffer (pH 6.6) and potassium ferrocyanide (1%) prior to incubation (50 °C, 20 min). Afterwards, TCA (2.5 mL) was added and centrifuged (3000 rpm, 10 min). Exactly 2.5 mL each of the resulting supernatant and distilled water were then mixed with 0.1% ferric chloride solution (0.5 mL). Finally, the absorbance of the mixture was spectrophotometrically (700 nm) determined against blank.

Iron chelating assay

For this experiment, 0.1 mL of either citrate (standard) or the samples (0.2–1.0 mg/mL) was added to 0.2 mM ferrous chloride solution (0.5 mL). To initiate the reaction, 5 mM ferrozine (0.2 mL) was added and the mixture incubated (25 °C, 10 min). The experiment was performed in triplicate, and the absorbance of the resulting solution was then taken against blank at 562 nm (Dinis et al. 1994).

Nitric oxide scavenging assay

For the NO radical inhibitory potential of MF, the procedure of Garrat (1964) was used. This involved 2 mL addition of 10 M sodium nitroprusside (in phosphate buffer saline (pH 7.4)) to varying concentrations (0.2–1.0 mg/mL) of the extract and ascorbic acid prior to incubation (25 °C, 2 h). Following this, 0.5 mL of the resulting solution was reacted with 1 mL sulfanilic acid reagent (0.3% in 20% glacial acetic acid). This was further incubated (25 °C, 5 min) before 1 mL of naphthylethylenediamine dihydrochloride (0.1%w/v) was added. Finally, the solution obtained was again incubated (25 °C, 30 min) and the absorbance was spectrophotometrically (540 nm) determined against blank.

2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid radical determination

The 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS+) scavenging potential of the MF extract was evaluated following a standard procedure (Re et al. 1999). In this assay, 50 mL each of 7 M ABTS and 2.45 mM potassium persulfate were mixed and incubated for 16 h in the dark. Subsequently, the concentration of solution was spectrophotometrically (734 nm) adjusted to 0.700 using ethanol. A portion (20 μL) of the extract (0.2–1.0 mg/mL) was thereafter reacted with the resulting ABTS solution (200 μL) in a 96-well microtiter plate prior to incubation (25 °C, 15 min) and final absorbance reading (734 nm) in a microplate reader (Model 680 Bio-Rad, USA).

Superoxide anion determination

The superoxide anion scavenging capability of the extract was determined as described by Liu et al. (2007). In this experiment, exactly 100 μL of the extract (0.2–1.0 mg/mL) was reacted with 50 μL each of NADH (0.936 mM), PMS (0.12 mM), NBT (0.3 mM), and tris-hydrochloric acid buffer (pH 8) (16 mM) in a 96-well microtiter plate. Afterwards, the resulting solution in each case was incubated (25 °C, 5 min) and the absorbance was read against blank on a microplate reader (560 nm).

Hydroxyl radical scavenging activity

The method of Mathew and Abraham (2006) was used in the determination of the OH* radical inhibitory potential of MF. Briefly, 0.1 mL of the extract (0.2–1.0 mg/mL) was added to a reaction medium containing 20 mM deoxyribose (0.12 mL), 0.1 M phosphate buffer (0.4 mL), 20 mM hydrogen peroxide (0.04 mL), and 500 μM ferrous sulfate (0.04 mL). Following this, sterile distilled water (0.1 mL) was added and the resulting solution incubated (37 °C, 30 min). To bring the reaction to a halt, 0.5 mL of 2.8% trichloroacetic acid and 0.4 mL of 0.6% thiobarbituric acid solutions were added. Thereafter, a portion (0.3 mL) was taken and dispensed into a 96-well microtiter plate, boiled for 20 min prior to absorbance reading at 532 nm. The percentage inhibitory potential of the extract was subsequently determined.

For each of the antioxidant assay, the inhibition/scavenging potential (I %) was calculated using the expression:

$$ \mathrm{Percentage}\ \mathrm{inhibition}\left(\mathrm{I}\%\right)=\left({\mathrm{A}}_{\mathrm{c}}-{\mathrm{A}}_{\mathrm{e}}\right)/\left.{\mathrm{A}}_{\mathrm{c}}\right]\times 100 $$

where Ac and Ae represents the absorbance of the control and the extract, respectively. The concentration of the extracts and ascorbic acid/citrate causing half maximal (50%) inhibition (IC50) for all the assays was estimated using a standard calibration curve.

In vitro anti-diabetic assays

α-Amylase inhibition and kinetic studies

The methods of Sabiu et al. (2016) were used for the determination of α-amylase inhibitory capacity of the extracts. Briefly, varying concentrations (0.2–1.0 mg/mL)) of the extracts and acarbose (control) were prepared and 500 mL of each was mixed with 500 mL of 0.02 M sodium phosphate buffer (pH 6.9) containing 0.5 mg/mL of α-amylase solution and incubated (25 °C, 10 min). After initial incubation, 500 mL of 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9) was added to each tube at timed intervals. The reaction mixtures were incubated (25 °C, 10 min) and stopped with 1.0 mL of dinitrosalicylic acid color reagent. The tubes were then incubated in a boiling water bath for 5 min and subsequently cooled to room temperature. The reaction mixtures were then mixed with distilled water (15 mL), and the absorbance readings were taken (540 nm) using a spectrophotometer (Biochrom WPA Biowave II, Cambridge) and the values compared with a control which contained 500 mL of the buffer instead of the extracts. The experiments were conducted in triplicate, and the α-amylase inhibitory activity was expressed as percent inhibition, using the following expression:

  • %inhibition = [(ΔAcontrol − ΔAextract)/ΔAcontrol] × 100, where Δ Acontrol and ΔAextract are the changes in absorbances of the control and extract samples, respectively. The concentration of the extract acarbose causes half maximal (50%) inhibition (IC50) in α-amylase assay estimated using a standard calibration curve.

For the kinetic experiments involving concentration-independent inhibition, hydroethanol extract (judging by the IC50 value) was selected and incubated with 5 mg/mL α-amylase with the starch concentration varying from 0.016 to 0.200 mg/mL. The reaction was then allowed to proceed as highlighted above. Maltose standard curve was used to determine the amount of reducing sugars released spectrophotometrically, before it is converted to reaction velocities (v). A Lineweaver-Burk double reciprocal plot (1/v versus 1/[S]) was constructed, and the kinetics of α-amylase inhibition by the extract was determined (Lineweaver and Burk 1934).

α-Glucosidase inhibition and kinetic studies

The method of Elsnoussi et al. (2012) and Sabiu et al. 2016 was used to determine the α-glucosidase inhibitory activity of the extracts. In brief, concentrations (0.2–1.0 mg/mL) of extracts and acarbose (control) were dilluted in distilled water. Then, 50 mL from the stock solution was mixed with100 mL of 0.1 M phosphate buffer (pH 6.9) containing 1.0 m of α-glucosidase solution. The mixtures were then incubated in 96-well plates at 25 °C for 10 min. Following this, 50 mL of 5 mM pNPG solution in 0.1 phosphate buffer (pH 6.9) was added to each well at timed intervals. The reaction mixtures were incubated (25 °C, 5 min) before the absorbance was taken using a microplate reader (405 nm). The absorbances were compared with a control which contained 50 mL of the buffer. Acarbose was prepared in distilled water at the same concentrations as the extract and used as control. The experiments were conducted in triplicate, and the α-glucosidase inhibitory activity was expressed as percent inhibition. The concentration of the extract acarbose causes half maximal (50%) inhibition (IC50) in α-amylase assay estimated using a standard calibration curve.

For the enzyme kinetics on inhibition of α-glucosidase activity by hydroethanol, a modified method of Dnyaneshwar and Archana (2013) was adopted. Briefly, 50 μL of 5 mg/mL hydroethanol was initially incubated with100 μL of α-glucosidase solution (10 min, 25 °C) in one group of tubes. In another separate group of tubes, α-glucosidase was pre-incubated with 50 μL of phosphate buffer (pH 6.9). Fifty milliliters of pNPG at varying concentrations (0.016–0.2 mg/mL) was added to both groups of reaction mixtures to start the reaction. The mixture was then incubated (10 min, 25 °C), and 500 mL of Na2CO3 was added to halt the reaction. A p-nitrophenol standard curve was used to evaluate the amount of reducing sugar released. Reaction rates (v) were then calculated, and double reciprocal plots (1/v versus 1/[S]) of enzyme kinetics were constructed to study the nature of inhibition (Lineweaver and Burk 1934). Km and Vmax values were also calculated from the curve.

Statistical analysis

Except otherwise stated, the evaluated antioxidant potentials were presented as percentage (%). The remaining data were presented as mean ± standard deviation (SD) of replicate experiments. The data were analyzed using analysis of variance (one way) complemented with Duncan’s multiple range test (Statistical Analysis System (SAS) Model 9.13) to detect significant differences between mean values. Differences were considered significant at p < 0.05 confidence interval value.

Results

Phytochemicals

The phytochemical screening of M. flabellifolius extracts (aqueous, ethanol, and hydroethanol) revealed that alkaloids, flavonoids, glycosides, phenol saponin, tannin, phytosterol, and triterpene were present in the three extracts. Anthraquinone and sterol are absent in all the extracts (Table 1). The quantitative phytochemical screening revealed that hydroethanol has the highest phenolic (91.95 ± 4.04% w/w) and flavonol (9.48 ± 1.67% w/w) contents, while ethanol extract has the highest flavonoid (48.86 ± 2.31% w/w) content (Table 2).

Table 1 Phytochemicals detected in the M. flabellifolius extracts
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Table 2 Total phenolic, flavonoid, and flavonol contents of the M. flabellifolius extracts
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Antioxidant activity

The in vitro antioxidative capabilities of M. flabellifolius extracts are shown in Figs. 1, 2, 3, 4, 5, 6, and 7, and their respective IC50 values are presented in Table 3. All extracts inhibited oxidative radicals in a dose-dependent manner. Except for the most potent effect elicited by the ethanol extract of MF, the other extracts compared favorably with vitamin C for the significant scavenging of DPPH radical. For the ABTS assay, vitamin C had better and significant inhibitory effect at p < 0.05 than the extracts. Ethanol extract compared favorably with vitamin C for the significant scavenging of nitric oxide while hydroethanol elicited the most potent effect on ferric chelation than other extracts and vitamin C. For superoxide radical, vitamin C showed significant effect than the extracts while hydroethanol compete favorably with vitamin C for hydroxyl radical scavenging at p < 0.05. Lastly, hydroethanol significantly reduced ferric than other extract and citrate at all concentrations except at 1 mg/mL.

Fig. 1
figure 1

DPPH scavenging effect of M. flabellifolius extracts. Values are mean ± standard deviation (SD)

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Fig. 2
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ABTS scavenging effect of M. flabellifolius extracts. Values are mean ± standard deviation (SD)

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Fig. 3
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Superoxide anion scavenging potential of M. flabellifolius extracts. Values are mean ± standard deviation (SD)

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Fig. 4
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Nitric oxide scavenging ability of M. flabellifolius extracts. Values are mean ± standard deviation (SD)

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Fig. 5
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Iron chelation potential of M. flabellifolius extracts. Values are mean ± standard deviation (SD)

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Fig. 6
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Hydroxyl radical scavenging potential of M. flabellifolius extracts. Values are mean ± standard deviation (SD)

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Fig. 7
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Reducing power potential of M. flabellifolius extracts. Values are mean ± standard deviation (SD)

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Table 3 IC50 (mg/mL) for the antioxidant capabilities of M. flabellifolius extracts
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In vitro anti-diabetic studies

The inhibitory effects of MF extracts on the specific activity of α-amylase are presented in Fig. 8. The extracts dose-dependently inhibited the activity of the enzyme with the best effect observed in all the extracts at the highest concentration (1.0 mg/mL). At the lowest concentration (0.2 mg/mL), the ethanolic extract had the highest inhibitory activity (13.14%) when compared with the other extracts and acarbose. In contrast, the hydroethanol extract exhibited the best effect at the highest investigated concentration. Based on the IC50 values (Table 4), all the extracts showed mild inhibitory effect on the activity of α-amylase when compared with acarbose. Similarly, the hydroethanol extract also had the highest inhibitory effect against α-glucosidase at the highest tested concentration (Fig. 9). Based on this and coupled with the estimated IC50 values (Table 4), the hydroethanol extract of MF was selected for the tentative kinetics of inhibition. The kinetics of inhibition using the Lineweaver-Burk double reciprocal plot revealed uncompetitive inhibitory modes against the two enzymes (Figs. 10 and 11). For α-amylase, the respective Vmax values for the extract and the control were 1.3 and 1.84 μM/min, while the Km was 409.604 and 503.0 mg, whereas for α-glucosidase, the Vmax and Km values for the control were 0.0056 μM/min and 22.690 mg, respectively, while those of the extract were 0.012 μM/min and 8.504 mg, respectively.

Fig. 8
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Inhibitory potential of M. flabellifolius extracts on α-amylase activity. Values are mean ± standard deviation (SD)

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Table 4 IC50 (mg/mL) for the α-amylase and α-glucosidase inhibitory potentials of M. flabellifolius extracts
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Fig. 9
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Inhibitory potential of M. flabellifolius extracts on α-glucosidase activity. Values are mean ± standard deviation (SD)

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Fig. 10
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Lineweaver-Burk plot of M. flabellifolius hydroethanol extract eliciting an uncompetitive mode of inhibition on α-amylase. Results represent mean ± standard deviation (n = 3) (p > 0.05)

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Fig. 11
figure 11

Lineweaver-Burk plot of M. flabellifolius hydroethanol extract eliciting an uncompetitive mode inhibition on α-glucosidase. Results represent mean ± standard deviation (n = 3) (p > 0.05)

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Discussion

As the pathogenesis of diabetes becomes clearer, exciting new targets for new potential herbal anti-diabetic agent is important. The conventional therapy for diabetics includes insulin injections and oral hypoglycemic drugs which elicit various pharmacokinetics to regulate the plasma sugar status. However, besides being very expensive, most patients using these conventional therapies are prone to side effects such as lactic acid intoxication, gastrointestinal upset, and hypoglycemia (Li et al. 2004). Therefore, various traditional therapies with natural carbohydrate-catabolizing enzyme inhibitors are sought as alternatives. Natural inhibitors of these enzymes are beneficial in reducing hyperglycemia by delaying the digestion of carbohydrates and, consequently, the absorption of glucose (Henda et al. 2014). In diabetics, PP hyperglycemia usually occurs following a meal due to the absorption of glucose from the gastrointestinal tract. Regulating sugar absorption in the intestines and/or stimulating sugar disposal in the tissues may be important for the control of DM in the postprandial state (Ellappan et al. 2013). The key enzymes for carbohydrate digestion are the intestinal α-glucosidase and pancreatic α-amylase. The two enzymes have been recognized as the main therapeutic targets for the modulation of PP hyperglycemia (Kim et al. 2005).

In this study, the results revealed that aqueous extract exhibited the strongest inhibitory effect against alpha amylase with an IC50 value of 0.75 mg/mL. Nevertheless, it is not enough to say that aqueous extract is the best for the control of postprandial glucose because excessive inhibition of pancreatic α-amylase could result to irregular fermentation of undigested carbohydrates by bacteria in the colon and only mild α-amylase inhibition activity is beneficial for the control of PP hyperglycemia (Apostolidis et al. 2007).

Since α-glucosidase inhibitors cause a reduction in the rate of sugar uptake and the postprandial blood glucose state, the reduction is key to modulation of non-insulin-dependent hyperglycemia (Hamdan and Afifi 2004). In view of these assertions, hydroethanol exhibits the most potent inhibition towards the activity of α-glucosidase and relatively mild inhibition towards α-amylase judging from its IC50 values on the two enzymes. Hence, the hydroethanol extract of MF could be a promising therapeutic agent for the control of postprandial hyperglycemia. Our findings concur with the reports of Krentz and Baile (2005), Kwon et al. (2008), Kajaria et al. (2013), and Sabiu et al. (2016), where mild α-amylase inhibition with potent α-glucosidase inhibition was canvassed as a desirable approach to minimize availability of carbohydrate substrate for sugar production in the gut. The kinetics of inhibition on the enzymes showed that the hydroethanol extract of MF exerted uncompetitive inhibition against both α-amylase and α-glucosidase. This implies that the extract binds exclusively to the enzyme-substrate complex yielding an inactive enzyme-substrate-inhibitor complex (Bachhawat et al. 2011; Sabiu et al. 2016), and this will consequently delay the digestion of carbohydrate and cause reduced glucose absorption rate (Kim et al. 2005).

Oxidative stress is known to play a definite role in the pathological manifestations of DM (Srivatsan et al. 2009). Antioxidants are saddled with the responsibility of scavenging the free radicals or protecting reactive oxygen species (ROS) defense mechanisms (Umamaheswari and Chatterjee 2008). The results showed that all the extracts of M. flabellifolius had a significant antioxidant activity when compared with the respective standard. The hydroethanol extract showed the best activity on hydroxyl radical, metal chelating, ABTS, and superoxide ion radical assays and relatively good activity on other assays when compared with the standard. This might be a probable reason for the α-amylase and α-glucosidase inhibitory prowess of the extract in this study.

Since free radicals have been implicated in the pathogenesis of diabetes, ameliorating deleterious influence of ROS mainly by augmenting antioxidant defense mechanism may be considered as one of the ways of preventing diabetes (Amaral et al. 2008). This was significantly elicited by the hydroethanol extract in this study. Besides being antioxidants, the presence of phytochemicals such as alkaloids, flavonoids, glycosides, saponin, tannin, phenols, triterpene, and phytosterol may be another justifiable reason for the elicited α-amylase and α-glucosidase inhibitory activities of the hydroethanol extract in this study. Many phytochemicals have been reported as potential anti-diabetic agents because they exert a good inhibitory action against α-amylase and glucosidase. For instance, phenolic compounds and specially flavonoids (Cazarolli et al. 2008), flavonols (Miliauskas et al. 2004), phytosterol (Suba et al. 2004), saponin (Song et al. 2011), alkaloid (Kiyoteru et al. 2005), and glycosides (Yarnell et al. 2009) have all been established as pharmacologically potent hypoglycemic agents.

In conclusion, it can be inferred from this study that the hydroethanol extract of M. flabellifolius displayed the most effective inhibitory effect against the specific activity of α-amylase and α-glucosidase, and this might be due to the phytochemical constituents of utmost pharmacological importance present in the plant. Moreover, this study has also lent credence to the use of MF in the management of diabetes among the people of Southern Africa.