In Vitro Hypoglycemic and Antioxidant Activities of Dichloromethane Extract of Xerophyta spekei

Diabetes mellitus is a chronic metabolic disorder which has greatly led to an increase in morbidity and mortality globally. Although Xerophyta spekei is widely used for the management of diabetes among the Embu and Mbeere communities in Kenya, it has never been empirically evaluated for its hypoglycemic activity. This study was carried out to verify the hypoglycemic activity of dichloromethane (DCM) extract of Xerophyta spekei as well as its antioxidant activity using various in vitro techniques. Phytochemicals associated with its antioxidant activity were identified through GC-MS. Data were subjected to descriptive statistics and expressed as mean ± standard error of the mean (X̄ ± SEM). Comparison between various variables was performed by using unpaired Student's t-test and one-way analysis of variance (ANOVA), followed by Tukey's post-hoc test. The confidence interval was set at 95%. The obtained results were presented in tables and graphs. Results showed that there was no difference in α-amylase inhibition activity between the plant extract and the standard (IC50 525.9 ± 12.34 and 475.1 ± 9.115, respectively; p  >  0.05). Besides, the glucose adsorption activity of the extract increased with an increase in glucose concentration (from 5.89 to 32.64 mg/dl at 5 mmol and 30 mmol of glucose, respectively; p  <  0.05). The extract also limited the diffusion of glucose more than the negative control (7.49 and 17.63 mg/dl, respectively; p  <  0.05). It also enhanced glucose uptake by yeast cells. In addition, the studied plant extract showed notable antioxidant activities. The therapeutic effects exhibited by this plant in managing diabetes mellitus and other ailments could be due to its antioxidant as well as its hypoglycemic activity. The study recommends the evaluation of X. spekei for in vivo hypoglycemic and antioxidant activities. Besides, the isolation of bioactive phytochemicals from the plant may lead to the development of new hypoglycaemic agents.


Introduction
Diabetes mellitus is a metabolic disorder characterized by high blood glucose as a result of insulin defciency or poor insulin-directed utilization of glucose by target cells.Diabetes can be classifed into type 1, type 2, gestational diabetes, and other type-specifc diabetes mellitus [1].Type 2 diabetes mellitus is the most common.It results from relative insulin resistance, inadequate insulin secretion, and excessive or inappropriate glucagon secretion.On the other hand, type 1 diabetes mellitus is characterized by absolute insulin defciency as a result of the immune destruction of pancreatic beta cells.Diabetes mellitus is a serious problem that afects the quality of life and life expectancy.Chronic hyperglycemia leads to abnormal carbohydrate, fat, and protein metabolism, and as the disease progresses, it results in both microvascular and macrovascular complications [2].According to the International Diabetes Federation, the prevalence of the disease among adult population was 9.3% in 2019, accounting for 463 million people worldwide.Tis number is expected to increase to about 700 million people by the year 2045 [3].
Several studies implicate oxidative stress as a major cause to the pathogenesis of diabetes mellitus.Tis is through altering enzymatic systems, causing lipid peroxidation, impairing the metabolism of glutathione, as well as decreasing vitamin C levels [4].Complications related to increased oxidative stress include neuropathy, retinopathy, and nephropathy [5].Besides, insulin insensitivity caused by mitochondrial dysfunction is one of the outcomes triggered by oxidative stress [6].Metabolic abnormalities in diabetes mellitus results in an increase in superoxide anion production in the mitochondrion of endothelial cells.As a result, there is activation of polyol pathway fux, increase in advanced glycation end products (AGEs), and activation of protein kinases as well as overactivity of the hexosamine pathway [7].
With the rising prevalence of diabetes, the search for new alternative interventions is critical.In resource-limited regions, particularly in developing nations, medicinal plants play a crucial role in the treatment of diabetes.Tis is because they are arguably cheap, safe, and readily available [8].On the other hand, conventional medicines used in the treatment of diabetes mellitus are marked with adverse effects including hepatotoxicity, nephrotoxicity, hypoglycemia, and gastrointestinal disturbances [9].Furthermore, medicinal plants are rich sources of bioactive compounds with the ability to ameliorate oxidative stress resulting from diabetes.For instance, antioxidant properties associated with medicinal plants have been shown to decrease expressions of intracellular cell adhesion molecule-1 protein involved in infammatory reactions, as well as improve diabetes state [10].
Excess production of ROS in pancreatic beta cells causes the activation of infammatory and apoptotic transcription factors resulting in their death.Furthermore, proinfammatory cytokines produced as a result of ROS causes infammation, atherosclerosis, vascular dysfunction, and diabetes-related kidney diseases [11].However, medicinal plants can decrease proinfammatory cytokines and slow down the development of kidney diseases [12].
Te search for new hypoglycemic agents is ever on, with medicinal plants providing new leads in fnding previously unearthed phytochemicals with hypoglycemic and antioxidant efects.Currently, more than 410 medicinal plants have been reported to have antidiabetic properties [13].
Oxidative stress is a state of imbalance between free radicals and antioxidants.It results from an increase in oxidative radicals, which include reactive oxygen species and reactive nitrogen species, with the subsequent weakening of the natural antioxidant system [14].Many studies have demonstrated that oxidative stress plays a signifcant role in the development of several degenerative illnesses including diabetes mellitus [15].
Both reactive oxygen species (ROS) and reactive nitrogen species (RNS) cause oxidative damage to proteins, lipids, and DNA through nitrosylation, peroxidation, carboxylation, and nitration [16].Besides, they alter the protein structure through amino acid oxidation, free radical-induced breakage, and cross-linking.Also, peroxyl radicals and Fenton-generated OH radicals oxidize purines, pyrimidines, and deoxyribose moieties, causing the biomolecules to degrade [17].
Antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase play a crucial role in humans in response to oxidative stress [16].Besides, substances such as ascorbic acid, alphatocopherol, beta-carotene, and uric acid are involved in the scavenging of oxidative radicals [18].Also, metalbinding proteins, such as albumin, ceruloplasmin, lactoferrin, and ferritin, can sequester free ions of copper, zinc, and magnesium involved in redox processes in the mitochondrial matrix [19].
Medicinal plants have antioxidant properties, which work in synergy with the endogenous antioxidants to bring about a redox balance in disease states [21].Some of the mechanisms involved in stabilizing reduced radicals entails halting the initiated chain reactions by using chain-breaking antioxidants, disintegrating the oxidants into harmless products, and stabilizing transition metals involved in oxidative processes.Enzymatic antioxidants reduce the rate of chain reaction initiation by oxidants by scavenging the initial free radicals [15].
Tis current study aimed to evaluate the in vitro antioxidant and antidiabetic activities of the DCM extract of Xerophyta spekei.Te plant Xerophyta spekei belongs to the family of Velloziaceae and is common in Kenya, Tanzania, Zambia, Zimbabwe, and Ethiopia [22].Te shrub measures 2-5 m in height by 6-12 cm in thickness, and its leaves are congregated at one edge.Te plant is also well adapted to dry climates.It is used in South Africa by herbalists to manage pain and infammation [22].Among the Mbeere and Embu communities, it is used to treat wounds, snake bites, and diabetes mellitus [23].In the Kamba community, the plant is used to treat burns [24].Previous studies have shown that X. spekei has antibacterial activities against S. aureus and B. subtilis [22].
Despite its extensive use, the ability of Xerophyta spekei to neutralize free radicals and its hypoglycemic potential is not well known.Besides, identifying its chemical constituents is crucial not only for the development of new medicinal products but also for the discovery of valuable phytocompounds with economic benefts as well as the validation of the plant's traditional use.Terefore, the purpose of this study is to determine the antioxidant activity, in vitro hypoglycemic activity, and phytochemical profles of dichloromethanolic (DCM) extract of X. spekei, as a potential antioxidant and hypoglycemic agent.

Sample Preparation and Extraction.
After air drying the plant material under shade for three weeks, the plant was ground into a fne powder using an electric mill.Five hundred grams of the ground powder of X. spekei were soaked in 1.5 L of dichloromethane with regular eddying for 24 hours.Te extract was then decanted and fltered by using Whatman's flter paper no. 1 into a clean conical fask.A rotary evaporator at 40 °C was used to concentrate the fltrate, and the weight of the resultant semisolid residue was assessed by using a weight balance.Te extract was then stored at −20 °C until the bioassay.

Determination of In Vitro α-Amylase Inhibition.
Te α-amylase inhibitory activity was performed as described by Wickramaratne et al. [25] with slight modifcations.Extract concentrations ranging from 0 to 1000 μg/ml were made by dissolving the plant extract in 0.02M Na 2 HPO 4 /NaH 2 PO 4 and NaCl (0.006 M) bufer at pH 6.9.A volume of 200 μl of the plant extract was mixed with an equal volume of α-amylase solution (2 units/ml) and incubated at 30 °C for 10 minutes.Tis was followed by the addition of 1% starch solution to each tube and incubated for 3 minutes.To terminate the reaction, 200 μl of DNSA reagent (12 g of sodium potassium tartrate tetrahydrate in 8.0 mL of 2M NaOH and 20 mL of 96 mM of 3,5-dinitrosalicylic acid solution) was added, and the mixture was boiled at 85-90 °C.
After cooling, 5 ml of distilled water was added and the absorbance was read at 540 nm using a UV-Visible spectrophotometer.Te blank was prepared at each concentration with the absence of enzyme solution.A positive control was also prepared using acarbose at similar concentrations as the extract.Te % α-amylase inhibition was plotted against extract concentration and the IC 50 values were calculated.

Determination of In Vitro Glucose Adsorption
Capacity.Glucose adsorption capacity was determined as described by Harish et al. [8].In brief, X. spekei's sample extract (250 mg) was added to each of the 25 ml glucose solution of increasing concentrations (5, 10, 15, 20, and 30 mmol), prepared in 50 ml conical fasks.Te mixture of each concentration was stirred and incubated in a shaker water bath at 37 °C for 6 h.Tis was followed by centrifugation for 20 minutes at 4000 × g and the glucose concentration in the supernatant determined.Glucose-bound was determined using the formula described by Harish et al. [8].
where G1 is the original glucose concentration and G6 is the glucose concentration after 6 hours

Determination of In Vitro Glucose Difusion.
Glucose difusion was carried out as described by Bhinge et al. [26].A volume of 25 ml of glucose solution (20 mM) and plant extract samples (1%) were dialyzed in a dialysis membrane against 200 ml of distilled water in a beaker at 37 °C using a shaker water bath.Contents of glucose in the dialysate were determined at intervals of 30, 60, 120, 180, and 240 minutes, using a glucose oxidase peroxidase kit.A control test was carried out without the extract.

Determination of Glucose Uptake by Yeast Cells.
A 10% (v/v) suspension of commercial baker's yeast was made by repeatedly washing commercial baker's yeast cells in distilled water through centrifugation (3000 r/m, 5 minutes) until the supernatant was clear.Various extract concentrations (1-5 mg) were added to 1 mL of glucose solution (5 and 10 mmol/L) and incubated for 10 minutes at 37 °C.A volume of 100 μL yeast suspension was added to the reaction, and the mixture vortexed and further incubated at 37 °C for 60 min.Te tubes were centrifuged (3,000 r/min, 5 min), and the glucose in the supernatant was determined as described by Cirillo [27].Te positive control was composed of metronidazole at similar concentration to the extract.

Scientifca
Glucose uptake by yeast cells was calculated using the following formula: Glucose uptake(%) � Absorbance of control − Absorbance of sample Absorbance of control × 100. (3)

Evaluation of DPPH Free Radical Scavenging Activity.
By using an analytical balance, 12 mg of DPPH salt was weighed and dissolved in 100 ml of analytical methanol to make a concentration of 0.3 mM of DPPH solution.Of this solution, 1 ml was added to 2.5 ml of each plant extract concentration (0.01, 0.1, 1, 10, 100, and 1000 μg/ml) and mixed.Tis was followed by incubation for 15 minutes at room temperature in a dark room.Finally, absorbance was read at 517 nm wavelength by using a Shimadzu UV-VIS (1600) microprocessor double-beam spectrophotometer.Ascorbic acid of similar concentrations as the extract was used as a positive control.To make a negative control, 2.5 ml of DPPH solution was added to 1 ml of methanol followed by reading of the absorbance.All the tests were performed in triplicates, and the percentage of radical scavenging activity (RAS) of the extract calculated as described by Kibiti and Afolayan [28].

% of radical scavenging activity � Absorbance of control of the sample
The absorbance of the control × 100. (4)

Evaluation of Ferric-Reducing Power.
Te ferricreducing activity of the plant extract was determined by using the method described by Moriasi et al. [21].Te standard and extract were prepared at various concentrations (0.01, 0.1, 1, 10, 100, and 1000 μg/mL).To each 1 ml of concentration of either the standard or the extract, 2.5 ml of 200 mM phosphate bufer (pH 6.6) and 2.5 ml of 30 mM potassium ferricyanide were added.Te mixture was incubated at 50 °C for 20 minutes.
In addition, 2.5 ml of 600 mM trichloroacetic acid was added and stirred.Tis was followed by a 15-minute centrifugation of the mixture at 3000 rpm.2.5 ml of the supernatant was diluted with an equal volume of distilled water.Finally, 0.5 ml of 600 mM ferric chloride was added, and the absorbance values of both the standard (ascorbic acid) and the extract measured using a spectrophotometer at 700 nm against the blank (Shimadzu UV-Vis 1600).Te blank solution included all of the reagents without the extract and the standard.All the tests were performed in triplicates and ascorbic acid was used as the standard.

Evaluation of Hydroxyl Radical Scavenging Activity.
Te test method was carried out as described by Arika et al. [14].A reaction mixture comprising 100 μl of 2-deoxy-2-ribose (28 mM), 20 mM KH 2 PO4-KOH bufer (pH 7.4), 200 μM FeCl 3 (1 :1 v/v), 1.04 mM 200 μl EDTA, 100 μl of 1.0 mM hydrogen peroxide, 100 μl of ascorbic acid (1.0 mM), and the extract of concentrate 0.1-1000 μg/ml to make a total volume of 1 ml, was incubated at 37 °C for 1 hour.A volume of 1.0 ml of 1% thiobarbituric acid (TBA) and 1.0 ml of 2.8% trichloroacetic acid (TCA) was added and incubated for 20 minutes at 100 °C, resulting in the formation of a pink color.After cooling the solution, the optical density was measured at 532 nm.Gallic acid was employed as a positive control and was processed in the same way as the extract.Te blank solution had all of the reactants but not the extract.All studies were carried out in triplicate, and the % hydroxyl radical scavenging activity was calculated as follows: % radical scavenging activity � The absorbance of control − Absorbance of sample absorbance of control × 100. (5)

Percentage Evaluation of Antilipid Peroxidation
Activity.A volume of 2 ml of trichloroacetic acid, thiobarbituric acid, and hydrochloric acid combination (15% (w/v) of TCA, 0.375% (w/v) of TBA, and 0.25 N of HCl) was added to 1 mL of various concentrations of the standard and extract (200, 400, 600, and 800 μg/ml).Te mixtures were incubated in a 90 °C water bath for 15 minutes before being centrifuged at 10,000 revolutions per minute for 5 minutes.Finally, using a UV-Vis spectrophotometer (Shimadzu UV-Vis 1600), the absorbance of the various supernatants was determined at 4 Scientifca 532 nm against the blank (the blank contained all the reactants apart from the extract and the standard).All the tests were performed in triplicates and the antilipid peroxidation was determined using the formula described by Moriasi et al. [21].
% anti-lipid peroxidation � Absorbance of control − Absorbance of the sample The absorbance of the control × 100.(6)

Evaluation of Iron Chelating Power.
A volume of 1 ml of the sample extract at diferent concentrations (50,100,150,200, and 250 μg/ml) was mixed with an equal volume of 0.125 mM iron (II) sulfate.To begin the reaction, 1 ml of 0.3125 mM ferrozine was added and vortexed.Tis was followed by incubation at room temperature for 10 minutes.
Te absorbance was measured at 562 nm.EDTA was used as the positive control.Te blank was composed of the reagents without the plant extract and EDTA.Te tests were performed in triplicates, and the percentage of iron chelating power was determined as described by Ebrahimzadeh et al. [29].[30].A solution of 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate (3 mL) was mixed with 0.3 mL of the extract solution.Tis was followed by a 90-minute incubation at 95 °C.Te absorbance of the solution was measured at 695 nm after cooling using a Shimadzu UV-VIS (1600) microprocessor double-beam spectrophotometer.Methanol was used in the blank solution (0.3 mL).Te total antioxidant activity was calculated as the number of grams of ascorbic acid equivalent.

Gas Chromatography-Mass Spectrometry
Analysis of the DCM Extract of X. spekei.Te GC-MS analysis of X. spekei was performed using a procedure previously described by Gitahi et al. [31].Te sample was analyzed using a GC-MS (7890/5975, Agilent Technologies, Inc., Beijing, China) system, which consists of a gas chromatograph connected to a mass spectrometer.Te GC-MS was outftted with a 30 m long, 0.25 mm diameter, and 0.25 μm flm thickness HP-5 MS (5% phenyl methyl siloxane) low-bleed capillary column.An electron ionization system with an ionization energy of 70 Ev was used for GC-MS detection.In the split mode, the carrier gas was helium (99.99%) at a constant fow rate of 1.25 ml/min.

Results
3.1.Efect of the DCM Extract of X. spekei on α-Amylase Enzyme Activity.Te DCM extract of X. spekei demonstrated concentration-dependent α-amylase inhibition activity as shown in Figure 1.In addition, there was no signifcant diference in α-amylase inhibitory activity between extract concentrations of 37.5 μg/ml and 75 μg/ml, 125 μg/ml, and 250 μg/ml as well as between 250 μg/ml and 500 μg/ml (p > 0.05).Besides, an extract concentration of 1000 µg/ml demonstrated the highest inhibitory activity than the rest (p < 0.05).Also, there was no notable diference between the extract and the standard at similar concentrations (p > 0.05).Moreover, there was no signifcant diference between the IC 50 of the extract and the standard (acarbose) (p > 0.05; Table 1).

In Vitro Glucose Adsorption Activity of DCM Extracts of X. spekei.
Te fndings of this study indicated that the X. spekei extract was efective in binding glucose at both low and high concentrations.As demonstrated in Figure 2, the extract adsorbed glucose in a concentration-dependent manner.However, the adsorption capacity varied signifcantly between glucose concentrations (5-30 mmol/L) (p < 0.05).

Efects of the X. spekei's Extract on Glucose Difusion.
As shown in Figure 3, the rate of difusion of glucose across the dialysis membrane was found to be time-dependent.
Besides, the inhibitory activity of the extract on glucose difusion was signifcantly high compared to the control (p < 0.05; Figure 3).

Efects of the DCM Extract of X. spekei on % Glucose
Uptake by Yeast Cells.As indicated by the result in Table 2, glucose uptake by yeast cells at glucose concentrations of 5 mmol and 10 mmol difered signifcantly in all tested extract concentrations (p < 0.05; Table 2).Besides, the percentage of glucose uptake by the yeast cells in both glucose concentrations (5 mmol and 10 mmol) increased remarkably with an increase in extract concentration (p < 0.05; Table 2).However, it was noted that the percentage of increase in glucose uptake by yeast cells was inversely proportional to the molar concentration of glucose.

In Vitro DPPH Radical Scavenging Activity of the DCM
Extract of Xerophyta spekei.Te six tested concentrations of the DCM extract of X. spekei displayed concentrationdependent in vitro DPPH radical scavenging activities as shown in Figure 4.Moreover, in all concentrations examined, the standard (ascorbic acid) demonstrated greater DPPH radical scavenging activity than the X. spekei extract (p < 0.05).Besides, the capacity of the extract to scavenge DPPH radicals at diferent concentrations varied signifcantly (p < 0.05), with the greatest concentration being the most efcient.Te concentrations of the DCM extract of X. spekei and the standard (ascorbic acid) necessary to inhibit 50% of DPPH radicals (IC 50 ) were also measured.Te IC 50 of the standard was found to be substantially greater than that of the extract (p < 0.05; Table 1).

In Vitro Ferric-Reducing Activity of the DCM Extract of
Xerophyta spekei.In Figure 5, the efciency of ferric-reducing activity of X. spekei extract was demonstrated by a rise in absorbance with increasing extract concentration.Moreover, the ferric-reducing activity of the extract changed considerably across all concentrations examined (p < 0.05).Nevertheless, in all concentrations examined, there was a signifcant difference in ferric-reducing activity between the standard (ascorbic acid) and the plant extract (p < 0.05).Te halfmaximal efective concentration (EC 50 ) of the plant extract and standard was also determined in this investigation.Te EC 50 of the standard and the extract was found to be signifcantly diferent (p < 0.05; Table 1).

In Vitro Hydroxyl Radical Scavenging Activity of the DCM Extract of Xerophyta spekei.
Te hydroxyl radical scavenging ability of the X. spekei extract was concentrationdependent, as shown in Figure 6.In addition, there was a signifcant diference in hydroxyl radical scavenging activity between the plant extract and the standard (gallic acid) in all tested concentrations (p < 0.05), except at 1000 μg/ml (p > 0.05).Similarly, hydrogen radical scavenging activity across plant extract concentrations difered considerably (p < 0.05), except at 100 μg/ml and 1000 μg/ml (p > 0.05).
Te IC 50 of the extract was similar to that of the standard (gallic acid) (p > 0.05; Table 1).

In Vitro Antilipid Peroxidation of the DCM Extract of
Xerophyta spekei.As demonstrated in Figure 7, the DCM extract of X. spekei inhibited lipid peroxidation in a concentration-dependent manner.All extract concentrations tested demonstrated diferent antilipid peroxidation 6 Scientifca activities (p < 0.05).However, the antilipid peroxidation activity of the DCM extract of X. spekei and L-ascorbic acid at comparable concentrations revealed that the standard had signifcantly higher lipid peroxidation inhibition activity than the extract at all tested concentrations (p < 0.05), except at 200 μg/ml (p > 0.05).In addition, the extract   Values are expressed as X̄± SEM.Mean values (n � 3) which do not share superscript small letters along the rows and superscript capital letters along the columns difer signifcantly from each other (p < 0.05).Scientifca concentration necessary to inhibit 50% of lipid peroxidation activity (IC 50 ) was signifcantly higher than that of the standard (p < 0.05; Table 1).8, the DCM extract of X. spekei became more efcient in chelating iron as the extract concentrations increased.Moreover, the chelating activity varied signifcantly across all extract concentrations (p < 0.05).All of the tested extract concentrations were considerably less efcient in iron chelating than the standard (p < 0.05), except at 250 μg/ml (p > 0.05).Te capacity of the extract and the standard to chelate 50% of radicals (IC 50 ) was also assessed.Notably, the IC 50 of the plant extract was markedly lower than that of the standard (EDTA) (p < 0.05; Table 1).

In Vitro Hydrogen Peroxide Scavenging Activity of the DCM Extract of Xerophyta spekei.
In all concentrations examined, the DCM extract of X. spekei was efcient in scavenging hydrogen peroxide radicals in a concentrationdependent manner.Te capacity of the standard (ascorbic acid) to scavenge these radicals was considerably higher than that of the extract (p < 0.05; Figure 9) at all tested concentrations, except at 500 μg/ml (p > 0.05; Figure 9).Also, the efectiveness of extract concentrations in scavenging hydrogen peroxide radicals difered signifcantly (p < 0.05; Figure 9).However, the IC 50 of the DCM extract of X. spekei was signifcantly higher than that of the standard (p < 0.05; Table 1).

In Vitro Total Antioxidant Capacity of the DCM Extract of Xerophyta spekei.
In Figure 10, it was observed that the total antioxidant capacity of the extract increased with an increase in extract concentration.Besides, the total antioxidant capacity of the extract calculated from the curve equation (y � 0.1934x-0.2872;R 2 � 0.9329) at 1000 μg/ml was found to be 15.75 ± 0.035 μg/mg.
From the study, it was found that the IC 50 value of the extract in inhibiting α-amylase was similar to that of acarbose.Inhibition of the alpha-amylase activity by the plant extract could have been caused by the presence of an inhibitor in the extract fbers or encapsulation of the enzyme and starch by the extract [46].Similar fndings were reported in a study on in vitro hypoglycemic efects of ripe and unripe M. sapientum [26].
Furthermore, the fnding from this study indicates that the extract can delay carbohydrate breakdown by inhibiting the α-amylase enzyme.Alpha-amylase inhibition activity is associated with bioactive compounds such as favonoid and alkaloid [48].Several studies have documented a positive correlation between phytochemicals and alpha-amylase inhibition activity [2].
Glucose difusion retardation predicts the efect of fbers on the delay of glucose absorption in the gut [26].In this study, the delay in glucose difusion was greater than that of the negative control.Studies have shown that soluble dietary fbers form gel-like substances when in solution, which traps glucose molecules and prevent them from being absorbed too quickly [49].Similarly, glucose adsorption capacity by X. spekei may be attributed to both the insoluble and soluble fber contents of the extract.Previous studies have demonstrated the ability of the extracts to adsorb and retard glucose difusion in a similar manner to that of X. spekei.A study on in vitro antidiabetic efects and antioxidant potential of Cassia nemophila pods showed a concentrationdependent activity on glucose adsorption [50].
Although glucose transport across the yeast cells is a facilitated process by membrane carriers [8], in this study, it was found that an increase in extract concentration resulted in an increase in glucose uptake by yeast cells.Some studies have also reported similar results.Te extracts of Cassia nemophila pods were shown to increase glucose uptake by yeast cells in a concentration-dependent manner [50].Furthermore, the in vitro hypoglycemic efects of Albizia lebbeck and Mucuna pruriens yielded comparable results [46].Glucose uptake by yeast cells may be due to increased facilitated transport by the extract or increased cellular glucose metabolism [8].Although glucose transport across yeast cell membranes may difer from human transport, plant extracts have been shown to increase the expression of glucose receptors in human cells, as well as enhance insulin secretion and increase the number of glucose transporters [51,52].
Terefore, this study suggests that the X. spekei extract may lower postprandial hyperglycemia probably by increasing the viscosity of glucose, slowing its difusion, and binding to glucose molecules, thus resulting in a decrease in its concentration as well as retarding the activity of alphaamylase enzyme [25].
Tis study also evaluated the antioxidant potential of X. spekei using various in vitro assays.One of the assay methods included 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity.Te steady diamagnetic radical is frequently used to assess the antioxidant capabilities of both lipophilic and hydrophilic substances due to its sensitivity [14].
Te extract of X. spekei scavenged DPPH radicals in a concentration-dependent manner.However, in all concentrations examined, the ability of the extract to scavenge these radicals was lower than that of the standard.Also, the IC 50 of the extract was substantially greater than that of the standard.A greater IC 50 indicates less radical scavenging activity.However, an IC 50 value of less than 50 μg/ml indicates substantial antioxidant activity [20].Te plant extract showed an IC 50 value of 1.66 μg/ml and hence demonstrated considerable activity.Te presence of bioactive phytoconstituents in the plant extract was linked to its ability to scavenge DPPH radicals [53].Similar results on DPPH radical scavenging activities were reported on L. cornuta aqueous root extract and methanolic extracts of B. pinnatum [54,55].A study by Arika et al. on the DCM extract of Gnidia glauca also revealed the ability of the extract to scavenge DPPH radicals in a concentration-dependent manner [14].
Te capacity of biomolecules to contribute electrons to an oxidized substance or their oxidized intermediates determines their reducing power [14].In the ferric-reducing experiment, the quantity of ferrous complex (Fe 4 [Fe(CN) 6 ] 3 ) formed, served as a demonstration of the ability of X. spekei extract to donate electrons.Te content of the antioxidants in the Antioxidant and anticancer [39,44] 46.6 Purin-2,6-dione, 1,3-dimethyl-8-[2-nitrophenethenyl] 6.71 ± 1.34 Antioxidant [45] RT, retention time.Results are expressed as mean ± SEM for replicate measurement (n � 3). 10 Scientifca extract correlated with the degree to which the complex was formed following the reduction of Fe 3+ to Fe 2+ Te capacity of a plant extract to transfer electrons, as indicated in this experiment, shows that it has the potential to halt oxidative chain reactions as well as the potential to decrease oxidized lipid peroxidation intermediates [14].In parallel to this investigation, Onoja et al. established concentration-dependent ferric reduction activity in methanol leaf extract of Bryophyllum pinnatum [55].Also, research on the ferric reduction activity of several chosen polyphenols yielded similar results [56].
Chelation involves combination of metal ions with organic or inorganic compounds.Tis allows them to be removed from intracellular and extracellular regions, enabling excretion [57].Ion chelators scavenge reactive oxygen species and decrease accessible ions, thus reducing hydroxyl radicals produced through Fenton reactions [58].Te chelating activity of the X. spekei extract was concentration-dependent. Similar ion chelation activity by plant extracts was reported by Ebrahimzadeh et al. and Arika et al. [14,29].Furthermore, an investigation of the pulp, seeds, and fruits of Tetrapleura tetraptera showed that the presence of phytochemicals correlated with metal ion chelating activity [57].
Among free radicals, hydroxyl radicals are thought to be extremely reactive and capable of destroying most of the biomolecules present in the cells [59].Hydroxyl radicals are produced via the Fenton reaction (Fe 2+ + H 2 O 2 ⟶ F e 3+ + OH + OH•) from hydrogen peroxide or superoxide anions in the presence of metal cations.By oxidizing thiol (-SH) groups in the body, these radicals denature enzymes.Moreover, they harm cell membranes by oxidizing polyunsaturated fatty acid moieties of phospholipids [14].Tey also cause lipid peroxidation, as well as protein and DNA damage.
In this study, it was found that the capacity of X. spekei extract to scavenge hydroxyl radicals produced through Fenton's reaction was concentration-dependent.Both the extract and the standard had equivalent hydroxyl radical scavenging activity at the highest tested concentrations.Besides, the IC 50 of the DCM extract of X. spekei was equivalent to that of the standard.Tese fndings complement those of Sasikumar who demonstrated similar efects in hydroxyl radicals scavenging activity of Kedrostis foetidissima leaf extracts [60].Likewise, research studies on G. glauca leaf extract, diterpenoid extract of M. glyptostroboides, and ethanolic extract of T. serpyllum have also reported concentration-dependent hydroxyl radicals scavenging activity [14,57,61].
Lipid hydroperoxides are produced by unsaturated fatty acids, cholesterol, and esters [62].Te hydrogen atoms on methylene carbon of these intermediates actively take part in radical chain reactions that alter lipid membranes by oxidation and covalent bonding, ultimately resulting in cell death.Te lipid peroxidation inhibitory activity of the DCM extract of X. spekei was concentration-dependent. Tese fndings correlated with the fndings of Arika et al, on the antilipid peroxidation activity of G. glauca [14].Moreover, the outcome of this investigation was similar to the concentration-dependent lipid peroxidation inhibitory activity of unripe fruit of R. steudneri [63].Te antilipid peroxidation activity in this study can be ascribed to the phytochemicals present in the plant extract.Hydrogen peroxide is a nonradical oxygen species that afects a variety of biological functions.It can permeate biological subsequently producing hydroxyl radicals in the cells.Hydrogen peroxide can be transformed into highly reactive hydroxyl radicals in the presence of transition metals such as iron.In Fenton's reaction, soluble Fe 2+ transfers an electron to hydrogen peroxide, causing it to break down, and eventually produce hydroxyl radicals [58].
In this study, the X. spekei extract scavenged hydrogen peroxide in a concentration-dependent manner.A similar hydrogen peroxide scavenging ability of Kefe cumin extract was previously established by Ebrahimzadeh et al. [28].Moreover, research by Arika et al. using the DCM extract of G. glauca produced equivalent results [14].Te presence of phytocompounds that donate electrons to hydrogen peroxide neutralizing it into water, may be the cause of the concentration-dependent hydrogen peroxide radical scavenging activity witnessed in this study.
On the other hand, some investigations have found a concentration-dependent decline in the hydrogen peroxide radical scavenging ability [28,64].Tis could be as a result of high extract concentrations saturating the reactive centers of hydroxyl radicals [64].However, an increase in hydrogen peroxide scavenging activity of the X. spekei extract with an increase in extract concentration can be attributed to an increase in its active principles.
Te total antioxidant capacity of an extract refects the total amount of its bioactive constituents .At an acidic pH, the antioxidant activity of X. spekei extract was determined by its ability to reduce Mo (VI) to Mo (V).Tis is a redox reaction that occurs when an antioxidant oxidizes at the expense of an oxidant.With increasing concentration, the total X. spekei antioxidant capacity increased.Te results are consistent with the fndings of Babu et al.who reported on the antioxidant and free radical scavenging activities of Triphala [53].
Te antioxidant potential demonstrated by the DCM extract of X. spekei can be ascribed to its constituent phytochemicals as revealed by GC-MS analysis.Tese included polyphenols, fatty acids, favonoids, terpenes, phytosterols, and alkaloids.
Previous research has connected phenol content to antioxidant activity.According to El Jemli et al., the antioxidant activity of J. thurifera, J. oxycedrus, J. phoenicea, and T. articulate extracts is strongly correlated to their phenolic contents [65].Bajpai et al. [59], reported that phenolic compounds with aromatic and hydroxyl groups are efective at scavenging hydroxyl radicals.Besides, the ability of plant extracts to efectively scavenge hydrogen peroxide is also associated with phenolic compounds, which donate electrons, thereby reducing hydrogen peroxide to water [60].
Terpenes are primarily found in essential oil hydrocarbons and are classifed according to their isoprene unit (C 5 H 8 ).Terpenes have been found to have a variety of health benefts in humans against diseases associated with oxidative stress [69].
Phytosterols are plant steroids similar to cholesterol in structure and function.Tey are also antioxidants and physical stabilizers of cell membranes [72].Te antioxidant activity mediated by phytosterols has been shown to protect against atherosclerosis by reducing levels of oxidized LDL-C in both in vitro and in vivo studies [44].
Additionally, the DCM extract of X. spekei contained stigmasterol.Tis is one of the most common unsaturated plant sterols with antioxidant activity and belongs to the class of tetracyclic triterpenes.Hassanein et al. reported that pretreating V. faba extracts with stigmasterol before being exposed to salt stress reduced oxidative damage and increased catalase (CAT), ascorbate peroxidase (APX), and glutathione (GSH) antioxidants [72].Besides, the radical scavenging abilities of stigmasterol have been linked to its anticancer properties [39].
Various studies have also reported the antioxidant activities of hydrocarbons.Te hydrocarbon tetracosane which was found in X. spekei extract was reported as having antioxidant properties [73].In a similar study, the antioxidant activity of the alkene was also documented by Faridha Begum et al. [74].
Among compounds with antioxidant properties found in the X. spekei extract are fatty acids including methyl stearate, E-15-heptadecenal, dodecanoic acid, and undecyl ester.Plantderived unsaturated fatty acids, as well as saturated fatty acids, are strongly linked to antioxidant activity [75].Hussein and Mohammed Hamad found that extracted oil with compounds including methyl stearate and hexadecanoic acid methyl ester had signifcant antioxidant activity [32].Besides its antibacterial activity, the fatty acid compound E-15-heptadecenal has also been reported to have antioxidant activity [76].12 Scientifca Flavonoids found in plant extracts have been linked to high DPPH radical scaveging as well as hydroxyl radical scavenging activity [77,78].In this study, a benzofuran favonoid coumaran-6-ol-3-one, ybenzylidene) was identifed in the DCM extract of X. spekei.Several biological activities, including antioxidant activity, have been associated with benzofuran derivatives [79].In a similar study, Baliyan et al. associated the presence of favonoids with the antioxidant activity of the extract of Ficus religiosa [78].Also, Bajpai et al. linked the antioxidant activity of Adhatoda vasica to the favonoids in the plant extract [59].
Due to the presence of multiple phytochemicals, antioxidant activity from the plant extract is thought to be a synergistic task.Te radical scavenging activity of the plant extract is also mediated through multistep processes.Plant-derived antioxidants not only combat oxidative radicals directly but also indirectly through mechanisms such as upregulating antioxidant enzymes and regulating transcription of so-called vitagenes.Furthermore, some phytochemicals have been shown to have a direct efect on the mitochondrion, an organelle susceptible to oxidative stress [82].

Conclusion
From this study, it is inferred that the DCM extract of X. spekei has in vitro hypoglycemic activity, as demostrated by its ability to inhibit α-amylase enzyme, prevent glucose adsorption, impede glucose difusion, and enhance glucose transport across the cell membrane.Besides the extract was found to possess antioxidant properties which could potentially combat free radicals and restore redox homeostasis.Furthermore, the GC-MS analysis confrmed the presence of phytochemicals rich in hypoglycemic and antioxidant activities.However, there is a need to isolate bioactive compounds and determine their precise hypoglycemic and antioxidant mechanisms of action in vivo.Besides, isolated compounds may provide a potential lead in developing alternative therapeutic agents in future.

Figure 1 :
Figure 1: α-amylase enzyme % inhibition activity by the DCM extract of X. spekei.Bar graphs which do not share a letter across the tested concentration are statistically diferent (p < 0.05).Bar graphs within the same concentration are not signifcantly diferent from each other (p > 0.05).

Figure 2 :Figure 3 :
Figure 2: Glucose binding capacity of the DCM extract of X. spekei at diferent concentrations of glucose.Means which do not share a letter across the tested concentration are statistically diferent (p < 0.05).Data are presented as mean ± standard error of the mean (n � 3).

Figure 6 :Figure 7 :Figure 8 :
Figure 6: Hydroxyl radical scavenging activity of the DCM extract of X. spekei and standard antioxidant compound, gallic acid.Data are presented as mean ± standard error of the mean (n � 3).

Figure 11 :
Figure 11: Chromatograms obtained from the gas chromatography-mass spectrometry (GC-MS) analysis of the DCM extract of X. spekei.
Before the collection of the plant material, permission was sought from the National Commission for Science Technology and Innovation.Te license number issued was NACOSTI/P/21/9972.Xerophyta spekei leaves and stems were collected through the help of 2.1.Collection of Plant Materials.
Te injector and mass transfer line temperatures were set to 250 °C and 200 °C, respectively, with a 1 μl injection volume.With a run time of 70 minutes, the oven temperature was programmed to start at 35 °C for 5 minutes and increase by 10 °C/minute to 280 °C for 10.5 minutes and then by 50 °C/minute to 285 °C for 29.9 minutes.Te ion source temperature was 230 °C, solvent cut time was 3.3 minutes, scan speed was 1666 Hz, scan range was 40-550 m/z, and interface temperature was 250 °C.Te central database of the National Institute of Standards and Technology was used to interpret the GC-MS results.
mean (X̄± SEM) after it was found to conform to basic assumptions of parametric data.For inferential statistics, one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test for pairwise separation of means was performed.Te confdence interval was set at 95%.Diferences in antioxidant activities between the extract and the standard Scientifca at diferent concentrations were deduced using the unpaired Student's t-test.Te obtained results are presented in tables and graphs.

Table 1 :
IC 50 values of the DCM extract of X. spekei against antioxidant activity and α-amylase activity.Values are expressed as mean ± standard error of the mean (n � 3).Values which do not share uppercase superscript letters along the rows are signifcantly diferent (p < 0.05).Data are analyzed through the unpaired t-test.

Table 2 :
Percentage of glucose uptake by yeast cells.