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Antidiabetic potential of corn silk extracts, identification and drug properties of bioactive compounds

Adewole E

Department of Chemical Sciences, Afe Babalola University Ado, Ekiti State, Nigeria

Department of Pharmacy, Centre for Advanced Drug Research (CADR), COMSATS Institute of Information Technology, Pakistan

E-mail : bhuvaneswari.bibleraaj@uhsm.nhs.uk

Ojo A

Department of Chemical Sciences, Afe Babalola University Ado, Ekiti State, Nigeria

Omoaghe AO

Department of Physiology, Afe Babalola University Ado, Ekiti State, Nigeria

Enye LA

Department of Anatomy, Afe Babalola University Ado, Ekiti State, Nigeria

Guy Njateng S

Department of Pharmacy, Centre for Advanced Drug Research (CADR), COMSATS Institute of Information Technology, Pakistan

Laboratory of Microbiology and Antimicrobial Substances, Faculty of Science, University of Dschang, P.O. Box 67 Dschang, Cameroon

Sumera Z

Department of Pharmacy, Centre for Advanced Drug Research (CADR), COMSATS Institute of Information Technology, Pakistan

Jamshed I

Department of Pharmacy, Centre for Advanced Drug Research (CADR), COMSATS Institute of Information Technology, Pakistan

DOI: 10.15761/TiM.1000166

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Abstract

Background and objective: The aim of the research work was to investigate the anti-diabetic potential inhibitory activities of Corn Silk (Zea Mays) crude extracts against selective enzymes which are well known to be connected with diabetes mellitus; α-glucosidase, maltase glucoamylase, β-glucosidase and the two isoforms of reductase namely aldose and aldehyde reductases and the extracts were characterized and the compounds were screened for various drug properties.

Materials and methodology: The corn silk was harvested from fresh corn and air dried for five days at room temperature and blended into powdered form using electric blender. It was subsequently subjected to extraction using analytical grade of hexane and methanol solvents. Enzymatic reaction assays were performed using standard recommended protocol with slight modifications and the extracts were characterized using Gc-Ms and finally some of the identified compounds were screened for various degrees of drug properties using Online OSIRIS property explorer.

Results: Hexane and methanolic crude extracts were found to be promising inhibitors of α-glucosidase with an IC50 range of 31.6±0.4 μg/mL to 35.7±0.6 μg/mL. The inhibitory effect of the methanolic extract of IC50 (5.9±0.4 μg/mL and that of hexane extract (1.61±0.01 μg/mL) against maltase glucoamylase indicated good selective inhibitor against glucoamylase. The β-glucosidase screening of the extracts showed that they do not have good selective inhibitor properties. IC50 of Aldose reductase (ALR2) of methanolic extract (0.4±0.02 μg/mL) was better than that of hexane extract of IC50 (0.6±0.19 μg/mL). These ALR2 values showed very promising selective inhibitor activities when compared with the standard positive control, sorbinil of IC50 (3.10±0.20 μM). The aldehyde reductase (ALR1) of methanolic extract IC50 (0.27±0.10 μg/mL) was better than that of hexane extract of IC50 (0.36±0.04 μg/mL) and when compared with standard 10 mM vaproic acid IC50 (57.4±10 μM).

Some of the compounds identified when screened computationally using OSIRIS Online server explorer for their drug characteristics showed that D-Allose has drug properties of -2.3409, cLogS 0.331, cLogP (-3.3581), mutagenic(none), tumorigenic (none) and irritability (none); Thymol has cLogS (-2.535), cLogP(2.8448), drug properties (-2.3359), mutagenic (high), tumorigenic (none) and irritability (none).

Conclusion: The results of the enzymatic investigations, GC-MS and revelation of drug properties of some of the identified compounds could serve as an eye opener for the search for novel therapeutic agent for the treatment of diabetes mellitus. 

Key words

diabetes mellitus, α-glucosidase, glucoamylase, β- glucosidase, ALR1, ALR2 and GC-MS

Introduction

Diabetes mellitus is said to be a group of metabolic disorders characterized by innate or acquired inability to transport glucose from blood stream to cells. Natural product chemists have severally examined plants for different therapeutic properties such as for the treatment of diabetes mellitus in lowering blood glucose level [1] as a result of various bioactive compounds presents in the plant extracts.

Maize (Zea mays L) is an annual plant that is widely consumed, and it has been reported that it does has various pharmacological properties such as reduction of blood sugar level [2]. The inhibitory activities of aqueous extract of corn silk has been previously examined to be linked to the presence of phytochemicals present and this include; flavonoid, phenols, tannin and phytosterols [3].

For the management of diabetes mellitus, inhibitors of glucoamylase and α-glucosidase offer an effective strategy to modulate levels of post prandial hyperglycaemia via control of starch metabolism [3]. α- glucosidase is considered as a carbohydrate –hydrolase which releases α-glucose as against β-glucose. Equally, β-glucose can be released by glucoamylase; a functionally similar enzyme. The substrate selection of α-glucosidase is dependent on the subsite affinities of the enzyme’s active site [4]. The α-glucosidase inhibitor like acarbose, competitively and reversibly prohibit α-glucosidase in the intestines and it lowers the rate of glucose absorption by delaying carbohydrate digestion process.

Acarbose as an inhibitor, is capable of preventing diabetes development symptoms [5], this justifies the use of acarbose for diabetes treatment drug particularly by combining it with other effective diabetes control drugs. Moreover, it has been widely reported that Aldose reductase having two isoforms (ALR1 and ALR2) has been implicated in the cause of diabetic problems that may be connected to a much significance flux of glucose through the polyol pathway, caused in tissues such as lens, kidney, retina and nerves at high blood glucose levels. Therefore, the inhibitory property of aldose reductase is gaining recognition as a major therapeutic tool for the treatment of hyperglycemia-induced cardiovascular pathologies [6]. Long term drawbacks of diabetes include among others; cataractogenesis and microangiopathy (including nephropathy, retinopathy and neuropathy) thought to be linked to excess free glucose in corresponding tissues [7]. Widely research work has provided evidence of aldose reductase (AR) implication in diabetes severalty [8,9]. Scientific investigation to provide inhibitors of AR has been a major task for the chemists in order to effectively treat various degrees of complications associated with diabetes

Justification

The corn silk, after extensive literature search, it has been found that the hexane and methanolic crude extracts of the plant have not been analysed completely for α-glucosidase, β-glucosidase, maltase glucoamylase and aldose reductase (ALR1 and ALR2) for its in vitro anti-diabetic efficacy.

Present study

Research laboratory: The research work was carried out at the centre for the Advanced Drug Research (CADR), Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad, Pakistan in the month between July and August 2017.

Materials and instruments

All the reagents, solvents used are of analytical grade, α-glucosidase (from Saccharomyces cerevisiae), substrate p-nitrophenylα-D-glucopyranoside (pNPG), β-glucosidase (from sweet almonds) and 96 well plates were purchased from Sigma Aldrich.

Plant source

The corn silks (zea mays) were collected in a local farm during harvesting of the maize corn in Ado Ekiti, Ekiti State, Nigeria on the 10th of May 2017 and were air dried for five days. The silk was blended to powdered form and stored for analysis (Figure 1).

Figure 1. Corn silk (Zea mays).

Crude extract preparation

The extraction was done by soaking two hundred grams of powdered corn silk in 1000 ml of hexane and methanol for a period of seven days and filtered through whatman filter paper. The extract was concentrated using a rotary evaporator at 35°C and the dried extract was stored at room temperature for further use. The stock sample was prepared by dissolving 10 mg of dried crude extracts in 1 mL of 100% dimethyl sulfoxide (DMSO) and labelled as stock (10 mg/ml), working solution was made as 1 mg/ml.

Extraction of intestinal maltase-glucoamylase enzyme

Intestinal maltase-glucoamylase was extracted by following the literature reported procedure [10,11]. The enzyme was extracted from white male rats (1-2 months) weighing 150-250 g and starved for 12 h before death. Rats were killed by cervical dislocation. Whole intestines were gently removed and washed with ice cold 0.9% NaCl. For extraction, intestines were cleaned and cut longitudinally, and mucosal scrapings of 5-6 rats were combined and homogenized in 50 volumes of 5 mM EDTA; pH 7.0. All the steps were carried out at 4°C. Homogenized intestines were centrifuged at 15,000 rpm for 45 min at 4°C and the supernatant was discarded. Pellet was re-suspended in 90 mL of ice-cold water followed by 5 mL of 0.1 M-EDTA/0.2 M potassium phosphate, pH 7, and 5 mL of 0.1 M cysteine was added. Mixture was incubated at 37°C for 30 min. Pellet was collected by centrifugation at 15,000 rpm for 45 min and the supernatant was discarded. Intestinal suspension was re-dissolved in 10 mM potassium phosphate buffer, pH 6.8, to which 4 mg papain and 0.4 mg cysteine were added. The activated papain solution was immediately used. Mixture was incubated at 37°C for 40 min with continuous shaking. It was then centrifuged at 15,000 rpm for 90 min. Supernatant was collected and precipitated with ammonium sulphate to 80% saturation. It was dialyzed overnight against distilled water with three changes of water (40 volume every time). The residue was re-dissolved in 4 mL of 10 mM-potassium phosphate (pH 7.0) and protein concentration was determined. The extracted enzyme was stored at ‒80°C until further use.

Maltase-glucoamylase inhibition assay

Maltase-glucoamylase inhibition assay was carried out using the substrate p-nitrophenyl α-d-glucoside using reported procedure [12]. The reaction mixture contained 70 µL buffer, 10 µL extracted enzyme (25.0 µg of protein) and 10 µL of test compounds and incubated at 37°C for 5 min. After the incubation, 10 µL of p-NPG (10 mM, prepared in assay buffer) was added to each well of a 96 well plate and further incubated at 37°C for 30 min. The activity of the test compounds against maltase glucoamylase was determined by measuring p-nitrophenol at a wavelength of 405 nm. Acarbose was used as a positive control. Each experiment was performed in triplicates.

α-glucosidase inhibition study

The inhibitory effect of the 1 mg/ml was performed by slight modification of a previously published method [13]. Briefly, solutions of α-glucosidase (from Saccharomyces cerevisiae) and its substrate p-nitrophenyl α-D-glucopyranoside (pNPG) were prepared in phosphate buffer (70 mM, pH 6.8). Buffer was used for the preparation of inhibitor solutions. The inhibition assays were conducted by adding inhibitor solution (10 μL) to 70 μL buffer and 10 μL of enzyme solution (2.5 unit/mL) in 70 mM phosphate buffer (pH 6.8) followed by pre-incubation at 37℃ for 5 min. After pre-incubation, 10 μL of 10 mM substrate (pNPG) prepared in phosphate buffer was added to the mixture to initiate enzymatic reaction. The reaction mixture was incubated at 37°C for 30 min. Acarbose was used as a positive control. The α-glucosidase activity was determined by measuring the p-nitrophenol released from pNPG at 405 nm using an Elx 800 Micro plate reader.

β- glucosidase inhibition study

The evaluation of inhibitory activity against β-glucosidase was performed with slight modification of the previously published method [14]. Briefly, β-glucosidase (from sweet almonds) enzyme and p-nitrophenyl β-D-glucopyranoside (pNPG) as substrate were prepared in 0.07 M phosphate buffer (pH 6.8). The inhibition assays were conducted by adding inhibitor solution (10 μL) to 70 μL buffer and 10 μL of enzyme solution (2.0 unit/mL) in 0.07 M phosphate buffer (pH 6.8) followed by pre-incubation at 37°C for 5 min. Following pre-incubation, 10 μL of 10 mM p-nitrophenyl glucopyranoside (pNPG) in phosphate buffer was added as a substrate to the mixture to start the reaction. The reaction mixture was then incubated at 37°C for 30 min. Negative control contained 10 μL of 10% DMSO instead of inhibitor. Castanospermine was used as a positive control.

Extraction of aldose enzymes

Material and instruments: All the chemicals needed for the enzyme extraction were of analytical grade. Substrates (D, L-glyceraldehyde and sodium-D-glucoronate), and nicotinamide adenine dinucleotide phosphate (NADPH) as co-factor were purchased from Sigma Aldrich. Eliza microplate reader was used with a UV range of 340 nm for the enzymatic reaction.

Isolation of enzymes

Isolation and purification of Aldose reductase enzyme (ALR2): This was done according to [15] with minor modification. Briefly, ALR2 was isolated from fresh calf lenses. The lenses were removed from the eyes immediately after slaughtering and were frozen until used. Lenses (100-200 g) were homogenized in cold distilled water and the homogenate was centrifuged at 10,000×g for 15 minutes to remove the insoluble materials. Precipitated material was discarded as it contained lipids. Supernatant layer was separated, and ammonium sulphate salt was added to make the saturation up to 40% (for 52 ml), 11.752 g of (NH4)2SO4. Additional inert protein was removed by increasing the concentration of ammonium sulphate up to 50% saturation (for 48 ml), 2.784 g of (NH4)2SO4 was added. Pure ALR2 was precipitated by addition of powdered (NH4)2SO4 to 75% saturation (for 45.5 ml), 7.2345 g (NH4)2SO4 was added. After centrifugation, supernatant was discarded, and precipitate enzyme was re-dissolved in 50 mM NaCl and dialyzed over night against 4 litres of mM NaCl. The volume of the suspension was recorded and the sample was dialyzed overnight against 50 mM NaCI (double replacement of dialysis solution). After dialysis, the volume of the sample was recorded, treated with liquid nitrogen and samples stored in 1 mL aliquots in the eppendorf tubes at ‒80°C.

Isolation and purification of ALR1 (Aldehyde reductase): Kidneys were removed from the calf soon after slaughtering and dissolved in 3 volumes of 10 mM sodium phosphate buffer, pH 7.2, containing 0.25 M sucrose, 2.0 mM EDTA dipotassium salt and 2.5 mM β-mercaptoethanol. The homogenate was Centrifuged the at 12,000×g at 0-4°C for 30 minutes. The precipitate was discarded as it contained insoluble lipids. Collected supernatant layer was subjected to 40% ammonium sulphate saturation to isolate ALR1. This 40% saturated liquid was centrifuged at 12,000×g at 0-4°C for 30 minutes. Again, precipitate was discarded, and supernatant was subjected to 50% saturation with ammonium sulphate salt. The same procedure was repeated with this and in the last step; ammonium sulphate was added to increase saturation up to 75%. Centrifuge the supernatant at 12,000×g at 0-4°C for 30 minutes, causing the precipitation of ALR1. Now precipitated material was collected and supernatant discarded. The precipitated material containing enzyme was re-dissolved in 10 mM sodium phosphate buffer, pH 7.2 containing 2.0 mM EDTA dipotassium salt and 2.0 mM β-mercaptoethanol and dialyzed overnight using the same buffer. The dialyzed material containing ALR1 enzyme was stored at -80°C before use [16-18].

Total protein evaluation in the extracted enzymes

Test Procedure using total protein kit (Laboratory kit made by: (Chemelex, S.A. Pol.ind.Can,Castells. Industrial 113 NAu J.08420, Canovelles, Barcelona [Spain])

  • Assay Conditions
    • Wavelength -------------------- 540 nm (530-550) nm
    • Cuvette-------------------------- 1 cm light path
    • Temperature------------------37°C
  • The instrument was adjusted to zero with distilled water
  • Pipette into a cuvette

(Table 1)

Table1. Protein assay protocol

 

Blank

Calibrator

Sample

R1a (mL)

0.25

0.25

0.25

Calibratorb (μL)

-

6.25

-

Sample(μL)

--

----

6.25

aTotal protein solution (Ref No: 30350-125 ml)

bTotal protein calibrator (AP: 0314, Ref No 30941-5 ml)

  1. The mixture was done and incubate for 5 minutes at 37°C
  2. Absorbance reading was taken, and colour change was stable for 30 minutes.

Calculation:

Optimization of aldehyde and aldose reductase enzymes concentration in the assay

For optimization of enzyme aldehyde reductase, different volume of enzyme crude was taken ranging from 5 μL to 50 μL in each well along with 100 mM potassium phosphate buffer ranging from 50-5 μL in each well, 20 μL of substrate (D, L-glyceraldehyde for ALR2 and Sodium-D-glucoronate for ALR1 50 mM) and 25 μL of cofactor NADPH (0.5 mM) was used. One unit of enzyme is defined as “an amount of enzyme required catalyzing the oxidation of 1 μM of NADPH per minute (Table 2) (Figure 2).

Table 2. Optimization protocol of purified enzymes

96 well

plate

Buffer

(μL)

Enzyme

(μL)

Substrate

 (μL)

Cofactor

(NADPH) (μL)

A

50

5a

20

25

B

45

10

20

25

C

40

15

20

25

D

35

20

20

25

E

30

25

20

25

F

25

30

20

25

G

20

35

20

25

H

15

40

20

25

amost consistent with highest absorbance difference

Figure 2. Absorbance reading of first optimization protocol using FLUO OMEGA micro plate reader.

Further optimization was done using 5 μL of enzyme with stable absorbance and amount of protein was quantified in 5 μL of the enzyme and found to be 130 μg for ALR2 and 1094 μg for ALR1. Further dilution of 5 μL enzyme was perfor med using buffer (6.2 pH for ALR2 and pH7.2 for ALR1) and another optimization was performed to obtain the most consistent and stable μL of enzyme to be adopted for the screening and determination of IC50 of the extracts with % inhibition >50% (Table 3) (Figures 3 and 4).

Table 3. Optimization protocol of diluted 5 μL of enzyme

96 well

plate

 

Buffer

 (μL)

Enzyme

 (μL)

Substrate

(μL)

Cofactor

(NADPH) (μL)

A

55

0

20

25

B

50

5

20

25

C

45

10

20

25

D

40

15

20

25

E

35

20

20

25

F

30

25

20

25

G

25

30

20

25

H

20

35a

20

25

 

aadorpted as most consistent absorbance after 5 minutes and 10 minutes reading

Figure 3. Absorbance reading of second optimization protocol using FLUO OMEGA micro plate reader showing consistency of 35 μL of enzyme after 5 minutes incubation.

Figure 4. Absorbance reading of second optimization protocol using FLUO OMEGA micro plate reader showing consistency of 35 μL of enzyme after 10 minutes incubation

Determination of aldose reductase (ALR2) inhibitory activity

UV spectrophotometer was used at 340 nm in order to determine the activity of aldose reductase by measuring the NADPH consumption. Each well of the 96-well plate contains exactly 100 μL of assay mixture containing phosphate buffer 100 mM at pH 6.2 (10 μL), with 10 μL of 1 mg/ml of crude extracts followed by addition of 35 μL of enzyme and 20 μL of substrate. The mixture was incubated at 37°C for 5 min and for the enzymatic reaction to run properly 0.5 mM NADPH (20 μL) as a cofactor was added and reading was taken at 340 nm. The mixture was incubated again at 37°C for 10 min and reading was taken at the respective UV range in ELIZA plate reader. As positive and negative control, 10 μL of 10 mM Sorbini and 20 μL buffer solution, respectively, were used. The enzymatic reaction was run in triplicate with a final volume of 100 μL in each well. Absorbance was noted, and results were analysed.

Determination of aldehyde reductase (ALR1) inhibitory activity

UV spectrophotometer was used at 340 nm in order to determine the activity of aldehyde reductase by measuring the NADPH consumption. Each well of the 96-well plate contains exactly 100 μL of assay mixture containing phosphate buffer 100 mM at pH 7.2 (10 μL), with 10 μL of 1 mg/ml of crude extracts followed by addition of 35 μL of enzyme and 20 μL of substrate (sodium D-glucoronate). The mixture was incubated at 37°C for 5 min and for the enzymatic reaction to run properly 0.5 mM NADPH (20 μL) as a cofactor was added and reading was taken at 340 nm. The mixture was incubated again at 37°C for 5 min and reading was taken at the respective UV range in ELIZA plate reader. As positive and negative control, 10 μL of 10 mM vaproic acid and 20 μL buffer solution, respectively, were used. The enzymatic reaction was run in triplicate with a final volume of 100 μL in each well. Absorbance was noted, and results were analysed.

Statistical analysis

The total percentage inhibitions were calculated by the formula:

The IC50 values were calculated using non-linear curve fitting program PRISM 5.0 (Graph pad, San-Diego, California, USA)

GC/MS analysis

GC-MS analysis of extracts of corn silk was performed using TurboMass GC System, fitted with an Elite-5 capillary column (30 m, 0.25 mm inner diameter, 0.25 μm film thickness; maximum temperature, 350°C coupled to a Perkin Elmer Clarus 600C MS. Helium was used as gas carrier at a constant flow rate of 1.0 ml/min. The injection, transfer line and ion source temperatures were 280°C. The ionizing energy was 70 eV. The oven temperature was programmed from 70°C (hold for 2 min) to 280°C (hold for 10 min) at a rate of 5°C/min. The crude extract was solubilised with chloroform and filtered with syringe filter (Corning, 0.45 μm). Volumes of 1 μl of the crude extracts were injected with a split ratio 1:20. The data were obtained by collecting the mass spectra within the scan range 50-550 m/z. The identification of chemical compounds in the extracts was based on GC retention time; the mass spectra matched those of standards available at NIST library.

Results

(Tables 4-8)

Table 4. Amount of protein in ALRI and ALR2 enzymes

Enzyme

Protein content

(μg/μL)

ALR1

22.88

ALR2

26.00

Table 5. Inhibitory effect of crude extracts

Extracts

α-glucosidase

 IC50±SEM

 (μg/mL)

glucoamylase

IC50±SEM (μg/mL)

β-glucosidase

% inhibition ±SEM

ALR2

IC50±SEM

(μg/mL)

ALR1

IC50±SEM

(μg/mL

Hexane extract

31.6±0.4

1.6±0.01

9.60±1.24

0.6034±0.19

0.36±0.37

MeOH extract

35.7 ± 0.6

5.9±0.4

13.76±0.88

0.4±0.20

0.27±0.07

Acarbosea

234.6±2.01(μM)

234.6±2.01(μM)

Not Tested

Not Tested

Not Tested

Castonospermineb

Not tested

Not tested

59.98%

Not tested

Not tested

Vaproic acidc

Not tested

Not tested

Not tested

57.4 ±10

Not tested

Sorbinild

Not tested

Not tested

Not tested

Not tested

3.10 ±0.20

SEM=standard mean error

aα-glucosidase standard

bβ-glucosidase standard

cALR1 standard

dALR2 standard (dReported IC50 of 3.42 μM of Sorbinil by Rakowitz et al. [17]a and Ali et. al. [17]b)

Table 6. Identified compounds in hexane crude extract

Compound name

Molecular formula

Retention time (min.)

CAS

Molecular weight

Thymol

C10H14O

14.763

89-83-8

150

Phenol 3,5-Bis (1,1 dimethylethyl)

C14H22O

22.281

1138-52-9

206

D-mannitol, 1-0-(22-hydroxylocosyl)

C28H58O7

46.641

119049-22-8

506

β-D-ribopyranoside, methyl

C6H12O5

8.658

17289-61-1

164

9-Oxime-3,6-dichloro-2,7-Bis
(2-piperidinoethoxy) fluorine

C27H33O3N3Cl2

33.731

900111-06-2

517

D-allose

C6H12O6

8.596

2595-97-3

180

Fucose, cyclic ethylene mercaptal

C8H16O4S2

10.842

3650-70-2

240

Sulfurous acid,butyl hexadecyl ester

C20H42O3S

21.141

900309-18-3

362

[BI-1,4-cyclohexadien-1-yl]-3,3’6,6’-tetrone,
4,4’-dihydroxy-2,2’,5,5’-tetramethyl

C16H14O6

21.146  

10493-51-3

302

 Table 7. Identified compounds in methanolic crude extract

Compound name

Molecular formula

CAS

Retention time (min)

Molecular weight

L-ascorbic acid, 6-octadecanoate

C24H42O7

10605-09-1

19.375

442

Heptacosanoic acid,25-methyl ester

C29H58O2

900112-14-5

29.924

438

[BI-1-4, cyclohexadien-1-yl]-3,3’,6,6’-tetrone,4,4’ dihydroxy-2,2’,5,5’-tetra methyl

C16H14O6

10493-51-3

20.241

302

2-methyl-2-hydroxy-decalin-4A-carboxylic acid,2,4A-lactone

C12H18O2

900146-226-6

14.593

194

D-mannitol, 1-0-(22-hydroxylocosyl)

C28H58O7

119049-22-8

34.536

506

1-Octanone,1-(2-octylcyclopropyl)

C19H36O

54965-36-5

21.971

280

Table 8. Drug properties of some of identified compounds in the crude extracts determined by OSIRIS property explorer

Compound

Drug likeness

Mutagenic

Tumorigenic

cLogS

cLogP

Polar surface area

(A°)

%Absorption

H-bond Acceptor

H-bond

Donor

Irritability

D-Allose

-2.3409

None

None

0.331

-3.3581

118.22

68.21

6

5

None

Fucose

0.38132

None

None

-0.256

-1.6941

90.15

77.90

5

4

None

Thymol

-2.3359

High

None

-2.535

2.8448

20.23

102.02

1

1

None

(Figures 5 and 6)

Figure 5. Chromatogram of methanolic crude extract of corn silk (Zea mays)

Figure 6. Chromatogram of hexane crude extract of corn silk (zea mays)

Discussion

The tested extracts were highly potent inhibitors of α-glucosidase with IC50 values in μg/ml ranges. Hexane extract was found to be the most potent inhibitor of α-glucosidase with IC50 value as low as 31.6±0.4 μg/mL. To the best of my knowledge, after extensive literature search, this is the first report of α-glucosidase inhibition potency of this hexane extract of corn silk (zea mays) and interestingly it was much better than the standard inhibitor acarbose, methanolic extract equally showed good and comparable inhibition potencies against α-glucosidase with IC50 35.7±0.6 μg/mL. The inhibitory effect of the methanolic extract of IC50 (5.9±0.4 μg/mL) and that of hexane extract IC50 (1.6±0.01 μg/mL) against glucoamylase indicated good selective inhibitor against glucoamylase. In addition, to confirm the inhibitory activities of the extracts against β-glucosidase collected from sweet almonds, the two extracts showed very less inhibition with less than 12.77%.

The promising results of the inhibitory potentials against α-glucosidase and glucoamylase of the two extracts may not be unconnected to the presence of the various bioactive compounds as revealed by the gas chromatography mass spectrophotometer. The α-glucosidase inhibitory activity in the hexane extract was positively associated with the amount of thymol [19], also mannitol which is only partially absorbed by the body and significantly reduces the rise in blood glucose and insulin levels that occur following the ingestion of glucose.

IC50 of Aldose reductase (ALR2) of methanolic extract (0.4±0.02 μg/mL) was better than that of hexane extract of IC50 (0.6±0.19 μg/mL). These ALR2 values showed very promising selective inhibitor activities when compared with the standard positive control, sorbinil of IC50 (3.10±0.20 μM).

The aldehyde reductase (ALR1) of methanolic extract IC50 (0.27±0.10 μg/mL) was better than that of hexane extract of IC50 (0.36±0.04 μg/mL) and when compared with standard 10 mM vaproic acid IC50 (57.4±10 μM)

Osiris Drug properties of some identified compounds

Some of the identified compounds were screened computationally for their drugs properties using online OSIRIS property explorer server and compounds such as D-Allose, Thymol and Fucose possess different drug properties such as cytotoxicity (mutagenic, tumorigenic, irritability), solubility, H-bond donor, H-bond acceptor and drug likeness.

Conclusion

The results of GC-MS and enzymatic investigations support the usage as traditional anti-diabetic herb [20]. It is worthy to state further that the corn silk extracts can be subjected to further studies as potential anti-diabetic agents with selective inhibition against glucoamylase and α-glucosidase based on the proved evidence of potent and selective inhibitory potentials the plant has.

Statement of significance

This study discovered the promising and excellent anti-diabetic inhibitory potentials of the methanolic and hexane crude extracts of corn silk against α-glucosidase, glucoamylase and isoform of reductase (ALR1 and ALR2) which can serve as a tool for the discovery of inhibitors for the treatment of diabetes mellitus and most importantly, the various drug properties revealed by the online OSIRIS server explorer will be an eye opener for the chemists to look into the pharmacological application of the compounds in formulating drugs. This study could be explored by researchers and a new anti-diabetic agent may be arrived at.

Acknowledgement

Dr. Adewole E. sincerely appreciates ‘The world academy of science’ (TWAS) for the Postdoctoral fellowship opportunity granted under the supervision of Professor Jamshed Iqbal, Head, Centre for Advanced Drug Research (CADR), Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad, Pakistan in 2017.

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Editorial Information

Editor-in-Chief

Ying-Fu Chen
Kaohsiung Medical University
Taiwan

Article Type

Research Article

Publication history

Received date: September 28, 2018
Accepted date: October 15, 2018
Published date: October 18, 2018

Copyright

© 2018 Adewole E. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation

Adewole E, Ojo A, Omoaghe AO, Enye LA, Guy Njateng S, et al. (2018) Antidiabetic potential of corn silk extracts, identification and drug properties of bioactive compounds. Trends Med 18: DOI: 10.15761/TiM.1000166

Corresponding author

Adewole E

Department of Chemical Sciences, Afe Babalola University Ado, Ekiti State, Nigeria and Department of Pharmacy, Centre for Advanced Drug Research (CADR), COMSATS Institute of Information Technology, Pakistan

E-mail : bhuvaneswari.bibleraaj@uhsm.nhs.uk

Figure 1. Corn silk (Zea mays).

Figure 2. Absorbance reading of first optimization protocol using FLUO OMEGA micro plate reader.

Figure 3. Absorbance reading of second optimization protocol using FLUO OMEGA micro plate reader showing consistency of 35 μL of enzyme after 5 minutes incubation.

Figure 4. Absorbance reading of second optimization protocol using FLUO OMEGA micro plate reader showing consistency of 35 μL of enzyme after 10 minutes incubation

Figure 5. Chromatogram of methanolic crude extract of corn silk (Zea mays)

Figure 6. Chromatogram of hexane crude extract of corn silk (zea mays)

Table1. Protein assay protocol

 

Blank

Calibrator

Sample

R1a (mL)

0.25

0.25

0.25

Calibratorb (μL)

-

6.25

-

Sample(μL)

--

----

6.25

aTotal protein solution (Ref No: 30350-125 ml)

bTotal protein calibrator (AP: 0314, Ref No 30941-5 ml)

  1. The mixture was done and incubate for 5 minutes at 37°C
  2. Absorbance reading was taken, and colour change was stable for 30 minutes.

Table 2. Optimization protocol of purified enzymes

96 well

plate

Buffer

(μL)

Enzyme

(μL)

Substrate

 (μL)

Cofactor

(NADPH) (μL)

A

50

5a

20

25

B

45

10

20

25

C

40

15

20

25

D

35

20

20

25

E

30

25

20

25

F

25

30

20

25

G

20

35

20

25

H

15

40

20

25

amost consistent with highest absorbance difference

Table 3. Optimization protocol of diluted 5 μL of enzyme

96 well

plate

 

Buffer

 (μL)

Enzyme

 (μL)

Substrate

(μL)

Cofactor

(NADPH) (μL)

A

55

0

20

25

B

50

5

20

25

C

45

10

20

25

D

40

15

20

25

E

35

20

20

25

F

30

25

20

25

G

25

30

20

25

H

20

35a

20

25

 

aadorpted as most consistent absorbance after 5 minutes and 10 minutes reading

Table 4. Amount of protein in ALRI and ALR2 enzymes

Enzyme

Protein content

(μg/μL)

ALR1

22.88

ALR2

26.00

Table 5. Inhibitory effect of crude extracts

Extracts

α-glucosidase

 IC50±SEM

 (μg/mL)

glucoamylase

IC50±SEM (μg/mL)

β-glucosidase

% inhibition ±SEM

ALR2

IC50±SEM

(μg/mL)

ALR1

IC50±SEM

(μg/mL

Hexane extract

31.6±0.4

1.6±0.01

9.60±1.24

0.6034±0.19

0.36±0.37

MeOH extract

35.7 ± 0.6

5.9±0.4

13.76±0.88

0.4±0.20

0.27±0.07

Acarbosea

234.6±2.01(μM)

234.6±2.01(μM)

Not Tested

Not Tested

Not Tested

Castonospermineb

Not tested

Not tested

59.98%

Not tested

Not tested

Vaproic acidc

Not tested

Not tested

Not tested

57.4 ±10

Not tested

Sorbinild

Not tested

Not tested

Not tested

Not tested

3.10 ±0.20

SEM=standard mean error

aα-glucosidase standard

bβ-glucosidase standard

cALR1 standard

dALR2 standard (dReported IC50 of 3.42 μM of Sorbinil by Rakowitz et al. [17]a and Ali et. al. [17]b)

Table 6. Identified compounds in hexane crude extract

Compound name

Molecular formula

Retention time (min.)

CAS

Molecular weight

Thymol

C10H14O

14.763

89-83-8

150

Phenol 3,5-Bis (1,1 dimethylethyl)

C14H22O

22.281

1138-52-9

206

D-mannitol, 1-0-(22-hydroxylocosyl)

C28H58O7

46.641

119049-22-8

506

β-D-ribopyranoside, methyl

C6H12O5

8.658

17289-61-1

164

9-Oxime-3,6-dichloro-2,7-Bis
(2-piperidinoethoxy) fluorine

C27H33O3N3Cl2

33.731

900111-06-2

517

D-allose

C6H12O6

8.596

2595-97-3

180

Fucose, cyclic ethylene mercaptal

C8H16O4S2

10.842

3650-70-2

240

Sulfurous acid,butyl hexadecyl ester

C20H42O3S

21.141

900309-18-3

362

[BI-1,4-cyclohexadien-1-yl]-3,3’6,6’-tetrone,
4,4’-dihydroxy-2,2’,5,5’-tetramethyl

C16H14O6

21.146  

10493-51-3

302

 Table 7. Identified compounds in methanolic crude extract

Compound name

Molecular formula

CAS

Retention time (min)

Molecular weight

L-ascorbic acid, 6-octadecanoate

C24H42O7

10605-09-1

19.375

442

Heptacosanoic acid,25-methyl ester

C29H58O2

900112-14-5

29.924

438

[BI-1-4, cyclohexadien-1-yl]-3,3’,6,6’-tetrone,4,4’ dihydroxy-2,2’,5,5’-tetra methyl

C16H14O6

10493-51-3

20.241

302

2-methyl-2-hydroxy-decalin-4A-carboxylic acid,2,4A-lactone

C12H18O2

900146-226-6

14.593

194

D-mannitol, 1-0-(22-hydroxylocosyl)

C28H58O7

119049-22-8

34.536

506

1-Octanone,1-(2-octylcyclopropyl)

C19H36O

54965-36-5

21.971

280

Table 8. Drug properties of some of identified compounds in the crude extracts determined by OSIRIS property explorer

Compound

Drug likeness

Mutagenic

Tumorigenic

cLogS

cLogP

Polar surface area

(A°)

%Absorption

H-bond Acceptor

H-bond

Donor

Irritability

D-Allose

-2.3409

None

None

0.331

-3.3581

118.22

68.21

6

5

None

Fucose

0.38132

None

None

-0.256

-1.6941

90.15

77.90

5

4

None

Thymol

-2.3359

High

None

-2.535

2.8448

20.23

102.02

1

1

None