In-Vitro and In-Silico Alpha Glucucosidase Inhibitory activity of Oroxylum indicum

 

S. Gejalakshmi1, N. Harikrishnan1, Anas S. Mohameid2

1Faculty of Pharmacy, Dr. M.G.R. Educational and Research Institute, Velappanchavadi, Chennai-77,

Tamil Nadu 600095, India.

2Faculty of Pharmacy, Omar Al Mukthar University, Al - Bayda, Libya.

*Corresponding Author E-mail: gejampharm@gmail.com

 

ABSTRACT:

Background: Diabetes mellitus is a metabolic condition characterized by elevated blood glucose levels in the bloodstream. It occurs due to the inadequate amount of insulin secreted in the body or resistance of insulin receptors. Objective: In the present study, for its effect on alpha-amylase and alpha-glucosidase enzymes, Oroxylum indicuma flavone glycoside was assessed using in-vitro assays by removing the respective enzymes from whole wheat and barley in conjunction with in-silico analysis. Method: in-vitro alpha amylase inhibitory activity and in-vitro alpha glucosidase inhibitory activity was performed using acarbose as a standard drug. The molecular docking study was performed using Schrodinger (Maestro V 11.5) software. The parameters glide score, Lipinski rule for drug likeliness, bioactive scoring and ADME properties were assessed in the docking study. In addition, baicalein's antioxidant function was assessed using DPPH assay, nitric oxide scavenging activity. The cytotoxicity of Oroxylum indicumwas evaluated using the Brine shrimp lethality assay. Results: The alpha-amylase assay performed showed IC50 value of 48.40 g/ml for Oroxylum indicumwhereas alpha-glucosidase assay showed an IC50 value of 16.03 g/ml. Oroxylum indicumshows the glide score of-5.565 with 5EOF and glide score of -5.339 with 5NN8 in the molecular docking study. The highest percentage of DPPH radical scavenging activity and nitrous oxide scavenging activity were found to be.27% at160 g/ml and 50.02% at the concentrations of 160 g/ml respectively. Conclusion: Based on further in vivo and clinical trials, Oroxylum indicummay be used for the management of hyperglycaemia.

 

KEYWORDS: Diabetes mellitus, alpha-amylase, alpha-glucosidase, Oroxylum indicum, insilico.

 

 


INTRODUCTION:

There are many research works carried out on diabetes mellitus. Since, it is a lifestyle modification disorder in current population. The resistance of insulin receptors presents in the beta (β) cells of the pancreas cause type 2 diabetes mellitus which may produce serious effects in humans such as diabetic nephropathy, neuropathy, retinopathy, cardiovascular and cerebrovascular disease1,2.

 

It has been judged that about 592 million adults will enhance to diabetes by the year 2035 due to increase in population size, ageing and to the extent of calories rich fatty fast foods3. Postprandial diabetes mitigation is the diagnostic approach to treating diabetes. This can be achieved by inhibiting alpha amylase and alpha glucosidase hydrolysing carbohydrate enzymes. These two enzymes play a significant role in carbohydrate digestion. The breakdown of large insoluble starch to absorbable molecule is performed by an enzyme called alpha-amylase, whereas the hydrolysing enzyme catalysing the digestion of starch and disaccharides at the end-stage is called as alpha-glucosidase4. Therefore, inhibiting these enzymes delays the absorption or production of glucose in the blood. Thus, the inhibition plays a main role to decrease postprandial hyperglycaemia. Due to some gastrointestinal side effects of acarbose and voglibose, a synthetic inhibitor of this enzyme occurs here preferred over natural sources like flavonoids which have no side effects. Hence, in the present study a synthetic drug Oroxylum indicumis used to evaluate the hyperglycaemic activity against the specified target enzyme by In-Vitro and In-Silico analysis5

 

Scutellariabaicalensis is a Chinese herbal medicine that is used to treat diabetic mellitus. The active constituent Oroxylum indicumis yellow crystalline solid found in several species of Scutellariabaicalensis. Oroxylum indicum consists of three hydroxyl group at the position of (C-5,6,7). The dried root of Oroxylum indicumcontains thirty types of flavonoids, out of that three flavonoids namely baicalin, Oroxylum indicum and wogonin are the major components of Scutellariabaicalensis6. Oroxylum indicumpossesses several health beneficial properties such as antioxidant, anti-HIV, anticancer, anti-inflammatory, antiviraland free radical scavenging effects. Many literature reviews possess that dietary consumption of Oroxylum indicumenhances glucose tolerance in obese mice. Previous study by Yu Fu et al, identified that dietary consumption of Oroxylum indicumenhances the blood insulin level and hyperglycaemia in obese diabetic mice7. Another study by Zhang et al showed that the anti-diabetic activity of Oroxylum indicumis consistent with intestinal microbiota regulation in animal models.8

 

The current work is being performed using in vitro and in-silico research to test the antidiabetic efficacy of Oroxylum indicumagainst the target enzymes alpha-amylase and alpha-glucosidase.

 

MATERIALS AND METHODS:

Chemicals and instruments used:

Oroxylum indicum was purchased from Sigma Aldrich (Product ID: 465119). Allthe chemicals used in the study were obtained from Merck and Co chemicals Pvt Ltd. The absorbance of the in vitro assays was performed using Shimadzu UV spectrophotometer UV 1800 Model. The brine shrimp lethality assay was evaluated in a 24 well plate and the lethality was observed using Magnus MLX-B Plus (SP) Inclined Binocular Microscope. Aquatic Remedies Artemia Bhrime Shrimps was purchased from Amazon sold by Sagar aquarium.

 

In-vitro alpha amylase inhibitory activity:

The Wheat alpha amylase extraction was carried out by the previous method as defined by Nair et al. The assay mixture containing 200μl of 0.02M sodium phosphate buffer, 20μl of enzyme and 5-160μg/ml of plant extracts was incubated at room temperature for 10 minutes, followed by the addition of 200μl of starch to both test tubes. 400μl of DNS reagent was applied and put for 5 minutes in a boiling water bath, cooled and diluted with 15ml of distilled water, and the absorbance was measured at 540nm.

 

The % inhibition was calculated according to the formula:

 

The IC50 values were derived from the percent inhibition versus log inhibitor concentration plots and were estimated from the mean inhibitory values by non-linear regression analysis. As the reference inhibitor of alpha-amylase, Acarbose was used. Both studies were conducted in triplicates.9

 

In-vitro alpha glucosidase inhibitory activity:

The yeast alpha glucosidase was dissolved in 100mM phosphate buffer pH 6.8 and was used as an extract of the enzyme. As the substrate, P-Nitrophenyl-alpha-D-glucopyranoside was used. Oroxylum indicum was used at concentrations ranging from 20-100μg/mlThe different concentrations of Oroxylum indicumwere combined for 5 minutes with 320μl of 100mM phosphate buffer pH 6.8 at 30C. A mixture of 3ml of 50mM sodium hydroxide was applied and the absorbance was read at 410nm. Control samples were provided without any Baicalein. The % inhibition was calculated according to the formula. The IC50Values were determined from the percent inhibition of alpha-glucosidase. Acarbose is used as the reference drug9. The % inhibition was calculated according to the formula:

 

The IC50 values were derived from the percent inhibition versus log inhibitor concentration plots and were estimated from the mean inhibitory values by non-linear regression analysis. As the reference alpha-glucosidase inhibitor, Acarbose was used. Both studies were conducted in triplicates.10

 

Antioxidant Assay:

DPPH free radical scavenging activity:

The DPPH assay is utilized to anticipate antioxidant activity by determining the scavenging potential of DPPH radical. The method relays on the scavenging of DPPH by an antioxidant, which upon reduction decolourize the DPPH ethanol solution. The extent of discolouration depicts the scavenging of the antioxidant compound in terms of hydrogen donating ability. The DPPH assay was performed by adding 25l DPPH (1mm) dissolved in ethanol to 10l of each sample (0.2-1l) in DMSO (dimethyl sulfoxide). Then it was vortexed for about 30 minutes in the dark and maintained at room temperature. Finally, the DPPH assay was analysed by measuring the absorbance of the vortexed mixture at 490nm by using a spectrophotometer UV-spectrophotometer and it was compared with standard drug11.

 

Scavenging ratio DPPH assay was calculated as follows:

 

Nitric oxide scavenging activity:

Nitric oxide scavenging assay was done by adding a different concentration of test sample to different test tubes. Then, add 1.5ml of sodium nitroprusside (5mM) in phosphate buffer to each test tubes and incubated for 30 minutes at 25oC. Finally, 1.5ml of Griess reagent (1% sulphanilamide, 0.1% naphthyl ethylenediamine dichloride and 3% phosphoric acid) was added to each test tubes. Then, the absorbance of coloured solution was measured at 546nm using UV-spectrophotometer and it was compared with standard drug11.

 

Brine shrimp lethality assay:

Artemia salina lethality assay was carried out to assess the cytotoxicity of baicalein. Artemia salina (1g) cysts were incubated for hatching in a conical container (separating funnel) filled with sea water. 0.06% yeast solution was added to the hatching chamber to feed the larvae after 24 hours, filled with seawater under constant aeration for 48 hours. After 48 hours, active nauplii free from egg shells were collected from the hatching chamber and used for the assay.

 

From the hatching chamber, 10-15 nauplii were drawn using a Pasteur pipette and introduced into the Petri dish containing sea water along with a drop of yeast solution. Different concentrations (0.1, 1, 10, 100 and 1000 g/mL) of Oroxylum indicum and positive control (potassium dichromate) were prepared. 0.5ml was added to 4.5ml of sea water and maintained at room temperature for 24 hours, to remain in contact with the active nauplii in the petri dish. The number of surviving nauplii in each Petri dish was counted after 24 hours. The percentage death was calculated by comparing the mean surviving larvae of the test substance Oroxylum indicumand control system. LC50 values were obtained from the best-fit line plotted concentration versus percentage lethality12,13.

 

The number of survivors was counted and the vials were inspected by a magnifying glass after 24 h of treatment with the sample. For each dilution, the percent (%) mortality was projected.

 

Molecular docking:

Generally, docking is a process where the ligand and receptor molecules are combined together to form a stable complex structure. Here the molecular docking study was performed by using Schrodinger (Maestro V 11.5) software which shows a great recognition among researchers and the pharmaceutical field14. The maestro software tool relays on flexible visualization, 3D realism of a compound, Quantitative structural analysis, molecular properties such as vibrational modes, molecular orbits or electron density are easily visualized in Maestro.

 

Protein preparation:

The 3D structure of alpha-amylase (PDB ID: 5U3A) and alpha-glucosidase (PDB ID: 5NN8) were retrieved from the RCSB protein data bank. The selected protein was imported via the protein preparation wizard. Then the proteins are further pre-processed, refined, optimized and then minimized.

 

Ligand preparation:

The 3D structure of the ligand compounds is retrieved from Pubchem database (ie., Baicalein, Acarbose, Voglibose in .sdf format. Using LigPrep wizard in maestro 2018, the ligands were then prepared15.

 

Receptor grid generation:

For the developed protein, the receptor grids were enumerated such that individual ligands bind during docking to the existing active site. The default parameters of van der Waals scaling factor 1.00 and charge cut off 0.25 were initiated in Glide grids subjected to the OPLS 2005 force field. The cubic box of certain proportions focus around the centroid of the active site was produced for the receptor16.

 

Ligand docking:

The ligand docking was performed. The final score was obtained based on the energy minimizing poses and reveal as a glide score. For the corresponding ligands, the best-docked pose with the least glide score value was reported.

 

RESULTS AND DISCUSSIONS:

In-vitro Alpha Amylase inhibitory activity:

The alpha-amylase assay performed shows 92.3% of inhibition. The inhibitory concentration of Oroxylum indicumon alpha-amylase was found to be (IC50 = 46.78 g/ml) and the reference standard Acarbose was found to be (IC50 = 36.36g/ml) as represented in Table 1 and Figure 1.


Table 1: In-vitro Alpha Amylase inhibitory activity of Baicalein

Concentration g/ml

Sample Absorbance

% Inhibition

Standard Absorbance

Inhibition %

5

0.0096

4

0.6887

15.6

10

0.0125

25

0.5078

17.3

20

0.034

33

0.8299

39.3

40

0.0513

41.3

0.8899

49.3

80

0.0616

51.6

0.9468

58.9

160

0.1023

92.3

1.176

97.4

All values are expressed as % inhibition calculated using the formula. IC50 values are calculated by non-linear regression analysis of log dose vs %inhibition as represented in figure 1. All experiments were performed in triplicate (n=3) for a specific concentration.

 

Figure 1: In-vitro Alpha Amylase inhibitory activity of Baicalein and Acarbose

 

Figure 2: In-vitro Alpha Glucosidase inhibitory activity of Baicalein and Acarbose

 

In-vitro Alpha Glucosidase inhibitory activity:

The alpha-glucosidase assay performed shows 33.8% inhibition. The inhibitory concentration of Oroxylum indicumon alpha-glucosidase was found to be (IC50= 584.1 g/ml) and the reference standard Acarbose was found to be (IC50 = 67.00 g/ml) as represented in table 2 and figure 2. The key role of the alpha-glucosidase enzyme is to catalyse dietary carbohydrate and starch hydrolysis to generate glucose for intestinal absorption. Consequently, inhibiting the work of these enzymes can delay glucose production following dietary digestion, which in turn reduces hyperglycaemia18.

 

Antioxidant Assay:

In this study, antioxidant assay was performed in which the percentage inhibition of DPPH radical scavenging activity was found to be 21.86%, 37.82%, 54.46%, 62.26%, 72.12%, 89.76% at the concentrations of 5, 10, 20, 40, 80, 160 g/ml respectivelyas represented in table 3 and figure 3; nitric oxide scavenging shows an inhibition of 19.9%, 35.46 %, 41.4%, 48.24%, 48.2%, 50.02% at the concentrations of 5, 10, 20, 40, 80, 160 g/ml respectively as represented in table 4 and figure 4. The IC50 value of Oroxylum indicumfor DPPH and Nitric oxide scavenging activity was found to be 19.41 g/mland 92.30 g/ml respectively as compared to the standard drug Acarbose which showed an IC50 value of 31.06 g/ml and 48.42 g/ml respectivelyfor DPPH and Nitric oxide scavenging activity. (Table-3).

 


Table 2: In-vitro Alpha Glucosidase inhibitory activity of Baicaleinand Acarbose

Concentration (g/ml)

Sample Absorbance

% Inhibition

Standard Absorbance

Inhibition %

5

0.498

18.3

0.6887

12.4

10

0.9151

25.3

0.5078

15.6

20

0.9646

26.1

0.8299

31.4

40

1.419

27.8

0.8899

42.6

80

1.064

33.8

0.9468

54.6

160

1.4204

42.4

1.176

62.8

All values are expressed as % inhibition calculated using the formula. IC50 values are calculated by non-linear regression analysis of log dose vs %inhibition as represented in figure 2. All experiments were performed in triplicate (n=3) for a specific concentration.


 

Table 3: DPPH free radical scavenging activity of Baicalein:

Concentration (g/ml)

Absorbance sample

% Inhibition of sample

Absorbance standard

% Inhibition of standard

5

0.05

21.86

1.688

8.14

10

0.22

37.82

1.647

16.48

20

0.224

54.46

1.63

37.92

40

0.297

62.26

1.539

56.72

80

0.533

72.12

1.591

75.48

160

1.068

89.76

1.176

95.98

All values are expressed as % inhibition calculated using the formula. IC50 values are calculated by non-linear regression analysis of log dose vs %inhibition as represented in figure 3. All experiments were performed in triplicate (n=3) for a specific concentration.

 

Figure 3: DPPH free radical scavenging activity of Baicalein and Acarbose

 

Figure 4: Nitric oxide scavenging activity of Baicalein and Acarbose

 

Table 4: Nitric oxide scavenging activity of Oroxylum indicum

Concentration (g/ml)

Absorbance sample

% Inhibition of sample

Absorbance of standard

% Inhibition of standard

5

0.1843

19.9

0.2042

27.2

10

0.1909

35.46

0.2213

32.9

20

0.1908

41.4

0.2328

33

40

0.2161

48.2

0.2468

36.8

80

0.238

48.24

0.2471

39.9

160

0.2954

50.02

0.2682

44.6

 

All values are expressed as % inhibition calculated using the formula. IC50 values are calculated by non-linear regression analysis of log dose vs %inhibition as represented in figure 4. All experiments were performed in triplicate (n=3) for a specific concentration.

 


Brine shrimp lethality assay:

The brine shrimp assay has been performed forOroxylum indicumand the lethality dose was calculated by using the standard potassium dichromate solution. The lethality dose was calculated at 24 hours. The brine shrimps were taken in 24 well plate and observed under microscope after 24 hours of exposure as represented in figure 5a and 5b respectively. The values obtained were entered in GraphPad software and the probit analysis was observed for both potassium dichromate and MCFE and the results were tabulated in Table 5.


 

Figure 5a: 24 well plate exposure of 10 brine shrimps for a period of 24 hours.

 

Figure 5b: Observations were made under a microscope showing images of brine shrimp.

 

Table 5: Brine shrimp lethality assay of Baicalein

Concentration (g/ ml)

Log Concentration

% death of nauplii after 24 hours of exposure

0.10

-1

18.42+2.12

1.00

0

29.38+1.08

10.00

1

48.72+2.72

100.00

2

68.82+1.86

1000.00

3

92.36+1.78

Assays were performed in triplicate. Each value represents Mean S.E.M.

 


The associations of Baicalein, Acarbose andvoglibose with alpha-amylase target was comparatively analysed. Comparative analysis of docking scores, glide scores, H-Bonding and Hydrophobic pockets are seen in Table 6 and the docked view of the compound was represented in 6a, 6b and 6c respectively for Baicalein, Acarbose and Voglibose against PDB ID: 5U3A.

 

The associations of Baicalein, Acarbose andvoglibose with alpha-glucosidase target was comparatively analysed. Comparative analysis of docking scores, glide scores, H-Bonding and Hydrophobic pockets are seen in Table 7 and the docked view of the compound was represented in 7a, 7b and 7c respectively for Baicalein, Acarbose andVoglibose against PDB ID: 5NN8.


 

Table 6: Molecular docking study of Baicalein, Acarbose andvoglibose on alpha-amylase (PDB ID: 5U3A)

S. No.

Protein

Ligand

Docking Score

Glide Score

H Bond

Aminoacid Interaction site

 

1.

α - amylase

Baicalein

-5.513

-5.565

3

His 299, Asp 197

Acarbose

-6.602

-6.390

6

Trp 59, Thr 163, Arg 195, His 299, Glu 233

Voglibose

-6.047

-6.052

5

Hip 305, Arg 195, Asp 197

 

Figure 6A: Docked view 0f Oroxylum indicumon alpha-amylase (PDB ID: 5U3A)

 

Figure 6B: Docked view of Acarbose on alpha-amylase (PDB ID: 5U3A)

 

Figure 6C: Docked view of Voglibose on alpha-amylase (PDB ID: 5U3A)

 

Table 7: Molecular docking study of Baicalein, Acarbose and voglibose on alpha-glucosidase (PDB ID: 5NN8)

S. No.

Protein

Ligand

Docking Score

Glide Score

H Bond

Aminoacid Interaction Site

1.

glucosidase (PDB ID: 5NN8)

Baicalein

-5.287

-5.339

4

Asp 404, Phe 649, Trp 481, Arg 600, Asp 518

Acarbose

-5.590

-5.918

8

Tyr 110, Gly 123, Trp 126, Asp 91, Ala 93, Arg 275, Arg 331

Voglibose

-6.396

-6.401

7

Asp 91, Lys 96, Cys 127, Trp 126, Gly 123, Arg 331,

 

Figure 7A: Docked view of Oroxylum indicumon alpha-glucosidase (PDB ID: 5NN8)

 

Figure 7B: Docked view of Acarbose on alpha-glucosidase (PDB ID: 5NN8)

 

Figure 7C: Docked view 0f Voglibose on alpha-glucosidase (PDB ID: 5NN8)

 

The purpose of this work was to test the inhibitory effect at differing concentrations of the compound Oroxylum indicumon Wheat alpha-amylase and Barley alpha-glucosidase. For type 2 diabetes mellitus, the enzyme alpha-amylase and alpha-glucosidase inhibitors are used to gain better control over hyperglycaemia.A research to determine the hypoglycaemic effect of Oroxylum indicum(insulin sensitivity and glucose uptake) was performed by Singh et al and concluded that 50% aqueous ethanolic Oroxylum indicum stem bark containing Oroxylum indicumdemonstrated anti-hyperglycaemic effects through inhibition of alpha-glucosidase activity in the 3T3-L1 adipocyte cell line and diabetic rat models.The significant effect of Oroxylum indicumto suppress bovine serum albumin glycation, as stated by Singh et al, may be helpful in the management of anti -hyperglycaemia by maintaining beta cells and enhancing insulin sensitivity17.

 

The present research aims to screen novel inhibitors of alpha-amylase and alpha-glucosidase from natural sources such as flavonoids to minimize the side effects of the commonly used inhibitors to regulate hyperglycaemia.

 

The in-silico analysis was carried out for the ligand interaction and binding affinity of a compound. In alpha-amylase, Acarbose possesses the highest glide score of -6.390 with 5EOF having 3 hydrogen bonds with THR 163, HIS 299, GLU 233 amino acid and with 3 water molecules. Vogliboseshows the glide score of -6.052 with 5EOF having 3 hydrogen bonds with ASP 197, ARG 195, HIP amino acids and with two water molecules. Oroxylum indicumhas the glide score of-5.565 with 5EOF having 3 hydrogen bonds with one HIS 299, and with two ASP 197 amino acids, here the water molecules are absent.

 

In alpha-glucosidase, voglibose possesses the highest glide score of -6.401with 5NN8 having 7 hydrogen bonds with LYS 96, TRP 126, CYS 127, ARG 331, GLY 123 and with two ASP 91 amino acids. Acarbose has the glide score of-5.918 with 5NN8 having 8 hydrogen bonds with one TYR 110, ARG 331, ARG 275, ALA 93, TRP 126, GLY 123, and with two ASP 91. Oroxylum indicumhas the glide score of -5.339 with 5NN8 having 4 hydrogen bonds with one ARG 600, ASP 404, TRY 481, PHE 649 and with two ASP 518 amino acids. Here totally the water molecules are absent.

 

CONCLUSION:

Finally, the study depicts that the targeted enzymes are active against baicalein, based on the glide score and the binding affinity it states that Oroxylum indicum has an antidiabetic activity. Consequently, the findings of the study strongly demonstrate the potential of Oroxylum indicumfor the treatment of hyperglycaemia. This hypothesis is the product of the in vitro, in-silico prediction and further in vivo experiments are required to validate the prediction.

 

ACKNOWLEDGEMENT:

The authors would like to thank the management of Sri Ramachandra Institute of Higher Education and Research (DU) for providing the software and chemicals to complete this research work successfully.

 

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Received on 22.05.2021 Modified on 29.05.2021

Accepted on 03.06.2021 AandV Publications All right reserved

Res. J. Pharmacognosy and Phytochem. 2021; 13(3):119-125.

DOI: 10.52711/0975-4385.2021.00020