Molecular Modelling Technique on Interactivity between Human Carbonic Anhydrase 1 and Mangiferin for Antiulcer activity

 

Paramita Das, Anjali Nayak, Bhavani K, Deepika, Ranjitha, Md Afsar

Krupanidhi College of Pharmacy 12/1, Chikka Bellandur, Carmelaram Gunjur Road Varthur Hobli,

Off Sarjapur Rd, Bengaluru, Karnataka 560035.

 

ABSTRACT:

Mangiferin is obtained naturally from Mangifera indica which is used in treating various diseases including gastrointestinal tract diseases. Ulcer means the damage of the mucosal membrane which commonly occurs in the stomach and upper duodenum. In the study, an antiulcer activity was conducted and the interactivity of human carbonic anhydrase 1 was studied. The human carbonic anhydrase 1 enzyme belonging proteins were taken in the study for the molecular docking. It was observed that mangiferin showed better activity in treating ulcers. The human carbonic anhydrase enzyme also mentioned as CA1 is one of the major reasons for an increase in gastric acid which leads to ulcers. In this study, mangiferin was able to inhibit the action of the CA1 enzyme which was increasing the levels of hydrochloric acid and preventing the formation of ulcers. A protein of CA1 (PDB ID: 6G3V) was docked with mangiferin which showed good interaction with the mangiferin. Further albino rats were used for the study of ulcer activity. ADMET studies were performed for mangiferin which showed a low toxic effect. A detailed study has been depicted for ulceration and its prevention using mangiferin.

 

 

 

INTRODUCTION: 

Mangiferin an ancient medicine used in treating various diseases is extracted mainly from different parts of Mangifera indica L. and Anemarrhena asphodeloides belonging to the Anacardiaceae and Liliaceae family respectively^1,2. Mangiferin consists of C-glycosylxanthone belonging to polyphenols³. An ultrasonic-assisted method is used for the extraction of mangiferin from leaves of Mangifera indica and yields yellow precipitate⁴. The major activity of mangiferin is gastroprotective other minor activities include anti-inflammatory, antidiabetics, hypocholesterolemic, antimicrobial, antiallergic, immunomodulatory, and anticancer^5,6.   

 

An ulcer is one of the most common diseases usually in the stomach and duodenum due to mucosal damage and infection by Helicobacter pyroli.

 

 

Mucosal damage is due to an impaired defence mechanism against HCl by mucus and prolonged use of medicaments like NSAIDs such as aspirin, ibuprofen etc⁷.

 

A metalloenzyme Carbonic anhydrase containing zinc ion is responsible for the release of proton and bicarbonate by the reversible reaction of carbon dioxide and water⁸. The various crude drugs, herbs, vegetables, and plants metabolites are used to block the release of HCl through CA activity, one such compound selected in this study is mangiferin⁹.   

 

In this study, we use albino rats to estimate the gastroprotective activity of mangiferin by pyloric ligation and ethanol methods to determine the ulcer score and index were determined. In silico molecular docking is performed between mangiferin and CA1 protein (PDB ID: 6G3V) along with ADMET studies to estimate the antiulcer activity¹⁰.

 

MATERIALS AND METHODS:

Mangiferin was obtained from Vital herbs, New Delhi. All chemicals used in the experiment were of analytical grade. Analytical TLC plate with silica gel 60 was used to determine the TLC profile. The spots on TLC plates were visualized using a UV lamp at 254nm.

 

Selection and preparation of target:

Carbonic Anhydrase-I protein with PDB ID: 6G3V was downloaded from Protein Data Bank in a three-dimensional PDB format. The energy of the protein is minimized by removing all other non-proteinaceous parts such as water molecules and unique ligands present along with the selected protein. In AutoDock 4.2, polar hydrogens and Kollman charges are added to make it suitable for the docking process and saved in PDBQT format¹¹.

 

Selection and preparation of ligand:

The ligand mangiferin is selected for anti-ulcer activity as a potent flavonoid based on literature studies. The property of the ligand is depicted in table-1 based on the Lipinski rule of five. The ligand is prepared using Chemdraw and converted to a three-dimensional PDB format. In AutoDock 4.2, the torsions in the ligand are detected and are set free. The bonds to be rotated are selected and saved in PDBQT format¹².

 

Docking studies:

By using AutoDock 4.2, mangiferin is made to bind to the CA-I protein at specific binding sites by adjusting the grid box at 60, 60, 60 points in X, Y, Z dimensions respectively. The docking is carried for 10 genetic algorithm runs with and population size of 150. Suitable conformation is selected for the complex possessing the least binding energy in Kcal/mol. The docking score along with the 2D structure of the ligand is deprived in Table-1. The binding energy is inversely proportional to the affinity of the ligand to the protein. The interaction between the ligand and the amino acids in proteins is visualized in two-dimensional form using Discovery studio 202¹³.

 

Table-1: Property of ligand based on Lipinski rule of five.

Structure	Chemical name	Molecular weight (g/mol)	Total polar surface area (TPSA)	Hydrogen bond donor and acceptor	Rotatable bound’s	Docking score (Kcal/mol)
	2-β-D-glucopyranosyl-1,3,6,7-tetrahydroxy-9H-xanthen-9-one	408.31	201.28A²	8 and 11	2	-5.5

 

Antiulcer activity:

Experimental Animals:  

40 Adult albino rats (130-160g) of either sex were used from the Krupanidhi College of Pharmacy animal facility in Bengaluru, Karnataka, India. The Institutional Animal Ethics Committee (IAEC) approved the study protocol with reference no 378/PO/ReBi/S/01, further maintenance and experimentation were carried out by CPCSEA guidelines. The animals were kept under standard housing conditions with free access to water and food.  

 

Acute Oral Toxicity Study:  

By OECD guidelines acute oral toxicity study was performed with a limited dose of mangiferin (2000mg/kg) and observed continuously for any behavioral and mortality changes for an hour and then later for 72 hours respectively. 100 and 400mg/kg doses of mangiferin were chosen for anti-ulcer activity¹⁴.

 

Antiulcer study animal grouping and treatment schedule:

Rats were divided into 5 groups; each group consist of 6 animals. The feed was removed in healthy adult male albino rats for 24 hours¹⁵. Group 1 includes normal control rats that receive only distilled water; group 2 serves as a positive control in which rats receive Ulcerogen; groups 3 and 4 are treated with 100 and 400mg/kg dose of mangiferin respectively; group 5 serves as a standard control in which rats are treated with 50mg/kg dose of ranitidine¹⁶.   

 

Methodology:

Gastric Ulceration Caused by Pyloric Ligation:

Pyloric ligation is carried out on fasted rats for 24 hrs, that was kept in an individual cage to prevent cannibalism. The rats are administered with reference drugs and mangiferin. The animals were anesthetized with ether and midline incisions are made to open the abdomen beneath the xiphoid process. Without disturbing the blood supply, the pyloric portion is lifted and ligated carefully¹⁷. The abdomen wall was sutured by placing back the stomach. During the postoperative period, the animals were denied access to beverages and sacrificed after six hrs by giving a high dose of anesthesia¹⁸. The cardiac end of the stomach was dissected by opening the stomach and the contents were collected in a glass tube. Contents volume was estimated and centrifugation is carried out for 10 min. Total acidity, pH, gastric contents, and free acidity were determined by taking aliquots (1ml each) from the supernatant¹⁹.

Gastric ulceration caused by ethanol:

1.5ml of ethanol is administered orally to fasted animals (12hours) to induce ulcers. After 2 hours, animals are sacrificed and the stomach part is exposed along the greater curvature for evaluation of ulcers.  

 

Determination of anti-ulcer activity parameters:

Ulcer index, U=Un+ Us+ Up/10

Where Un is the average number of ulcers per animal; Us is the average severity score, and Up percentage of animals with an ulcer²⁰.  

 

It is determined by identifying the severity of sores and it is scored as follows: 0-normal-colored stomach, 0.5-red coloration, 1-superficial mucosal injury (spot ulcer), 1.5-hemorrhagic streak, 2-deep Ulcer, 3-perforation²¹.

 

Determination of total acidity:

To a 50 ml conical flask, 1 ml of gastric juice is diluted with 9 ml of distilled water and a few drops of indicator (phenolphthalein) are added. The above solution is titrated against 0.01 N NaOH until the endpoint is permanent pale pink colour ²². The acidity is determined by using the below formula:

 

                              V NaOH × N × 100mEqIL

Total Acidity = ---------------------------------------------

                                                    0.1

RESULTS:

With the help of result discovered in docking research Mangiferin (Anti-ulcer pastime respectively) proposed to proper inhibitory pastime on the basis of their binding interplay with the protein. The binding interplay poses of these hit compounds with protein pocket amino acids are given below:

 

Table 2: Drug-receptor interactions

SI. No	Compound name	Receptor	Interacting residues
1.	Mangiferin	Human carbonic anhydrase 1 Receptor (PDB ID: 6G3V)	His 64, Asp72, and Pro201

 

 

Figure-1: 3D and 2D Protein-Ligand interaction

 

Pharmacological screening:

Table-3: Gastric juice volume, pH, Acidity, pepsin activity of magneferin on ethanol-induces ulcer models.

Treatment	Gastric Juice Volume (ml)	pH	Acidity (mEq/It)		Pepsin activity (Per ml/h)
			Total	Free
Group I   (Negative control)	9.31± 0.25	7.18±0.17	78.32±1.55	21.66±1.93	6.16 ± 0.15
Group II (Positive control)	1.89± 0.89	1.47±0.23	54.19±2.13	23.35±0.78	1.53 ± 0.47
Group III (Mangiferin 100mg/kg)	8.94± 0.49	6.28±0.31	30.47±1.92	14.81±0.32	5.02 ± 0.96
Group IV (Mangiferin 400mg/kg)	7.56± 0.71	6.93±0.21	26.91±1.37	12.66±0.55	4.97 ± 0.88
Group V (Standard 80mg/kg)	9.17± 0.49	6.88±0.43	21.38±1.82	9.5± 0.42	5.99 ± 0.26

 

Effect of Mangiferin on gastric parameters by pyloric ligation induced ulceration in rats:

Values are expressed as Mean±SEM, n= 6, *p < 0.05 and **p< 0.01 when compared with vehicle control group. (Statistically analysed by one-way ANOVA followed by Dunnet’s t-test).

 

Table-4: Ulcer index of magneferin on ethanol-induces ulcer models.

Treatment	Ulcer Index
	Pyloric Ligation	Ethanol
Group I   (Negative control)	0 ± 0	0±0
Group II (Positive control)	3.19 ± 1.34	0.31±0.75
Group III (Mangiferin 100mg/kg)	2.86 ± 0.42	1.3±0.28
Group IV (Mangiferin 400mg/kg)	2.15 ± 2.61	0.83±0.17
Group V (Standard 80mg/kg)	1.58 ±0.89	1.3±0.21

 

 

Figure-2: a) positive control, b) standard, c) Mangiferin 100mg/Kg, d) Mangiferin 400mg/Kg

 

 

Figure-3: Ulcer index

 

Figure-4: Gastric Juice Volume, pH, and Pepsin activity

 

STATISTICS:

Table-5: statistical table of Magneferin and standard

Source	DF	Sum of Square	Mean Square	F Statistic	P-value
Sets of individuals (between groups)	2	1	0.5	2.1429	0.1519
Error (within groups)	15	3.5	0.2333	-	-
Total	17	4.5	0.2647	-	-

 

Using the F distribution df(2,15), perform a one-way ANOVA test (right-tailed)

1.   H0 hypothesis

H0 is accepted because the p-value is greater than.

All group averages are presumed to be equal.

To put it another way, there is no statistical difference here between the average of all groups.

2.   P-value

[p(x F ) = 0.848149 ] has a p-value of 0.151851. This suggests that if we reject H0, the risk of making a type 1 error (rejecting a correct H0) is too great: 0.1520 (15.19 percent)

The higher the p-value, the more strongly H0 is supported.

3.   The statistics

The test statistic F equals 2.142856 and is within the acceptable 95 percent critical value range: [-: 3] 0.6823]

4.   Effect size

The f effect size that has been observed is quite large (0.53). This shows that the disparity between the averages is considerable in magnitude.

The value of 2 is 0.22. This suggests that the group is responsible for 22.2 percent of the variation in the average (similar to R2 in the linear regression)

5.   Tukey HSD / Tukey Kramer

There is no statistically significant difference between any pair's means.

 

Figure-5: F distribution by ANOVA test

 

DISCUSSION:

The results of this study demonstrate that mangiferin inhibited the development of a variety of acute ulcers that were induced in the rat stomach and duodenum. In the pyloric ligation ulcer-induced model the ulcer index for mangiferin 100mg/kg has significantly decreased compared to mangiferin 400mg/kg, and in the ethanol ulcer-induced model the ulcer development for mangiferin 400mg/kg has decreased compared to mangiferin 100mg/kg. In addition, all treatment groups showed an increase in gastric juice volume, pH, and pepsin activity when compared to the positive control group. The results imply that the mangiferin may have a direct impact on how caustic the gastric juice discharged is. Mangiferin is currently thought to have a preventive effect against stomach ulcers. Rats that received mangiferin at the same pace as normal medication recovered more quickly from stomach ulcers caused by ethanol and pyloric ligation.

 

CONCLUSION:

The present results have been used to determine the mangiferin binding interactions with carbonic anhydrase 1 enzyme which were depicted through in silico molecular docking and antiulcer activity on rats. Toxicity studies were performed for mangiferin. A statistical study of gastric juice, ulcer index was detailed. An in vivo study for gastro protective action by mangiferin through pyloric ligation and ethanol method was performed. Ultimately mangiferin showed better activity against carbonic anhydrase-1 by reducing the ulcer formation

 

ACKNOWLEDGMENTS:

I would like to express my special thanks of gratitude to GOWTHAM S E (emergency consulting physician) [ER] for doing statistics in our project.

 

CONFLICT OF INTEREST: 

Conflict of interest declared none.

 

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Accepted on 13.01.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(5):2465-2469.