GC-MS Analysis of the Leaf Extract of Swertia chirata and its In-silico Binding Affinity against Toxicity Receptors

 

Jerine Peter S, Gayathri Ashok, Megha Treesa Saju, Mary Thomas, Evan Prince Sabina*

School of Biosciences and Technology, VIT, Vellore - 632014, India.

 

ABSTRACT:

Objective: Swertia chirata is a medicinal plant that is found in temperate Himalayas. Swertia chirata belongs to Gentinaceae family. It is widely used in pharmaceutical industries because of its medicinal use. It is used for treating fever and various skin diseases. They are also used for the hepatitis and inflammatory diseases in many parts of the world. They are rich in flavonoids, alkaloid and terpenoids. Traditionally Swertia chirata species are used against asthma. Methods: The leaf powder of Swertia chirata was subjected to GC-MS.  The 3D structure of the active ligands was obtained by using the Corina molecular network. The receptors for toxicity used are nuclear bile receptor FXR, LXR, Nf-kB. The 3D structures used here are obtained from RSCB protein databank. Patch dock server was used for carrying out the docking experiment and the docked complex obtained were analyzed by the Pymol molecular viewer. This software helps in predicting the number of hydrogen bonds and the length of ligand-receptor bond. The molecular docking is a valuable tool in drug discovery.
Results and conclusion: Our research has demonstrated the potential activity of S.chirata as antioxidant activity through assays and showed potential binding affinity against toxic receptors. Further analysis on in vivo models helps in confirming the use of S.chirata as a valuable drug against toxicity.

 

 

 

1. INTRODUCTION:

The major obligatory factor hidden under the success for health-care is the serviceable amount of acceptable drugs. The affordable and accessible origin of treatment begins with the use of traditional medicine. In the sub-continent of India there are numerous native system of medicinal plant growth which been described for its various affliction. In the temperate Himalayas there are presence of many local native medicinal plants, were main focus was given to plants that have high source of anti-oxidants as they have rich role in functions including physiological as well as in biotransformation such as detoxification process¹.

 

Swertia chirata plant is present in Himalayas which belongs to the family Gentianaceae. That has reported to be the best for its medicinal uses, which has been reported in the British, Indian and American pharmaceutical codex along with different system such as ayurveda, siddha. The plant is famous for its bitter nature; that is used as a tonic for the treatment of fever as well as various skin allergies. It has reported that the amarogentin is the factors that contribute to the properties such as bitterness, antipyretic and anti-helmintic nature. In Western India this plant is one among the herbs used for the treatment of liver sickness². Swertia chirata also called Swertia chiratiya also has different names around all over India suggesting according to its extensive use. Himalayas is its native which is present at a 1200-3000m altitude from Kashmir along Bhutan. Here annual and perennial herbs are included in the genus Swertia. They grow in soils rich in humus with sandy loam having carbon. Work done on the plants cytology is poor. Out of the different parts of the plants root is considered as the most useful part it has an immense informative molecules such as Xanthones as well as mangiferin³. The plant is well known for its anti-diarrhoeal, laxative, febrifugal properties in the system of Indian medicine. Because of the presence of flavonoids it is used as an antioxidant. It has also used as an asthma curative which is taken with the paste of sandalwood, were it blocks stomach hemorrhage internally⁴. The hypoglycemic activity has been possessed from the plants hexane extract⁵.

 

The predominant use of S. chirata from traditional to modern drug and its marketable system in current medicine gave rise to its exploration in scientific as well as in phytochemistry for the identification of active compounds in phytochemical determination which resulted in inquiring considerable constituents of chemical in this plant. The endless biological activity of the plant S. chirata are accredited for the presence of multiple group of bioactive synthesis of compounds pharmacologically which belongs to various classes such as lignans, flavonoids, iridoids and other major compounds like chiratin, palmitic acid and stearic acid⁶.

 

GC-MS is a widely used technique for the analysis of plant metabolomics because of its easy sample preparation procedure and also for the detection of primary as well as secondary metabolities in a single fast run along with its analysis quantitatively. In this literature in order to find out the bioactive compounds of S.chirata, GC-MS process has done were those ligands has been docked for the analysis for their toxicological effects checking their ability to bind with the receptors involved in toxicity, this process leads to the discovery of new drugs under a short life period under low cost. The ligand of the plant that shows high bond interaction is considered as the best compound that could be used drug preparation. Here Patchdock is the best known docking method for the analysis of this interaction⁷. The main aim under docking is the establishment of affinities and bonding of the tiny molecules of the plant with the receptor. The goals in docking include the virtual screening and pose prediction. The basic rules included in docking are search algorithm and scoring. The interaction between protein and the ligand binding site can be distinctly explained by docking technique ⁸.

 

2. MATERIALS AND METHODS:

2.1 GC-MS analysis on leaves of Swertia chirata:

The leaves of Swertia chirata was purchased from Neeraj Traders, Jhansi, Uttar Pradesh, India. The leaves of the plant were subjected to GC-MS to obtain the active compounds present in the leaves to be docked with the receptors of toxicity.

 

2.2 In silico analysis on leaves of Swertia chirata:

2.2.1 Receptors:

The receptors used here are activated during the course of toxicity. PDB was used in order to retrieve the structures of these receptors. The receptor and its PDB id are Apo Human Pregnane X Receptor-1ILG, Nuclear bile acid receptor FXR - 1OSH, Constitutive androstane receptor - 1XNX, LXRalpha - 5AVI and Nf-kB - 1NFK.

 

2.2.2 Ligands:

The ligands are active compounds present in the leaves. About twelve ligands were selected and used for docking with the receptors. The structures of the ligands were obtained from corina molecule net by submitting the canonical smile obtained from Pubchem.

 

2.2.3 In silico docking:

In silico Docking was performed using online server Patch dock. To the online server the receptors and ligands were submitted to obtain docked complex. The docked complexes are visualized using the visualization software Pymol.

 

2.2.4 Docked complex analysis:

The interaction between the active ligands and receptors are identified using Pymol software. The major characteristics taken to account are the Area, ACE and bond length. The atoms present in the receptors and the amino acid residues present in the ligands are labeled.

 

3. RESULTS:

3.1 GC-MS analysis on the leaf extract of Swertia chirata:

The active compounds of the plant leaves were obtained through GC-MS and the graph was shown in Figure 1. The list of compound are listed in Figure 2.

 

The active compounds present in the leaf extract are Tetracosamethyl-cyclododecasiloxane, Cyclononasiloxane, octadecamethyl, Heptasiloxane, hexadecamethyl, Hexasiloxane, tetradecamethyl, Cyclodecasiloxane, eicosamethyl, Cyclooctasiloxane, hexadecamethyl, 1, 1, 1, 5, 7, 7, 7-Heptamethyl-3,3-bis (trimethylsiloxy), 1, 1, 1, 3, 5, 7, 7, 7-octamethyl-3,5-bis (trimethylsiloxy), 1, 1, 1, 5, 7, 7, 7-heptamethyl-3,3,5-tris (trimethylsiloxy), Hexasiloxane 1, 1, 1, 3, 3, 5, 5, 7, 7, 9, 9, 11, 11-dodecamethyl, 1, 1, 1, 3, 5, 7, 9, 11, 11, 11-decamethyl-5-(trimethylsiloxy) and Benzeneacetic acid, alpha-3,4-tris(trimethylsilyl). The obtained active compounds are used for docking analysis.

 

Fig. 1: GC-MS analysis of Swertia chirata leaves

The graph shows the different peaks obtained during gas chromatography- mass spectrometry.

 

 

Fig. 2: List of compounds present in the Swertia chirata

 

3.2 In silico analysis on the leaves of Swertia chirata:

3.2.1 Interaction of the ligands with the receptor 1ILG:

The receptor 1ILG interacts with the ligands and the hydrogen bonds are showed in table 1 with ACE, score, area and bond length. Ligand called tetracosamethyl-cyclododecasiloxane showed high interaction with the receptor.

 

Table 1:  Area, ACE, score and bond length of docked complex with 1ILG

 

The higher hydrogen bond was seen between the receptor 1ILG and the ligand tetracosamethyl-cyclododecasiloxane (Figure 3). The amino acid residues and their respective bond length are GLY314-3.1, GLY313-1.9 and ALA312-3.3. The ligand cyclononasiloxaneoctadecamethyl showed three hydrogen bonds. The amino acid residue and their bond length are HIS407-2.6, HIS407-2.9 and SER247-2.4. Cyclononasiloxaneeicosamethyl showed two hydrogen bonds, the bonds are SER247-2.8 and SER 208-2.4. The ligand hexasiloxanedodecamethyl showed two hydrogen bonds, SER248-2.1 and SER208-3.5.

 

 

The docked complex of receptor (purple) with ligand (red) with bonding in yellow, b. the docked complex of receptor (purple) with ligand (blue) with bonding in yellow, c. the  docked complex of receptor (white) with ligand (red) with bonding in yellow, d. docked complex of receptor (purple) with ligand (red) with bonding in yellow.

Fig. 3: Docked complex of 1ILG with ligands

 

Table 2: ACE, score, area and bond length of the docked complex with 1XNX

3.2.2 Interaction of the ligands with the receptor 1XNX:

The receptor 1XNX interacts with the ligands and the hydrogen bonds are showed. The ACE, Area, score and bond length of the docked complex is given in table 2.

 

The higher hydrogen bond interaction was seen between the receptor 1XNX and ligand Tetracosamethyl-cyclododecasiloxane. The amino acid residue and their respective bond length are ARG316-2.7, ARG316-0.9, ARG316-3.0, ARG316-3.1 and ARG316-2.9 (Figure 4A).

 

3.2.3 Interaction of the ligands with the receptor 5AVI:

The receptor 5AVI interacts with the ligands and the hydrogen bonds are showed. The ACE, score, area and bond length of the docked complex is given in table 3.

 

Table 3: ACE, score, area and bond length of the docked complex 5AVI

 

The higher hydrogen bond interaction was seen between the receptor 5AVI and ligand cyclononasiloxaneoctadecamethyl. The amino acid residue and their respective bond length are SER422-3.2, SER422-2.6, SER418-3.1 and SER422-3.3 (Figure 4B).

 

3.2.4 Interaction of the ligands with the receptor 1OSH:

The receptor 1OSH interacts with the ligands and the hydrogen bonds are showed. The Ace, score, area and bond length of the docked complex are given in table 4.

1OSH receptor showed only single hydrogen bond interaction with the ligand hexasiloxane-1,1,3,5,7,9,9,11,11-dodecamethyl. The amino acid residue and their bond length is TYR365-3.3. The ligand 1,1,1,3,5,7,9,11,11,11-decamethyl-5-(trimethylsiloxy) interacted with 1OSH showed hydrogen bond with the amino acid residue ASN287 with bond length 2.9 (Figure 4C and 4D).

 

 

A.The docked complex of receptor (green) and ligand (red) with bonding in yellow, B. The docked complex of receptor (Yellow) with ligand (red) with bonding in yellow,  C. The docked complex of receptor (blue) with ligand (yellow) with bonding in red,  D. The docked complex of receptor (blue) with ligand (red) with bonding in yellow.

Fig. 4: Docked complex of 1XNX, 5AVI and 1OSH with ligands

Table 4: ACE, score, area and bond length of docked complex of 1OSH

Ligand	Score	Area	ACE	Bonds
				Residue	Atom	Length
Tetracosamethyl-cyclododecasiloxane	6688	773.70	-236.42	Arg459	O7	2.4
Heptasiloxane, Hexadecamethyl	6136	853.30	-281.08	TYR365	O21	3.3
Cyclooctasiloxane,Hexadecamethyl	5920	758.10	-250.24	SER336	O9	3.3
Hexasiloxane,1,1,3,3,5,5,7,7,9,9,11,11-dodecamethyl	5608	724.10	-255.87	TYR365	O9	3.3
1,1,1,3,5,7,9,11,11,11-Decamethyl-5-(trismethylsiloxy)	5872	830.70	-285.46	ASN287	O22	2.9

 

Table 5: ACE, score, area and bond length of the docked complex 1NFK

Ligand	Score	Area	ACE	Bonds
				Residue	Atom	Length
Tetracosamethyl-cyclododecasiloxane	7176	916.10	-262.78	ASN244	O21	1.2
				ASN244	O23	1.9
				GLN274	O25	2.2
				GLN274	O23	2.2
Heptasiloxane, Hexadecamethyl	6608	835.40	-78.83	ASN247	O9	2.6
Cyclononasiloxane,Eicosamethyl	6798	811.80	-148.56	LYS24	O7	1.9
				ARG305	O3	2.5
				ARG305	O3	1.9
Cyclooctasiloxane,Hexadecamethyl	6060	746.80	-128.24	LYS241	O13	1.3
				ARG305	O9	2.5
4,1,1,5,7,7-Heptamethyl-3,3-Bis(Trimethylsiloxy)	5490	728.60	-102.67	LYS249	O3	1.8
1,1,1,5,7,7,7-Heptamethyl-3,3,5-tris(trimethylsiloxy)	5488	800.50	-158.54	PRO243	O25	2.8
				ASN247	O20	2.5
Hexasiloxane,1,1,3,3,5,5,7,7,9,9,11,11-dodecamethyl	6100	709.60	-53.28	LYS249	O9	2.9
1,1,1,3,5,7,9,11,11,11-Decamethyl-5-(trismethylsiloxy)	6756	797.30	-109.27	ARG305	O3	2.4
Benzeneacetic Acid, Alpha,3,4-TRIS(Trimethylsilyl)	5040	871.70	-107.16	LYS249	O5	2.6

 

3.2.5 Interaction of the ligands with the receptor 1NFK:

The receptor 1NFK interacts with the ligands and the hydrogen bonds are showed. The ACE, score, area and bond length of the docked complex are given in the table 5.

 

The higher hydrogen bond interaction was seen between the receptor 1NFK and ligand tetracosamethylcyclododecasiloxane, the amino acid residues and their bond length are the following ASN244-1.2, ASN244-1.9, GLN274-2.2 and GLN274-2.2 and the ligand cyclononasiloxaneeicosamethyl, the amino acid residues and their bond length are the following LYS24-1.9, ARG305-2.5 and ARG305-1.9. The ligand cyclooctasiloxanehexadecamethyl showed two hydrogen bonds, LYS241-1.3 and ARG305-2.5. 1,1,1,5,7,7,7-Heptamethyl-3,3,5-tris(trimethylsiloxy) showed two hydrogen bonds, PRO243-2.8 and ASN247-2.5 (Figure 5).

 

 

A and B: The docked complex of  receptor(purple) and ligand (red) with bonding in yellow, C: The docked complex of receptor(blue) with ligand(red) with bonding in yellow, D: The docked complex of receptor (red) with ligand (pink) with bonding in yellow.

Fig. 5: Docked complex of 1NFK with ligands

4. DISCUSSION:

Use of various herbs for the treatment of different diseases including cancer is most commonly seen in different parts of the world. Swertia chirata is one such plant that has anti hepatotoxic activity. It shows activity like anti-inflammatory, antidiarrheal, antiviral and anticarcinogenic activity⁹. This work explored the anti hepatotoxic activity of Swertia chirata and obtaining active components of its leaf. The interactions between the active components are examined to understand the extent of hepatotoxic activity. Hepatotoxicity is a major disease that is common in developing countries. There are many ways of curing the disease, but in-silico docking is the method of understanding the activity to inhibit the ability of causing this disease¹⁰. There are five receptors that are prominent in causing hepatotoxicity which are 1NFK, 1OSH, 1XNX, 5AVI and 1ILG. The 1NFK is the bioactive compound of NF-κB¹¹. NF-κB plays a vital role in regulating cell proliferation, cell differentiation and apoptosis, which also help in regulating soluable types of pro-inflammatory chemokines and cytokines. This can be targeted and minimized to suppress the apoptosis¹². The active compound of the S. chirata was obtained with the help of the GC-MS analysis. It is the main technique used in plant metabolomics. We have obtained 12 main ligands. These ligands were then docked with each of the receptors. Patch dock was used for the docking purposes which is the most simple and convenient technique. Docking is an experimental analysis of the effective interaction between the ligand and the receptor. The ligand that interacted more effectively with the receptor is predicted to be the best compound in pre-occupying the receptor. The binding capacity of the ligands determines the efficiency of that particular ligand to be used as a drug for hepatotoxicity. The result thus obtained has to be then undergone in vivo tests to analyze the antihepatotoxic activity of the particular ligand. The ligand that is having most number of hydrogen interaction are the best for the further experiments¹³. This is widely used as a tool for the drug designing for different similar diseases. Search algorithm and score is the main in docking to understand the interactions between the ligand and the receptor¹⁴.

 

5. CONCLUSION:

Hepatotoxicity being a chemically driven disorder of the liver, the receptors responsible for the toxicity is docked with the active compounds of the leaf extract of the plant Swertia chirata. The docking results showed the inhibitory effects of the plant active compounds towards the toxicity. The active compounds such as tetracosamethylcyclododecasiloxane was seen to have high interaction with the receptors such as 1ILG,1NFK and 1XNX. The active compound like cyclononasiloxaneoctadecamethyl showed high interaction with the receptor 5AVI. Cyclononasiloxaneeicosamethyl was shown to have greater hydrogen bond with the receptor 1NFK. The binding of the active compound may lead to inactivation of the receptor and thereby preventing the toxicity to occur. From our current study it is predicted that the active compounds present in the leaf extract of Swertiachirata may have inhibitory effect on toxicity receptor. Further in-vivo experiments can prove the effect of the leaf extract on toxicity thereby confirming its effects.

 

6. ACKNOWLEDGEMENT:

We would like to thank the Vellore Institute of Technology for providing us this great opportunity to perform and carry out all the necessary work.

 

7. CONFLITS OF INTEREST:

Authors declare that they do not have any conflicts of interests.

 

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Accepted on 10.05.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(3):1622-1628.