UNRAVELING THE PHYTOCONSTITUENTS OF SCHLEICHERA OLEOSA AS AN ANTI-ARTHRITIC ACTIVITY: GC-MS ANALYSIS AND MOLECULAR DOCKING STUDIESHTML Full Text
UNRAVELING THE PHYTOCONSTITUENTS OF SCHLEICHERA OLEOSA AS AN ANTI-ARTHRITIC ACTIVITY: GC-MS ANALYSIS AND MOLECULAR DOCKING STUDIES
Shivani Singh, Shweta Paliwal, Tabish Qidwai and Sachidanand Singh *
Department of Biotechnology, Smt. S. S. Patel Nootan Science & Commerce College, Sankalchand Patel University, Visnagar, Gujarat, India.
ABSTRACT: Rheumatoid arthritis is a systemic autoimmune and inflammatory disease that primarily affects the synovial joints and eventually destroys them. Schleichera oleosa is a plant of Indian provenance that exhibits anti-rheumatoid arthritis activity. The phytochemical constituents in the methanol extract of barks from Schleichera oleosa were identified using gas chromatography-mass spectrometry (GC-MS) in the current study. The GC–MS analysis in bark extract identified 21 phytochemical compounds. The PASS server assessed the biological activity of the reported compounds and identified six compounds with anti-arthritic and anti-inflammatory activities. Subsequently, these compounds were screened through ADME/Tox and molecular docking approaches. Two compounds 2,4-Di-tert-butylphenol and 2,6-DI-Tert-butylphenol qualify for all the criteria of ADME/Tox. The molecular docking method identified compounds 2,4-Di-tert-butylphenol and 2,6-DI-Tert-butylphenol had binding affinity ranging between -4.89 to -6.49 kcal/mol with the active site of anti-arthritic target proteins Chitotriosidase-1 and Vascular cell adhesion molecule 1. The findings of this study will create a way to invent herbal medicines for Rheumatoid Arthritis using the Schleichera oleosa plant, potentially leading to the development of new drugs.
Keywords: Schleichera oleosa, Rheumatoid arthritis, CHIT1, VCAM-1
INTRODUCTION: Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that can affect numerous tissues and organs but is most commonly associated with synovial joints 1. RA affects approximately 1% of the world's population, and there is no cure 2. Rheumatoid arthritis can also produce diffuse inflammation of the lungs, pericardium, pleura, and sclera, as well as nodular lesions, which are most frequent in subcutaneous tissue 3.
Although this disease is prevalent worldwide, its pathogenesis is poorly known 4. Persistent inflammation causes swollen joints with severe synovitis, a lower nociceptive threshold, and significant sub-synovial infiltration of mononuclear cells, which leads to pannus development and angiogenesis. Pannus enlargement causes bone erosion and cartilage weakening, resulting in joint function loss 5.
The Indian subcontinent is rich in aromatic and medicinal plant flora. This is because India has a diverse range of climatic conditions. Plants with medicinal properties are known to have fewer adverse effects. The Sapindaceae family tree Schleichera oleosa (kusum tree) grows in the Himalayan foothills. It is common in many Indian states, including Uttar Pradesh, Madhya Pradesh, West Bengal, Bihar, and Chhattisgarh. This plant's bark and seed have high pharmacological activity. Central Indian tribes utilize it to relieve arthritis pain and rheumatoid arthritis. Other disorders treated include itching, acne, burns, other skin problems, rheumatism (external massage), hairdressing, and hair growth promotion 6. The bark is used as an astringent and to treat skin inflammations, ulcers, itching, acne, and other skin diseases 7. For over three decades, computer-aided drug design has been a popular method for screening, designing, and developing therapeutically useful molecular candidates 8. These methods combine many approaches, such as molecular docking, ADME (absorption, distribution, metabolism and excretion), and toxicity that aid in the optimization and screening of drugs. Nowadays, another prominent method for determining the interaction between a target protein and small molecule candidates is molecular docking 9. The approaches can identify promising compounds that can be used in drug design. The ADME specifies pharmacokinetics and pharmacology features, which are critical aspects in the drug design process because they allow us to understand the safety and efficacy of a molecule as a drug candidate. Toxicity analysis is another phase in the drug design process that can be performed using computer tools to determine an undesirable biological effect of a chemical substance or drug candidate. Therefore, the study aimed to identify bioactive compounds from Schleichera oleosa by Gas chromatography-mass spectrometry and computational methods that could be used as a therapeutic option for rheumatoid arthritis.
MATERIALS AND METHODS:
Collection of Plant Materials: The plant stem bark sample was collected from Dala Range, Obra Forest Division, Sonbhadra, Uttar Pradesh. The herbarium specimen number of this plant is 7883 10. Plant identification and authentication were done with the help of CSIR- National Botanical Research Institute, Lucknow, Uttar Pradesh, India.
Preparation of the Extract: The stem of the plant Schleichera oleosa (Lour.) Oken was washed in fresh water thoroughly 2-3 times and once finally with sterile water to remove adhering dust. The stem was then sun-dried for a day, the outer bark of stem was peeled off, and the filling of inner bark was done, and then the grinding of the filled inner bark was done to convert it into powder form. About 2g of the plant bark extract was weighed and placed inside a Soxhlet extractor. A mixture of n-hexane and methanol (50/50) v/v was placed within a round-bottom flask. The flask was attached to the main Soxhlet extractor, and the condenser was attached to the extractor, which was connected to a pipe with a continuous water flow. The extractor was heated using a heating chamber for about 2–3 h. The solvent was run through the tumble containing the sample, which traps all the possible extract into the solvent inside the round bottom flask. The extract was then cleaned by passing it through a column packed with silica gel that had already been saturated with methanol. The extract was dried using hydrous sodium sulphate. The cleaned extract was then concentrated to about 1 ml using nitrogen concentration before being introduced into the GC-MS analyzer.
Gas Chromatography-mass Spectrometry (GC-MS) Analysis: The gas chromatography-mass spectrometry (GC-MS) analysis of the bark Schleichera oleosa was performed using a GC-MS (Modal; Agilent technologies 7890A) equipped with a VF -5 ms fused silica capillary column of 30 m length, 0.25 mm diameter and 0.25 mm film thickness. An electron ionization system with an ionization energy of 70eV was used for GC-MS detection. Helium gas (99.99%) was used as a carrier gas at a constant flow rate of 1 ml/min. Injection and mass transfer line temperatures were set at 200 and 240 °C, respectively.
The oven temperature was programmed from 80 °C to hold for 2 mins@ 10 °C/min to 240 °C to hold for 6 mins. 2 ml of water solution of the samples was manually inserted in the split less mode, with a split ratio of 1:40 and with a mass scan of 50–600 amu. The total running time of the GC-MS was 35 min. The relative percentage of each extract constituent was expressed as a percentage with peak area normalization. The plant extract's mass spectrum was interpreted using the database of the National Institute of Standard and Technology (NIST) library, having more than 62,000 spectral patterns. The spectra of the compounds were compared with the spectra of the National Institute of Standard and Technology (NIST) library database.
Prediction of the Biological Activity of Compounds: The compounds were submitted to the Pass online server, which detects the biological activity of compounds 11. Its assistance was used to identify compounds with biological activity against Rheumatoid Arthritis.
Ligand Preparation: A review of the literature revealed that small molecules extracted from the inner bark of the plant Schleichera oleosa have anti-arthritic and anti-inflammatory activities. So, after Soxhlet extraction, the isolated bioactive compounds were subjected to the GC-MS method for identification. The 2D structure of identified compounds by GC-MS method was drawn using the PubChem Sketcher V2.4 (https://pubchem.ncbi.nlm.nih.gov//edit3/index.html). Open Babel was used to converting 2D to 3D structure of identified compounds 12.
In-silico ADME/Tox Prediction of Compounds: In-silico physicochemical, drug-likeness, and pharmacokinetics of identified bioactive compounds were predicted by the Swiss ADME web tool 13. The Pre ADMET tool was used to assess the toxicological properties of compounds 14.
Protein Preparation: Chitotriosidase (CHIT1) is a widely secreted protein mostly generated by activated macrophages and epithelial cells. Serum chitotriosidase levels were considerably higher in rheumatoid arthritis patients 15. Vascular Cell Adhesion Molecule-1 (VCAM-1) expression is closely associated with rheumatoid arthritis. Wang et al. discovered that serum VCAM-1 level was significantly higher in RA patients than in controls and that long-term use of aspirin decreased serum levels of rheumatoid factor and VCAM-1, suggesting that serum VCAM-1 level may be related to disease severity 16.
Crystal structure of human chitinase in complex with allosamidin (PDB Id: 1HKK) and Integrin-binding fragment of Vascular Cell Adhesion Molecule-1 (PDB Id: 1VCA) was retrieved from the Protein Data Bank (https://www.rcsb.org/). All the heteroatoms were removed, and polar hydrogen was added to proteins for the molecular docking process.
Molecular Docking: Binding site residues of Chitotriosidase-1 and Vascular Cell Adhesion Molecule-1 were retrieved from literature 17, 18. Docking was done on the MTi Open Screen server using AutoDock implemented in MTiAutoDock 19. Docking results were visualized using Python Molecular Viewer tool to show the 3D structure and position of compound binding to the protein 20.
RESULTS AND DISCUSSION: The GC-MS analysis was performed to identify the bioactive compounds in Schleichera oleosa bark methanol extract. GC-MS chromatogram of methanol extract of bark parts of Schleichera oleosa is shown in Fig. 1. A total of 21 bioactive compounds were obtained based on NIST libraries.
FIG. 1: GC-MS CHROMATOGRAM FOR METHANOL EXTRACT OF BARK PARTS OF SCHLEICHERA OLEOSA
The PASS server was used to predict the biological activity of identified 21 compounds by GC-MS analysis. These 6 compounds were found to have anti-arthritic activity, as shown in Table 1.
TABLE 1: BIOLOGICAL ACTIVITY OF COMPOUNDS IDENTIFIED FROM BARK OF SCHLEICHERA OLEOSA
ADME/Tox Analysis of Compounds: The Physicochemical and drug-likeness properties of six compounds were shown in Table 2. Compounds were accessed by Lipinski rule-of-five 21. Table 3 shows the ADME predictions for compounds for human gastrointestinal absorption (GI) and blood-brain barrier (BBB) penetration. The features of human intestine absorption are a determinant of drug synthesis and must be supplied orally. BBB plays a significant role in the pharmacology of drugs 22. The result presented in Table 2 shows that compounds 2, 4-Di-tert-butylphenol, 2, 6-Di-Tert-butylphenol and Methyl 14-methylpentadecanoate have high gastro-intestinal absorption and good BBB. Methyl 14-methylpentadecanoate is toxic as shown in Table 3. At the end of the entire ADME/Tox evaluation screening, two compounds 2,4-Di-tert-butylphenol and 2,6-Di-Tert-butylphenol satisfied all the criteria.
TABLE 2: THE PHYSICOCHEMICAL PROPERTIES AND DRUG-LIKENESS PREDICTION OF COMPOUNDS
|Sl. no.||Ligand Name||Physicochemical||Druglikeness|
nRot: Number of rotatable bonds, HBA: Hydrogen bond acceptor, TPSA: Topological polar surface area, MW: Molecular weight.
TABLE 3: THE PHARMACOKINETICS AND CARCINOGENICITY PREDICTION OF COMPOUNDS
|Sl. no.||Ligand Name||Pharmacokinetics||Carcinogenicity|
|GI||BBB||Log Kp (cm/s)||Ames test||Mouse||Rat|
GI: Gastrointestinal absorption, BBB: Blood brain barrier penetration, Log Kp-Skin Permeation Coefficient.
Analysis of Molecular Docking: Active site of Chitotriosidase-1 (PDB ID: 1HKK) was retrieved from the PDB sum. Chitotriosidase-1 active site residues were taken with the centroid of the residues for constructing the receptor grid box, taking into account the following amino acids: Tyr267(A), Asp213(A), Trp358(A), Tyr212(A), Met210(A), Trp99(A), Asp138(A), Gly98(A), Phe58(A), Ala183(A), Glu140(A), Tyr27(A), Met356(A). Integrin-binding motif (Q38IDSPL) of Vascular Cell Adhesion Molecule-1 was used as binding site residues for docking studies. The position of the grid box can be adjusted to cover the entire binding site residues GLN38, ILE39, ASP40, SER41, PRO42, LEU43 and LYS46 of VCAM-1 protein. 2,4-Di-tert-butylphenol and 2,6-DI-Tert-butylphenol were docked in the binding site of Chitotriosidase-1 (CHIT1) and Vascular cell adhesion molecule 1 (VCAM-1) (PDB Id: 1VCA) using MTiAutoDock server. The docking results of compounds with CHIT1 and VCAM-1 proteins were shown in Table 4.
TABLE 4: DOCKING RESULTS OF COMPOUNDS WITH CHIT1 AND VCAM-1 PROTEIN
|Sl. no.||Compound Name||CHIT1 (kcal/mol)||VCAM-1 (kcal/mol)|
On analysis of the binding energy of compounds, it was found that these had a low binding affinity with CHIT1 and VCAM-1 proteins Table 3. Predicted docked complexes were analyzed through Python Molecular Viewer for their interaction study shown in Fig. 2 & 3. Compounds were represented in sticks model. 2, 4-Di-tert-butylphenol interacted with residues GLY98, TRP99, GLU140, ASP138, PHE58, TRP31, TRP358, TYR212 and MET210 of CHIT1 protein Fig. 2A. 2, 4-Di-tert-butylphenol also interacted with residues SER34, TRP35, PHE33, LEU43 and VAL47 of VCAM-1 protein Fig. 2B. 2, 6-Di-tert-butylphenol interacted with residues GLU140, TRP99, ASN100, PHE58, TRP31, TRP358 and TYR212 of CHIT1 protein Fig. 3A. 2, 6-Di-tert-butylphenol also interacted with residues SER34, TRP35, PHE33, LEU43 and VAL47 of VCAM-1 protein Fig. 3B.
FIG. 2: (A) DOCKING POSE OF 2, 4-DI-TERT-BUTYLPHENOL IN BINDING SITE OF CHIT1 PROTEIN. (B) DOCKING POSE OF 2, 4-DI-TERT-BUTYLPHENOL IN BINDING SITE OF VCAM-1 PROTEIN
FIG. 3: (A) DOCKING POSE OF 2,6-DI-TERT-BUTYLPHENOL IN BINDING SITE OF CHIT1 PROTEIN. (B) DOCKING POSE OF 2,6-DI-TERT-BUTYLPHENOL IN BINDING SITE OF VCAM-1 PROTEIN. ONE H-BOND WAS FORMED BETWEEN AMINO ACID TRP35 OF PROTEIN WITH COMPOUND, RESPECTIVELY. THE HYDROGEN BOND IS REPRESENTED WITH SPHERICAL LINE
Plants have been used as a source of medicine for centuries and are an essential component of the Indian healthcare system. It has been reported that the bark of the Schleichera oleosa plant contains antibacterial, antiarthritic, antioxidant and anticancer properties, as well as phytochemicals such as terpenoids, betulin and betulinic acid 23. Kaur et al. (2012) reported on various herbs and plants with anti-arthritic and anti-inflammatory characteristics for reducing joint pain and inflammation in RA 24. Currently, nonsteroidal anti-inflammatory drugs, corticosteroids, disease-modifying anti-rheumatic medicines, and biological agents are employed in treating RA 25. However, taking these drugs together may cause constipation 26, stomach ulcer 27, dizziness, headaches, and dyspepsia 28. Therefore, exploration continues for alternative products and natural phytochemicals from medicinal plants with lesser side effects.
CONCLUSION: The identification of bioactive compounds in Schleichera oleosa was done by GC-MS analysis, which shows the presence of 21 compounds. Among the identified compounds, 2,4-Di-tert-butylphenol and 2,6-Di-Tert-butylphenol have a role in anti-arthritic and anti-inflammatory activity. In molecular docking experiments, these compounds also showed promising binding affinity toward RA target proteins CHIT1 and VCAM-1. From this study, it can be concluded that Schleichera oleosa may serve as a new potential source of drugs for Rheumatoid arthritis due to the presence of these bioactive compounds.
ACKNOWLEDGMENT: The authors extend heartfelt gratitude to the Guides and gratefully acknowledge the cooperation of the faculty of Biotechnology, Shri Ramswaroop Memorial University, Lucknow-Deva Road, Barabanki, India.
CONFLICTS OF INTEREST: The authors declare no conflict of interest.
- Debreova M, Culenova M, Smolinska V, Nicodemou A, Csobonyeiova M and Danisovic L: Rheumatoid arthritis: From synovium biology to cell-based therapy. Cytotherapy 2022; 24(4): 365-375.
- Almoallim H, Al Saleh J, Badsha H, Ahmed HM, Habjoka S, Menassa JA and El-Garf A: A Review of the Prevalence and Unmet Needs in the Management of Rheumatoid Arthritis in Africa and the Middle East. Rheumatol Ther 2021; 8(1): 1-16.
- O’Neil LJ and Kaplan MJ: Neutrophils in Rheumatoid Arthritis: Breaking Immune Tolerance and Fueling Disease. Trends in Molecular Medicine 2019; 25(3): 215-27.
- Giannini D, Antonucci M, Petrelli F, Bilia S, Alunno A and Puxeddu I: One year in review 2020: pathogenesis of rheumatoid arthritis. Clin Exp Rheumatol 2020; 38(3): 387-397.
- Scott DL, Shipley M, Dawson A, Edwards S, Symmons DP and Woolf AD: The clinical management of rheumatoid arthritis and osteoarthritis: strategies for improving clinical effectiveness. Br J Rheumatol 1998; 37: 546–554.
- Iwasa S: Schleichera oleosa (Lour.) Oken. In: Faridah Hanum I, van der Maesen LJG (Eds) Plant Resources of South-East Asia No. 11. Auxiliary Plants, Prosea Foundation, Bogor, Indonesia 1997; 227-229.
- Anjum N, Hossain MJ, Haque MR, Chowdhury A, Rashid MA and Kuddus MR: Phytochemical Investigation of Schleichera oleosa (Lour.) Oken Leaf. Bangladesh Pharmaceutical Journal 2021; 24(1): 33–36.
- Sabe VT, Ntombela T, Jhamba LA, Maguire G, Govender T, Naicker T and Kruger HG: Current trends in computer aided drug design and a highlight of drugs discovered via computational techniques: A review. Eur J Med Chem 2021; 224: 113705.
- Bouback TA, Pokhrel S, Albeshri A, Aljohani AM, Samad A, Alam R, Hossen MS, Al-Ghamdi K, Talukder M, Ahammad F, Qadri I and Simal-Gandara J: Pharmacophore-Based Virtual Screening, Quantum Mechanics Calculations, and Molecular Dynamics Simulation Approaches Identifed Potential Natural Antiviral Drug Candidates against MERS-CoV S1-NTD, Molecules 2021; 26(16): 4961.
- Shankar R, Lale SK and Mudaiya RK: Conservation and Sustainable Utilization of Medicinal Plants of Chandauli and Obra Forests of Uttar Pradesh. J Drug Res Ayurvedic Sci 2017; 2(2): 49-63.
- Filimonov DA, Lagunin AA, Gloriozova TA, Rudik AV, Druzhilovskii DS, Pogodin PV and Poroikov VV: Prediction of the biological activity spectra of organic compounds using the PASS online web resource. Chemistry of Heterocyclic Compounds 2014; 50(3): 444-457.
- O'Boyle NM, Banck M, James CA, Morley C, Vandermeersch T and Hutchison GR: Open Babel: An open chemical toolbox. Journal of cheminformatics 2011; 3: 33.
- Daina A, Michielin O and Zoete V: SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci. Rep 2017; 7: 42717.
- Lee SK, Lee IH, Kim HJ, Chang GS, Chung JE and No KT: The PreADME Approach: Web-based program for rapid prediction of physico-chemical, drug absorption and drug-like properties, EuroQSAR 2002 Designing Drugs and Crop Protectants: processes, problems and solutions, Blackwell Publishing, Massachusetts, USA 2003; 418-420.
- Sorić Hosman I, Kos I and Lamot L: Serum Amyloid A in Inflammatory Rheumatic Diseases: A Compendious Review of a Renowned Biomarker. Front Immunol 2021; 11: 631299.
- Wang L, Ding Y, Guo X and Zhao Q: Role and mechanism of vascular cell adhesion molecule-1 in the development of rheumatoid arthritis. Exp Ther Med 2015; 10: 1229–1233.
- Rao FV, Houston DR, Boot RG, Aerts JM, Sakuda S and van Aalten DM: Crystal structures of allosamidin derivatives in complex with human macrophage chitinase. The Journal of biological chemistry 2003; 278(22): 20110–20116.
- Jones EY, Harlos K, Bottomley MJ, Robinson RC, Driscoll PC, Edwards RM, Clements JM, Dudgeon TJ and Stuart DI: Crystal structure of an integrin-binding fragment of vascular cell adhesion molecule-1 at 1.8 Å resolution. Nature 1995; 373(6514): 539–544.
- Labbé CM, Rey J, Lagorce D, Vavruša M, Becot J, Sperandio O, Villoutreix BO, Tufféry P and Miteva MA: MTiOpenScreen: a web server for structure-based virtual screening. Nucleic Acids Res 2015; 43(1): 448-54.
- Sanner MF: Python: A Programming Language for Software Integration and Development. J Mol Graph Model 1999; 17(1): 57-61.
- Lipinski CA: Lead- and drug-like compounds: the rule-of-five revolution. Drug Discov Today Technol 2004; 1(4): 337–341.
- Ma X, Chen C and Yang J: Predictive Model of Blood-Brain Barrier Penetration of Organic Compounds. Acta Pharmacologica Sinica 2005; 26: 500-512.
- Anjum N, Hossain MJ, Aktar F, Haque MR, Rashid MA and Kuddus MR: Potential In vitro and In vivo Bioactivities of Schleichera oleosa (Lour.) Oken: A Traditionally Important Medicinal Plant of Bangladesh. Research Journal of Pharmacy and Technology 2022; 15(1): 113-1.
- Kaur AM, Nain PA and Nain JA: Herbal plants used in treatment of rheumatoid arthritis: a review. Int J Pharm Pharmaceut Sci 2012; 4(4): 44–57.
- Negrei C, Bojinca V, Balanescu A, Bojinca M, Baconi D, Spandidos DA, Tsatsakis AM and Stan M: Management of rheumatoid arthritis: impact and risks of various therapeutic approaches (Review). Experimental and Therapeutic Medicine 2016; 11(4):1177–83.
- Jones RH and Tait CL: Gastrointestinal side-effects of NSAIDs in the community. Br J Clin Pract 1994; 49(2): 67–70.
- Bjarnason I, Hayllar J, Macpherson AJ and Russell AS: Side effects of nonsteroidal anti-inflammatory drugs on the small and large intestine in humans. Gastroenterology 1993; 104(6): 1832–47.
- Weisman MH, Furst DE, Park GS, Kremer JM, Smith KM, Wallace DJ, Caldwell JR and Dervieux T: Risk genotypes in folate-dependent enzymes and their association with methotrexate-related side effects in rheumatoid arthritis. Arthritis Rheum 2006; 54(2): 607–12.
How to cite this article:
Singh S, Paliwal S, Qidwai T and Singh S: Unraveling the phytoconstituents of Schleichera oleosa as an anti-arthritic activity: GC-MS analysis and molecular docking studies. Int J Pharm Sci & Res 2023; 14(3): 1312-18. doi: 10.13040/IJPSR.0975-8232.14(3).1312-18.
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Shivani Singh, Shweta Paliwal, Tabish Qidwai and Sachidanand Singh *
Department of Biotechnology, Smt. S. S. Patel Nootan Science & Commerce College, Sankalchand Patel University, Visnagar, Gujarat, India.
12 July 2022
26 September 2022
19 November 2022
01 March 2023