Anti-inflammatory evaluation of Spatoglossum variabile by GC-MS, in silico and in vitro assessment

INTRODUCTION

A complicated biological reaction of vascular tissue in response to noxious stimuli, characterised by pathogens and irritants are known as inflammation.^[1] Chemical and physical factors such as bacteria, poisons, radiation, injuries and caustic chemicals are also responsible for inflammation.^[2] A defensive mechanism, the inflammatory response seeks to limit hazardous substances. Another objective is the removal of damaged cells to promote tissue or organ repair.^[3] The beginning, progression, modulation and eventual resolution of the acute stage of inflammation are all mediated by a variety of chemical mediators that are released from neutrophils and macrophages. The removal of cell debris is primarily accomplished by monocytes. There will be a chronic stage if the acute stage is not resolved.^[4] Rheumatoid arthritis, atherosclerosis, hay fever and ischaemic heart diseases are all caused by chronic inflammation^[5-7] and inflammation is a common symptom of infectious diseases such as leprosy, tuberculosis, syphilis, asthma, inflammatory bowel syndrome, nephritis, vascularitis, celiac diseases and autoimmune diseases. Recently, chronic inflammatory diseases have been identified as the leading cause of death worldwide, accounting for more than half of all fatalities. These conditions include ischaemic heart disease, chronic kidney disease, cancer, diabetes, neurodegenerative diseases and autoimmune diseases. However, non-steroidal anti-inflammatory medicines (NSAIDs) have a variety of side effects, including increased risk for heart disease and gastrointestinal discomfort.^[7] Therefore, an in-depth study was done on several plant species and their active components in an effort to find new chemicals with anti-inflammatory capabilities that are less expensive and side effect free. Furthermore, research was not just done to assess the anti-inflammatory efficacy. As a result, numerous studies were conducted to identify all possible therapies.

The nuclear factor kappa B (NF-kB) pathway has long been considered a prototypical proinflammatory signalling pathway, largely based on the role of NF-kB in the expression of proinflammatory genes, including cytokines, chemokines and adhesion molecules. NF-kB’s intricate role in inflammation is discussed in this article.^[8] However, these most recent results indicate that this pathway may prove to be a challenging target in the treatment of chronic disease. NF-kB has long been regarded as the ‘holy grail’ as a target for new anti-inflammatory medications. The activation of NF-kB by proinflammatory cytokines such as interleukin-1 and tumour necrosis factor-α, as well as the role of NF-kB in the expression of other proinflammatory genes such as cytokines, chemokines and adhesion molecules, which have been extensively reviewed elsewhere, has led to the long-held belief that NF-kB is the prototypical proinflammatory signaling pathway.^[9] However, the inflammatory response involves a complicated physiological process called NF-kB and inflammation is one of these processes. Through its capacity to trigger the transcription of proinflammatory genes, nuclear translocation of cytoplasmic complexes, which activate the NF-kB/Rel transcription family, plays a crucial role in inflammation.^[10] The proper cellular stimulation, which is typically caused by signals relating to infections or stress, causes this route to become active. That is how different NF-kB proteins are specialised, how they play a part in inflammatory diseases, how IκB proteins and IκB kinase control NF-kB activity and how NF-kB inhibition therapy techniques are being developed. In response to a specific stimulus, the production of NF-kB proteins can offer site- and event-specificity.^[8,11,12]

Spatoglossum variabile belongs to the Dictyotaceae family and has a wide range of beneficial benefits. Ninety per cent of the world’s living biomass is found in the oceans, with marine species comprising approximately half of the total global biodiversity. Marine vegetation is a potentially abundant source of highly bioactive secondary metabolites that could serve as helpful leads in the creation of novel pharmacological medicines.^[13] Algae can be divided into two primary categories: macroalgae (seaweeds), which include green, brown and red algae and microalgae (blue–green algae, dinoflagellates, Bacillariophyta).^[14] Marine algae are abundant in dietary fibre, minerals, lipids, proteins, vital amino acids, polysaccharides and Vitamins A, B, C and E. The main components of brown and red algal cell walls are phycocolloids, which are extensively employed in industry and include agar, alginic acid and carrageenan. In the last four decades, a large number of new compounds have been identified from marine creatures, and many of these compounds have been shown to exhibit fascinating biological activity.^[15] Their sizes range significantly, from microscopic unicellular organisms to huge kelps up to 70 m long that can grow as fast as 50 cm/day.^[16] Algae are present almost everywhere on the planet, including the ocean, rivers, lakes, soil and cliffs. Lately, microalgae metabolites have drawn a lot of attention, and several researchers have written on the subject. Many health-promoting properties, such as anti-oxidative, anti-inflammatory, antibacterial and anti-cancer effects, have been identified by studies on the bioactivities of marine algae.^[17] Anti-inflammatory properties are well-known for many marine-based natural products that contain antioxidants.^[18] Sargassum vulgare, Spatoglossum schroederi and S. variabile also have anti-inflammatory effects. Recent epidemiological and clinical research has demonstrated that consumption of plant-derived foods and beverages may lower the risk of oxidative damage-related diseases, such as aging and other lifestyle diseases.^[19] A lot of emphasis has been placed on the isolation and study of new bioactive components with biological activity from marine algae. According to their colour, marine algae were largely divided into three primary types known as brown, red and green algae, which are referred to as Phaeophyceae, Rhodophyceae and Chlorophyceae, respectively.^[20,21] It has been discovered that different pigments extracted from marine algae have a variety of biological activities and potential health perks. Due to this, there has recently been a surge in interest in adopting complementary and natural therapies to treat a variety of disorders; however, there is a lack of sufficient scientific data. Interest in medicines derived from nature has increased to create novel drugs with greater therapeutic potential.^[22,23]

MATERIALS AND METHODS

Gas chromatography/mass spectroscopy (GC/MS) analysis

The GC-MS analysis was conducted on a GC-MS QP2010 Plus equipped with a flame ionisation detector. The GC is equipped with a fused silica (30 m × 0.25 mm ID × 0.25 mm) capillary column. The oven temperature was programmed at 60°C for 2 min, then increased to 300°C for 6 min at the rate of 10°C/min. Helium was used as a carrier gas at a flow of 1.0 mL/min. The injector temperature was 250°C, injection size 1.0 µL neat, with a split ratio 10:1. Mass detector turbo mass gold-Perkin Elmer was used as the detector. The phytoconstituents were identified after comparison with those available in the WILEY 8. LIB is attached to the instrument and reported [Figure 1].^[26]

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Figure 1:

Graphical representation of protein denaturation.

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RESULTS

GC-MS analysis

The GC-MS chromatogram of the extracts of S. variabile in ethanol, ethyl acetate and chloroform revealed 155 compounds in ethanol and 112 peaks in the other two solvents. According to the bioactive compounds that were identified [Table 3] by comparing their mass spectral fragmentation patterns, peak retention times, peak areas (%) and heights (%) to those of the known compounds listed in the NIST collection. Results revealed that 13 compounds were found in chloroform and ethyl acetate; 14 compounds were found in ethanol extracts. Overall, the 39 main phytocompounds were identified in chloroform, ethyl acetate and ethanol. Overall, the successive extraction was effective in identifying the diverse range of compounds from the ethanol extract. The ethanolic extract contained more polar compounds when compared to chloroform and ethyl acetate. Moreover, several of these compounds have been shown to have potential anti-inflammatory properties. Overall presence of these compounds 2-HYDROXY-2-METHYL-4-PENTANONE (DIACETONE), Tetradecanoic acid, Neophytadiene, n-Hexadecanoic acid, Oleic acid, Octadecanoic acid, Behenic alcohol, 4,8,12,16-Tetramethylheptadecan-4-olide, 1-Heptacosanol, Oleoyl chloride, 9-Octadecenoic acid, 1,2,3-propanetriyl ester, (E,E,E)-, Pregnane Silane Derivatives in the ethanolic extract suggest that it has the potential to promote wound healing through several different mechanism, including reducing inflammation, promoting tissue repair, increasing collagen production and preventing infection.

Table 3: GCMS identified compounds.

Swiss ADME analysis

From the ChemDraw Ultra, all of the ligands’ smiles were obtained, and the Swiss ADME programme was then used. The representation of a boiled egg [Figure 2], physicochemical properties, lipophilicity, water solubility, drug similarity and pharmacokinetics are all provided. Drug-like compounds should have a good aqueous solubility, which is predicted by three methods: ESOL, (ALI) logs and (SILICOS-IT) logs.^[33]

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Figure 2:

Graphical representation of human red blood cell assay.

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In silico studies

Docking studies

In silico docking studies can be used to predict the binding affinity of small molecules to a target protein for anti-inflammatory purposes. The binding affinity of each compound for the target protein is expressed as a binding energy or score [Table 4]. The predicted binding mode of the compound to the target protein, including the amino acid residues involved in the interaction and the type of interaction (e.g., hydrogen bonds, van der Waals forces) [Supplementary 1]. Interpretation of the results and their significance in relation to the anti-inflammatory activity of the compounds studied. The results obtained from in silico docking studies should be considered as preliminary indications of a compound’s potential anti-inflammatory activity. Additional experimental data and validation are necessary to confirm these predictions.^[34]

Table 4: Binding energy of the compounds by in silico docking.

Compound name	Binding energy
	NF-kappa b
Diclofenac sodium	−7.1
2,6-Dimethyl-4-nitro-3-phenyl-cyclohexanone	−6.5
2-(2-aminoanilino)-5-methyl-2-thiazoline	−6.2
10-heneicosene, 11-phenyl-	−6.2
N-benzyl-2-([5-(2-furyl)-1,3,4-oxadiazol-2-yl] sulfanyl) acetamide	−7.3
2-((e)-[((e)-2-([(e)-(2-hydroxyphenyl) methylidene] amino) propyl) imino] methyl) phenol	−7.1
1,1’-(4-methyl-1,3-phenylene) bis[3-(5-isopropyl-1,3,4-thiadiazol-2-yl) urea]	−9.0
2-(3-hydroxy-1-propynyl)-2-adamantan-2-ol	−5.6

In vitro studies

Protein denaturation assay

EGG albumin

Egg albumin assay is a common in vitro test used to assess the protein content of egg white. The egg albumin assay is very important to assess the protein content of egg white. The results of the egg albumin assay, a graph showing the standard curve generated using known concentrations of albumin and their corresponding absorbance values [Figure 3]. The protein concentration of the egg white sample, expressed in mg/mL or percentage. The results from replicates of the assay, including mean values and standard deviations. The results from control samples, including positive and negative controls. Interpretation of the results and their significance in relation to the protein content of the egg white sample. Comparison with previously reported studies, limitations and future directions. The results obtained from in vitro egg albumin assays should be interpreted with caution, as there may be variations in the protein content of eggs due to factors such as diet, age and storage conditions. Additional experimental data and validation may be necessary to confirm the protein content of the egg white sample [Table 5].

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Figure 3:

GCMS chromatogram of the ethanolic extract of Spataglossum variabile

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Table 5: Egg albumin assay.

Concentration	Standard (%)	Test (%)
10 µg	20	13.6
20 µg	32	28
40 µg	54	46
80 µg	72	64
100 µg	95	92

HRBC assay

The HRBC assay is an in vitro test used to evaluate the anti-inflammatory activity of compounds. The HRBC assay is very important to evaluating the anti-inflammatory activity of compounds. The absorbance values obtained for the control group may consist of untreated HRBCs or HRBCs treated with a known pro-inflammatory agent [Figure 4]. The absorbance values obtained for the test groups, which consist of HRBCs treated with different concentrations of the test compound. The concentration of the test compound that inhibits 50% of haemolysis, calculated using a dose–response curve. Results and their significance in relation to the anti-inflammatory activity of the test compound were interpreted [Table 6]. Comparison with previously reported studies, limitations and future directions.

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Figure 4:

SwissADME BOILED-Egg model representing the predicted gastrointestinal absorption. (HIA) and blood–brain barrier (BBB) permeability of the identified phytoconstituents

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Table 6: HRBC assay values.

Concentration	Standard (%)	Test (%)
10 µg	25	17
20 µg	40	36
40 µg	62	57
80 µg	104	97
100 µg	129	113

HRBC: Human red blood cell

DISCUSSION

Microalgae contain several different types of potent, naturally occurring bioactive compounds. The extract from S. variabile was used in the current study to examine anti-inflammatory activity. An HRBC assay and albumin denaturation were used to measure the extract’s anti-inflammatory activity.^[35,36]

In addition to fucoxanthin, the most significant component found in marine brown algae, the spectral analysis of the extract from S. variabile revealed the existence of numerous other active elements.^[37] Using GC MS analysis, it is feasible to identify the major non-volatile chemicals. Unprocessed ethanolic extract displayed a high peak intensity molecule that was significant to numerous other essential compounds, including pentadecanoic acid, 14-methyl-, methyl ester and methyl stearate, among other derivatives.^[38,39] Minor substances such as 10-methyl-, methyl ester, methyl tetradecanoate, tetradecanoic acid, 12-methyl-, methyl ester, 9-hexadecenoic acid, methyl ester, (Z)- By using GC MS analysis, the main non-volatile chemicals are recognised.^[40] The high peak intensity compound dominating the presence of important compounds, including Pentadecanoic acid, 14-methyl-, methyl ester and methyl stearate among other derivatives, was indicated by the crude ethanolic extract.^[20] Minor compounds were also found. They were 10-methyl-, methyl ester, methyl tetradecanoate, tetradecanoic acid, 12-methyl-, methyl ester, 9-hexadecenoic acid, methyl ester, (Z)-, hexadecanoic acid, 14-methyl-, methyl ester, 10-octadecenoic acid, methyl ester and heptadecanoic acid. These substances have been shown to have antioxidant, antifungal, antimicrobial, nematicide, hypercholesterolemic and cancer-preventive properties.^[41] Furthermore, the compounds identified from GCMS analysis of the algae extract have revealed many active compounds which are responsible for anti-inflammatory activity. Certain literature has also reported that these active constituents have been present in certain plant species and have anti-inflammatory activity. By this, we are also confirming that the extract of S. variabile also has anti-inflammatory activity by performing the egg albumin and HRBC assay.^[42] Several investigations have led researchers to the conclusion that the ethanol extract of seaweeds contains phenolics, alkaloids and amino acids that may be the cause of the anti-inflammatory activity.^[43]

For in silico docking, the protein 1SVC was chosen based on the pathophysiological steps it is engaged in, and 7 compounds were chosen and docked with this protein. The compounds were chosen based on the peak produced from GCMS analysis. In addition, the standard drug Diclofenac was docked with these proteins, and the binding energies of the standard drug were compared to the binding energies of the seven compounds obtained from the extract. All 7 compounds have excellent binding energies compared to standard drugs with proteins. This was further evaluated by an in vitro anti-inflammatory protein denaturation assay in which most proteins lose their biological function when they are denatured, a process known as albumin denaturation. Inflammation can be predicted to be caused by the denaturation of proteins. The capacity of the ethanolic extract of S. variabile to inhibit protein denaturation was intended as a component of the research into the mechanism of the anti-inflammation activity. With a maximal inhibition of 92% seen at 100 µg/mL, it was successful in preventing albumin denaturation. Diclofenac, a common anti-inflammatory medication, had a maximal 95% inhibition at the same dose. Protein denaturation was intended to be avoided by the ethanolic extract of S. variabile. At 100 µg/mL, the maximum level of inhibition is 92%. It has shown efficacy in halting albumin denaturation. The common anti-inflammatory drug diclofenac demonstrated a maximal inhibition of 95% at the same dose. Since the erythrocyte membrane and the lysosomal membrane have a similar structural makeup, the HRBC membrane stabilisation method was used to evaluate the in vitro anti-inflammatory efficacy.^[26] Lysosomal stabilisation is essential for limiting the release of lysosomal contents, which in turn helps to regulate the inflammatory response. Lysosomal enzymes generated during inflammation are responsible for a wide range of diseases. At lesser concentrations like 100 g/mL, the standard medication had more activity (25%) than the test sample (17%), but at this concentration, the standard drug had more activity (129%) than the test drug’s 113%.

The ethanolic extract of S. variabile prevented heat-induced haemolysis at a number of doses, including 10, 20, 40, 60, 80 and 100. According to the results at a 100 g/mL concentration, the test sample shows a considerable inhibition of 113% to the common drug Diclofenac, which yields 129%. The inhibition values were found to be 113% and 129% for the test samples and the standard, respectively.

CONCLUSION

The evaluation of the ethanolic extract of S. variabile indicates the existence of an in vitro study that was utilised to demonstrate the potent anti-inflammatory activity of S. variabile and aids in the discovery of active metabolites. The current study’s findings indicate that S. variabile is a potent antibiotic that can successfully cure a wide range of bacterial infections as well as diseases such as cancer, neurological disorders and aging. It is also a potent anti-inflammatory drug that can be used. More in vivo testing on the chemical is required to determine the extract’s mode of action as a cutting-edge medicinal agent.