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In silico Investigation and BSA Denaturation Inhibitory Activity of Ethyl Acetate and N-butanol Extracts of Centaurea tougourensis Boiss. and Reut.



Mohamed Sabri Bensaad, Saliha Dassamiour, Leila Hambaba, Mohamed Akram Melakhessou, Rokayya Sami, Amina A. M. Al-Mushhin, Nada Benajiba and Luluah M. Al Masoudi
 
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ABSTRACT

Background and Objectives: Phytotherapy is considered nowadays an important discipline that allowed both the scientific community and pharmaceutical companies to innovate in the drug discovery and development process. In this study, we aimed to evaluate the anti-inflammatory activity of the n-butanol (n-BuOH) and ethyl acetate (EA) extracts of a plant species named C. tougourensis as well as to investigate for the first time the physicochemical and biological properties of some compounds identified in this plant using a bioinformatics approach. Materials and Methods: In this study, the in silico approach was performed to predict the physicochemical properties, ADMET profile, pharmacological capacities, cytotoxicity as well as the gene expression profiles of four compounds recently identified from Centaurea tougourensis via the gas chromatography-mass spectrometry (GC-MS) approach. Thus, two compound were tested from the n-BuOH extract named, respectively, Silanediamine, 1,1-dimethyl-N,N'-diphenyl- (compound 1), Methanone, 1,3-dithian-2-ylphenyl-(compound 2) and two compounds from EA extract, named, respectively, 4-Fluoro-1-methyl-5-carboxylic acid, ethyl (ester) (compound 3), propionic acid, 3-iodo-, tetradecyl ester (compound 4). The anti-inflammatory activity of these extracts was also tested using bovine serum albumin (BSA) denaturation approach. Results: The in silico investigation revealed that the four tested compounds could be a good candidate to regulate the expression of key genes and may also exert significant cytotoxic effects against several tumour cell lines. In addition, these compounds could also be effective in the treatment of metabolic disorders linked to diabetes and obesity, but also oxidative stress, immune system dysfunction and skin pathologies. The BSA assay revealed that both extracts have almost the same moderate inhibition effect on protein denaturation. The EC50 values were (335±0.03 μg mL–1) for n-BuOH and (338±0.07 μg mL–1) for EA extracts. The difference was considered highly significant (p<0.001) when compared to diclofenac sodium (115.76±0.19). Conclusion: These preliminary results indicate that the actual biocompounds contained in Centaurea tougourensis could be a good candidate for the elaboration of more effective and natural drugs.

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Mohamed Sabri Bensaad, Saliha Dassamiour, Leila Hambaba, Mohamed Akram Melakhessou, Rokayya Sami, Amina A. M. Al-Mushhin, Nada Benajiba and Luluah M. Al Masoudi, 2022. In silico Investigation and BSA Denaturation Inhibitory Activity of Ethyl Acetate and N-butanol Extracts of Centaurea tougourensis Boiss. and Reut.. International Journal of Pharmacology, 18: 1296-1308.

DOI: 10.3923/ijp.2022.1296.1308

URL: https://scialert.net/abstract/?doi=ijp.2022.1296.1308
 
Copyright: © 2022. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

Several bioinformatics means are now-a-days used to predict with high accuracy the physicochemical and pharmacological properties of specific compounds due to the emergence of a discipline called in silico, which is principally based on computer-aided drug design softwares and collaborative platforms1. This bioinformatics approach can be very helpful, especially before conducting in vitro and in vivo experiments2. Indeed, several fields of research including genetic and evolutionary biology have considerably evolved due to in silico and molecular docking process3, as well as the possible interactions of a molecule with a specific receptor at the atomic level, based on energy transfer process4, helping the scientific community to develop more effective therapies, especially against cancer.

Human health is constantly threatened by pathogens including; microorganisms, chemicals, radiations, physical agents, which may also have a negative repercussion on the genetic level5,6. However, our body has many natural means to maintain its physiological state. Among them, the inflammatory reactions are considered as a natural response of the body's immune system mainly orchestrated by immune cells via the secretion of key cytokines, interferons and chemokines to promote tissue repair and recovery7. However, in the pathological situation, a dysfunction of the immune system can lead to an excessive inflammatory response which can result in the development of chronic pathologies such as ulcer, tuberculosis, asthma and rheumatoid arthritis8.

Plants have always played a central role in human history and the bioactive compounds found in Centaurea species such as flavonoids, saponins, terpenes, tannins and other phenolic compounds could explain their remarkable abilities to treat several illnesses related to oxidative stress, diabetes, inflammation, cancer, infections and neurological disorders9-12. This study is a continuation of the previous works in which demonstrated the antioxidant, anti-inflammatory, hepatoprotective, antidiabetic, haemostatic, neuroprotective, photoprotective and antimicrobial activities of Centaurea tougourensis13-15.

The present study aimed to predict the physicochemical and biological properties of four identified compounds using in silico approach as well as to evaluate in vitro the anti-inflammatory effect of both extracts.

MATERIALS AND METHODS

Study area: The study was carried out in collaboration between the Laboratory of Biotechnology of Bioactive Molecules and Cellular Physiopathology (LBMBPC), University Batna 2, Algeria and the Department of Food Science and Nutrition, College of Sciences, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia from October, 2021 to January, 2022.

Plant collection: Centaurea tougourensis was collected from the commune of Fesdis (Algeria) (Lat 35.621975, long 6.241327) and conserved under a voucher specimen (CT/2019/LPTPCMB).

Plant extraction method: The plant was dried, covered away from sun’s rays and then ground for future use. Maceration was carried out in triplicate with EtOH-H2O mixture (70:30) at ambient temperature for 3 days then evaporated using solvents with different polarities.

In silico study

Generation of canonical smiles: For this approach, Open Babel (version 2.0.2)16 and In Draw webserver (http://in.indraw.integle.com/) were used. Indeed, two compounds were selected from the n-BuOH extract and named, respectively, Silanediamine, 1,1-dimethyl-N, N'-diphenyl-(compound 1), Methanone, 1,3-dithian-2-ylphenyl- (compound 2) while two compounds were selected from the EA extract, named, respectively, 4-Fluoro-1-methyl-5-carboxylic acid, ethyl(ester) (compound 3), Propionic acid, 3-iodo-, tetradecyl ester (compound 4).

Physicochemical properties: SwissADME web-service (http://www.swissadme.ch/) was used for this context and six physicochemical properties were taken into account; lipophilicity, size, polarity, solubility, flexibility and saturation17.

ADMET profile: The absorption, distribution, metabolism, excretion and toxicity of the four compounds was tested using admetSAR 2.0 (http://lmmd.ecust.edu.cn/admetsar2)18.

Pharmacological properties: The bioactivity score of each compound was predicted using the online platform of PASS online (http://way2drug.com/passonline/)19.

Cytotoxicity prediction: To estimate the possible cytotoxicity on human tumour cell lines, the online platform of CLC-Pred made it possible, based on the structural formula of each compound previously obtained20.

Gene expression profiles: The in silico prediction of the possible influence of the four compounds on gene expression has been considered using DIGEP Pred (http://www.way2drug.com/ge/), which is primarily based on Upregulation and Downregulation processes21,22.

Bovine albumin denaturation assay: The principle of this assay consists to evaluate the possible anti-inflammatory effect of our plant on bovine serum albumin (BSA) denaturation caused by heat (72°C). For this purpose, 1 mL of each concentration of extract or diclofenac sodium was added to 1 mL of BSA (0.2%) solution previously prepared in Tris-HCl buffer solution (pH = 6.6). The mixture was then incubated at ambient temperature for 15 min, then in a water bath at 72°C for 5 min. After cooling, the turbidity was measured at 660 nm23. In addition, the EC50 value was estimated and was calculated by this equation:

Image for - In silico Investigation and BSA Denaturation Inhibitory Activity of Ethyl Acetate and N-butanol Extracts of Centaurea tougourensis Boiss. and Reut.


Abscontrol = Represent the absorbance of the control group
Abssample = Represent the absorbance of plant extract or standard drug

Statistical analysis: Data were expressed as Mean (±SD) for in vitro tests and ±SEM and the statistical analyses were performed by ANOVA test using GraphPad Prism version 8. Results were considered highly significant at p<0.001.

RESULTS AND DISCUSSION

In silico study
Canonical smiles generation: In order to estimate the physicochemical and biological properties of the tested compounds, the corresponding canonical smile of each compound was first generated as follow:

Compound 1: (Silanediamine, 1, 1-dimethyl-N,N'- diphenyl-): C[Si](C)(NC1=CC=CC=C1)NC 2=CC=CC=C2
Compound 2: (Methanone, 1, 3-dithian-2-ylphenyl-): C1CSC(SC1)C(=O)C2=CC=CC=C2
Compound 3: (4-Fluoro-1-methyl-5-carboxylic acid, ethyl(ester)): CCOC(=O)C1=C(N=CN1C )F
Compound 4: (Propionic acid, 3-iodo-, tetradecyl ester): CCCCCCCCCCCCCCOC(=O)CCI

Table 1 provides detailed information about the molecular formula and structure of the tested compounds.

Physicochemical properties: The evaluation of the possible therapeutic effect of candidate compounds is directly linked to their physicochemical properties. This approach can help in the design process of new drugs to treat several diseases related to infections24. Indeed, scientists solved some physicochemical characteristics related to mucus, considered as a complex hydrogel biopolymer barrier which is found in several areas including airway epithelium, gastrointestinal tract or the ocular surface of eyes. Indeed, the mucus act by preventing direct access of microorganisms to these zones via its viscoelasticity property, which nowadays helped in the development of transmucosal drug delivery system25,26.

Figure 1 shows the degree of unsaturation of compound 1 was considered the best, which could mean that this compound may be more reactive, since the degree of unsaturation is proportional to the number of carbons present in the molecule which contain generally double or triple covalent bonds between adjacent carbon atoms to add more hydrogens27. This information is very important since unsaturated compounds are giving very promising results in the treatment of human carcinogenicity28. In term of polarity, compound 2 showed the best results and the degree of unsaturation of this compound were also very important. Compound 4 exhibited the best flexibility, insolubility and lipophilicity properties and was considered remarkable. Lipophilicity and polarity are important parameters that allowed scientists to treat several infections affecting kidneys via the identification of physicochemical determinants of human renal clearance29 and to develop more effective pesticides that are less harmful to human health and ecosystem30. A compound with high flexibility means an increase in the dynamics molecular binding process with respective receptors which will considerably strengthen the action of drugs on their biomolecular targets to generate the therapeutic effect31. Noting that, compound 3 showed also a non-negligible unsaturation property.

ADMET profile: Table 2 shows that, results clearly indicate that the four compounds may cross blood-brain barrier (BBB), Caco-2 cells but may also be absorbed by human intestine with a optimum oral bioavailability, except for compound 4, suggesting that most of these compounds may easily transit from the gastrointestinal tract into the bloodstream to reaches their site of action32. This process helps also in the development of more effective drugs with less risk of side effects and toxicity33. The majority of molecules with an important molecular weight are excluded from the central nervous system (CNS) via BBB membrane, which suggests that all compounds may be considered a suitable candidate for the elaboration of more effective neurotherapeutics and CNS drug delivery programs34. The data also revealed that these compounds may not be a P-glycoprotein (P-gp) substrates, excluding also a probable attenuation on the activity of P-gp, which suggest that these compounds will not interfere with the biotransformation and excretion of drugs35.

Table 1: Generation of the canonical smiles of the four tested compounds
Extracts Compounds
Molecular formula
Structure
Canonical smiles
nBuOH Compound 1
C14H18N2Si
Image for - In silico Investigation and BSA Denaturation Inhibitory Activity of Ethyl Acetate and N-butanol Extracts of Centaurea tougourensis Boiss. and Reut.
C[Si](C)(NC1=CC=CC=C1)NC2=CC=CC=C2
Compound 2
C11H12OS2
Image for - In silico Investigation and BSA Denaturation Inhibitory Activity of Ethyl Acetate and N-butanol Extracts of Centaurea tougourensis Boiss. and Reut.
C1CSC(SC1)C(=O)C2=CC=CC=C2
EA Compound 3
C7H9FN2O2
Image for - In silico Investigation and BSA Denaturation Inhibitory Activity of Ethyl Acetate and N-butanol Extracts of Centaurea tougourensis Boiss. and Reut.
CCOC(=O)C1=C(N=CN1C)F
Compound 4
C17H33IO2
Image for - In silico Investigation and BSA Denaturation Inhibitory Activity of Ethyl Acetate and N-butanol Extracts of Centaurea tougourensis Boiss. and Reut.
CCCCCCCCCCCCCCOC(=O)CCI


Image for - In silico Investigation and BSA Denaturation Inhibitory Activity of Ethyl Acetate and N-butanol Extracts of Centaurea tougourensis Boiss. and Reut.
Fig. 1: Prediction of the physicochemical properties of the 4 tested compounds, (a) Compound 1, (b) Compound 2, (c) Compound 3 and (d) Compound 4
LIPO: Lipophilicity, SIZE, POLAR: Polarity, INSOLU: Insolubility, INSATU: Unsaturation and FLEX: Flexibility


Table 2: Prediction of the pharmacokinetic characteristics of the 4 compounds
n-BuOH extract EA extract
Parameters/compounds Compound 1 Compound 2 Compound 3 Compound 4
Absorption
Human oral bioavailability HOB+ HOB+ HOB+ HOBG
Human intestinal absorption HIA+ HIA+ HIA+ HIA+
Blood brain barrier BBB+ BBB+ BBB+ BBB+
Caco-2 permeability Caco2+ Caco2+ Caco2+ Caco2+
P-glycoprotein substrate Non-substrate Non-substrate Non-substrate Non-substrate
P-glycoprotein inhibitor
Distribution and metabolism Non-inhibitor Non-inhibitor Non-inhibitor Non-inhibitor
Subcellular localization Mitochondria Mitochondria Mitochondria Mitochondria
CYP450 3A4 substrate Non-substrate Non-substrate Non-substrate Non-substrate
CYP450 2C9 substrate Non-substrate Non-substrate Non-substrate Non-substrate
CYP450 2D6 substrate Non-substrate Non-substrate Non-substrate Non-substrate
CYP450 3A4 inhibition Non-inhibitor Non-inhibitor Non-inhibitor Non-inhibitor
CYP450 2C9 inhibition Inhibitor Non-inhibitor Non-inhibitor Non-inhibitor
CYP450 2C19 inhibition Inhibitor Inhibitor Inhibitor Non-inhibitor
CYP450 2D6 inhibition Non-inhibitor Non-inhibitor Non-inhibitor Non-inhibitor
CYP450 1A2 inhibition
Excretion and toxicity Inhibitor Inhibitor Inhibitor Inhibitor
Acute oral toxicity Class III Class III Class III Class III
Hepatotoxicity Non-hepatotoxic Non-hepatotoxic Hepatotoxic Non-hepatotoxic
Carcinogenicity Carcinogens Non-carcinogens Non-carcinogens Non-carcinogens
Ames mutagenesis Non-mutagenic Non-mutagenic Non-mutagenic Non-mutagenic
Eye corrosion Non-corrosive Non-corrosive Non-corrosive Corrosive
Eye irritation Irritant Irritant Irritant Irritant
Honey bee toxicity Non-toxic Toxic Non-toxic Toxic
Crustacea aquatic toxicity Toxic Non-toxic Non-toxic Toxic
Fish aquatic toxicity Toxic Non-toxic Non-toxic Toxic
Estrogen receptor binding Binding Non-binding Non-binding Non-binding
Androgen receptor binding Non-binding Non-binding Non-binding Non-binding
Thyroid receptor binding Non-binding Non-binding Non-binding Non-binding
Glucocorticoid receptor binding Non-binding Non-binding Non-binding Non-binding
Biodegradation
ADMET predicted profile (regression) Non-biodegradable Biodegradable Non-biodegradable Biodegradable
Water solubility (log S) -3.416 -2.396 -4.247 -3.491
Plasma protein binding (%) 1.115 0.977 0.44 0.759
Acute oral toxicity (kg mol–1) 2.88 2.303 2.211 2.816
Tetrahymena pyriformis (pIGC50, μg L–1) 0.517 1.522 0.56 3.007

Concerning distribution, the mitochondrion seems to be the targeted organelle for all tested compounds which suggest that these compounds could modulate mitochondrial functions since this organelle is considered the main source of cellular energy via the production of adenosine triphosphate (ATP) by oxidative phosphorylation process36, which contribute to the maintenance of optimal tissue homeostasis. It is also important to note that dysfunction in mitochondrial energy balance could contribute to the emergence of several pathologies associated with diabetes, immunity and aging37,38. In term of metabolism, the four compounds may be classified as non-substrates for CYP450 3A4, 2C9 and 2D6 isoforms. It was also observed that the compounds 2, 3 and 4 can be considered as non-inhibitors of CYP450 3A4, 2C9 and 2D6 isoforms while the four compounds could be considered potential inhibitors of CYP450 1A2 isoform. This information is very important since Cytochrome P450 enzymes are essential for the metabolism of many medications but also to avoid the formation of a variety of toxic products39.

Concerning the Ames mutagenicity, the results suggest that all compounds could be considered non-mutagenic and only compound 1 may be carcinogenic. These non-mutagenic compounds could be considered as potential antitumoral agents since the development of more effective anticancer drugs is crucial in the actual era40. The acute oral toxicity for all tested compounds was very high (class III) and excepting the compound 3, the other compounds seem to be non-toxic for the liver. The compounds appear to be irritant to the eyes, however, only compound 4 could corrosive. The toxicity prediction revealed that compounds 1 and 4 could express high toxicity on fish and Crustacea, while compounds 2 and 3 could be non-toxic. Concerning the toxicity to the honey bee, compounds 1 and 3 could be non-toxic.

Table 3: Bioactivity prediction of the 4 compounds
Extracts Compounds
Pa
Pi
Biological activities
n-BuOH Compound 1
0.749
0.012
Fragilysin inhibitor
0.742
0.030
Antiseborrheic
0.562
0.028
Antiviral (Picornavirus)
Compound 2
0.669
0.020
Anti-inflammatory
0.594
0.044
Kidney function stimulant
0.512
0.047
Oxygen scavenger
EA Compound 3
0.723
0.005
Cognition disorders treatment
0.698
0.007
Antiobesity
0.614
0.011
Antidiabetic
Compound 4
0.800
0.003
Leukopoiesis stimulant
0.729
0.010
Macrophage colony-stimulating
0.734
0.035
Factor agonist antieczematic
Pa: Probability of activity, Pi: Probability of inactivity

The data also revealed that the four compounds may not interact with androgen, triiodothyronine (T3), thyroxine (T4) and glucocorticoid receptors. However, concerning estrogen receptors, data showed that compound 1 could interact with this receptor suggesting that compound 1 may mimic the activity of estrogen. This information is very important, since this steroid hormone plays a key role in mammalian reproduction, regulate uterus function and contribute to the development of women's secondary sex characteristics41. This hormone is also involved in the treatment of breast cancer and cardiovascular disease42,43.

Compound 3 exhibited the lowest value of water solubility (-4.247) while compound 1 showed the highest affinity for plasma proteins (1.115%) and also the most important acute oral toxicity (2.88 kg moL–1). Finally, compound 4 was the most active on Tetrahymena pyriformis by expressing a median population growth of (3.007 μg L–1).

Pharmacological properties: Table 3 shows that, compound 1 could be useful as fragilysin inhibitor as well as a potent antiseborrheic agent with corresponding values (Pa = 0.749, Pa = 0.742). Fragilysin is an enterotoxin produced by pathogenic microbial strains including Bacteroides fragilis that is responsible for diarrhoeal disease in humans44. It is well known that an excess of sebum could generate scaly patches, stubborn dandruff and red skin45. Compound 4 may also exert a significant effect against eczema (Pa = 0.734), which suggests that compounds 1 and 4 could be considered good candidates for the treatment of seborrheic dermatitis and other conditions affecting the skin.

Data also revealed that compound 2 may exert a moderate anti-inflammatory effect (Pa = 0.669), but also a non-negligible stimulation on kidney function and oxygen scavenging process with corresponding values (Pa = 0.594, Pa = 0.512). This information is very important since the deregulation of the inflammatory response could break body homeostasis and generate long-term severe pathologies46,47.

It is also interesting to underline that compound 3 could be a potential candidate to treat cognitive disorders (Pa = 0.723), which can be temporary or progressive disorders48. A decrease in cognitive performance will have a bad repercussion on intellectual function including reasoning, memory and speech but also on spatiotemporal perception which will directly affect motor skills since these elements are interconnected49. The compound 3 showed also moderate antiobesity and antidiabetic effects with respective values (Pa = 0.698, Pa = 0.614). There is an important relationship since obesity increases the risk of developing type 2 diabetes50, but also cardiovascular diseases and certain types of cancers51. This information suggests that compound 3 could be useful to treat metabolic disorders related to diabetes and obesity52.

Compound 4 showed a remarkable stimulation on leukopoiesis process (Pa = 0.800) but also on the expression of a cytokine called macrophage colony-stimulating factor (Pa = 0.729) which suggest that this compound could boost the hematopoiesis phenomenon to accelerate the production and differentiation of immune system cells53, especially macrophage, lymphocytes, granulocytes and natural killer to fight infections especially intercellular viral infections54.

Cytotoxicity prediction: Table 4 shows that, compounds 1 and 4 are active on brain tissue. Indeed, these two compounds exhibited a moderate cytotoxic activity in which compound 1 was active on the oligodendroglioma (Hs 683) cell line (Pa = 0.734), while compound 2 was on glioblastoma (SF-539) cell line (Pa = 0.507). This information is very important since neurodegenerative diseases are rapidly rising in prevalence55 and the actual treatment especially antibiotic used to treat pathologies related to the nervous system presents a lot of side effects56, which explain the urgent situation to elaborate more effective drugs.

Table 4: Probable cytotoxic activities of the 4 compounds on some tumour cell lines
Extracts Compounds Cell-line Cell-line full name Tissue Tumour type
Pa
Pi
n-BuOH Compound 1 Hs 683 Oligodendroglioma Brain Glioma
0.734
0.007
MDA-MB-468 Breast adenocarcinoma Breast Adenocarcinoma
0.563
0.009
HOP-18 Non-small cell lung carcinoma Lung Carcinoma
0.454
0.018
Compound 2 HeLa Cervical adenocarcinoma Cervix Adenocarcinoma
0.669
0.007
Hs 683 Oligodendroglioma Brain Glioma
0.537
0.044
HOP-18 Non-small cell lung carcinoma Lung Carcinoma
0.311
0.069
EA Compound 3 NCI-H838 Non-small cell lung cancer stage 3 Lung Carcinoma
0.648
0.013
DMS-114 Lung carcinoma Lung Carcinoma
0.566
0.015
SK-MES-1 Squamous cell lung carcinoma Lung Carcinoma
0.541
0.004
Compound 4 SF-539 Glioblastoma Brain Glioblastoma
0.507
0.012
SN12C Renal carcinoma Kidney Carcinoma
0.505
0.013
NCI-H838 Non-small cell lung cancer stage 3 Lung Carcinoma
0.430
0.103
Pa: Probability of activity, Pi: Probability of inactivity


Table 5: Possible effect of the 4 compounds on mRNA expression level of some genes
Extracts Compounds
Genes (upregulation)
Pa
Pi
Genes (downregulation)
Pa
Pi
n-BuOH Compound 1
SAT
0.887
0.014
MYO5C
0.862
0.014
POR
0.878
0.028
IFIH1
0.852
0.037
HTATIP2
0.866
0.009
TEP1
0.848
0.011
Compound 2
TNNT1
0.724
0.030
IFI27
0.663
0.079
GAS6
0.687
0.056
TFAM
0.616
0.131
SLC2A4
0.629
0.088
TEP1
0.582
0.124
EA Compound 3
LST1
0.866
0.036
ALDH18A1
0.897
0.010
SQLE
0.747
0.061
SLC15A1
0.876
0.014
SFRP1
0.610
0.095
SLC2A1
0.505
0.023
Compound 4
TNNT1
0.776
0.023
ELAVL1
0.628
0.037
FTL
0.763
0.070
RARB
0.583
0.101
FECH
0.706
0.067
SPAST
0.501
0.092
Pa: Probability of activity, Pi: Probability of inactivity

Compound 1 also showed a moderate cytotoxic effect (Pa = 0.563) on breast adenocarcinoma (MDA-MB-468) cell line. Noting that this type of cancer is in increase now-a-days, in 2020, records reported that 2.3 M women diagnosed with breast cancer57.

Data also revealed that compound 2 may exert a noticeable cytotoxic effect on the cervical adenocarcinoma (HeLa) cell line (Pa = 0.669). Noting that, the statistics established in 2020 for 185 countries, reported that this type of cancer is ranked fourth most common cancer in women and represents 6.5% of all female cancers58. It is also very interesting to note that all tested compounds were active on lung tissue, especially compound 3 which showed a non-negligible cytotoxic effect on non-small cell lung cancer stage 3 (NCI-H838) cell line (Pa = 0.648), which is important since lung cancer is considered the leading cause of death among both men and women, representing almost 25% of all cancer deaths58.

Compound 4 demonstrated also a modest cytotoxic effect on renal carcinoma (SN12C) cell line (Pa = 0.505), which is important since kidneys ensure vital functions, especially the regulation of extracellular fluid volume and the filtration and excretion of waste products and toxins out of the blood via urine59.

Gene expression analysis: Data indicated that compound 1 may remarkably upregulate the mRNA expression of SAT, POR and HTATIP2 genes with corresponding values of (Pa = 0.887, Pa = 0.878, Pa = 0.866) (Table 5). These genes play key roles, indeed, a study revealed that SAT gene may suppress tumor growth in xenograft tumor models60 and may also play a significant role in the cell survival process by acting as a rate-limiting enzyme in the pathway of polyamine metabolism61. The POR gene is crucial for the biosynthesis of cytochrome P450 enzymes62, which means that this gene could be a precursor for the synthesis of steroid hormones63. Another study suggested that the HTATIP2 gene may act as a metastasis suppressor via an antiangiogenic effect to prevent the formation of new blood vessels to block the blood supply into the tumor zone64.

Concerning the downregulation process, compound 1 showed also a great decrease in the mRNA expression of MYO5C (Pa = 0.862), IFIH1 (Pa = 0.852) and TEP1 (Pa = 0.848) genes and this information is crucial since the emergence of several illnesses is linked to the overexpression or mutation of these genes; in which MYO5C is associated with an abnormal spreading of neurofibrillary tangles in Alzheimer’s disease65, while IFIH1 may generate neuro-immunological disorders caused by an abnormal elevation of interferon due to excessive inflammatory reactions66, also TEP1 gene activity could be associated with an infectious disease called campylobacteriosis and prostate cancer67. Noting that compound 2 exerted a moderate downregulation on this TEP1 gene (Pa = 0.582).

Among the four tested compounds, results revealed that compound 2 could possibly increase the mRNA expression of TNNT1 (Pa = 0.724), GAS6 (Pa = 0.687) and SLC2A4 (Pa = 0.629) genes.

Human physiology depends a lot on these genes, the TNNT1 gene plays a key role in the regulation of actin thin filament function in skeletal muscle68, while the GAS6 gene may enhance hemostatic response by enhancing platelet aggregates and clot formation processes69. The expression of the SLC2A4 gene could significantly stimulate glucose uptake by adipose tissue and skeletal muscle due to an important increase in the expression of glucose transporter protein type-4 (GLUT4) mediated by SLC2A4 gene70. Noting that compound 4 showed a better result in the expression of the TNNT1 gene (Pa= 0.776). The results also indicated that compound 2 may act with a moderate way to down-regulate the gene expression of IFI27 (Pa = 0.663) and TFAM (Pa = 0.616). A recent study made by Zhao et al.71 revealed that the IFI27 gene could be closely associated with Hepatitis C and Oral Leukoplakia while an alteration in the TFAM gene may increase the risk of developing Parkinson’s disease due to a mitochondrial DNA depletion syndrome generated by the excessive activity of this gene72.

Its also important to underline that compound 3 may considerably upregulate the mRNA expression level of the LST1 gene (Pa = 0.866), since the activity of this gene may regulate leukocyte abundance in lymphoid organs but also the inflammatory response in the gut73. This compound may also increase the mRNA expression of SQLE (Pa = 0.747) and SFRP1 (Pa = 0.610) genes, respectively. Recently, a study showed that the SQLE gene could be used as a potential prognostic biomarker for the identification of head and neck squamous cell carcinoma74, while SFRP1 may prevent renal damage in a mouse model through the non-canonical Wnt/planar cell polarity pathway75. On the other side, compound 3 showed a considerable decrease in the mRNA expression of ALDH18A1 (Pa = 0.897) and SLC15A1 (Pa = 0.876) respectively. Some clinical and genetic investigations reported that a mutation in the ALDH18A1 gene could generate cerebellar ataxia and cognitive impairment76, while SLC15A1 polymorphisms were associated with dyslipidemia, considered as an important risk factor for stroke and coronary heart disease77. A modest don regulation process was also exerted by this compound on the SLC2A1 gene (Pa = 0.505), noting that a mutation in this gene could lead to a deficiency in glucose transporter type 1 (GLUT1), which will generate severe metabolic disorders78.

Results also revealed that compound 4 may be useful to increase the mRNA expression process of FTL and FECH genes with corresponding values (Pa = 0.763, Pa = 0.706). Some studies revealed that the FTL gene may act as an iron detoxifier agent since, during reactions involving iron such as electron transfer, there is an important generation of free, highly toxic radicals, that’s why the elimination of iron excess by this gene is vital79. The FECH gene is vital for the synthesis of ferrochelatase, a key enzyme mandatory for the production of heme80. This information is fundamental since heme is an important component of hemoglobin that ensures the fixation of oxygen (O2) in red blood cells and allows the transport of O2 from the lungs to the rest of the body81. In contrast, compound 4 showed a modest down-regulation process on the mRNA expression level of ELAVL1 gene (Pa = 0.628). It was reported that this gene is the central oncogenic driver for several malignant peripheral nerve sheath tumors82. A non-negligible down-regulation process was also exerted by this compound on RARB and SPAST genes with a respective value (Pa = 0.583, Pa = 0.501). This information is very important since a mutation in the RARB gene may cause microphthalmia or diaphragmatic hernia83, while a mutation in the SPAST gene may cause a rare inherited disorder called hereditary spastic paraplegia which is characterized by weakness and stiffness in leg muscles84.

In vitro anti-inflammatory assay (BSA): The test of the ability of C. tougourensis to inhibit bovine albumin denaturation revealed a moderate effect for both extracts. Thus, the n-BuOH extract showed a maximum inhibitory activity of (68.94±0.01%) at the maximum tested dose of (800 μg mL–1) while (63.47±0.01%) for EA extract. Those data were considered highly significant (p<0.001) when compared to standard diclofenac sodium which showed an inhibition percentage of (90.23±0.81%) at the same tested concentration in Fig. 2. It is also interesting to see that the anti-inflammatory effect of the two tested extracts was almost the same at the tested concentrations of 200 and 400 μg mL–1.

Image for - In silico Investigation and BSA Denaturation Inhibitory Activity of Ethyl Acetate and N-butanol Extracts of Centaurea tougourensis Boiss. and Reut.
Fig. 2: Anti-inflammatory activity (Bovine albumin denaturation assay) of C. tougourensis extracts
All values are expressed as Mean±SD (n = 3). One-way ANOVA followed by multiple Dunnettʼs test. Level of significance ap<0.05, bp<0.01 and cp<0.001 is statistically significant with comparison to diclofenac sodium


Table 6: EC50 values of standard and C. tougourensis extracts from the bovine serum albumin denaturation assay
Extracts/standard EC50 (μg mL–1)
n-BuOH extract 335.00±0.03
EA extract 338.00±0.07
Diclofenac sodium 115.76±0.19

The median effective concentration (EC ) of each sample is represented in Table 6 and its important to note that comparable EC50 values were also recorded for the two tested extracts and were (335±0.03 μg mL–1) for n-BuOH and (338±0.07 μg mL–1) for EA extract while (115.76±0.19 μg mL–1) for diclofenac sodium.

Albumin, the most abundant circulating protein (35-50 g L–1)85, ensures vital functions like the transport of hormones, vitamins and fatty acids86. It is also crucial to maintain normal osmotic pressure in blood vessels by stabilizing extracellular fluid volume via contribution by its pression known as oncotic pressure87. Hypoalbuminemia may have serious repercussions on kidneys and liver functions88.

The results are linked with Banerjee et al.89 and Marrassini et al.90, in which they clearly showed the ability of their plant to inhibit albumin denaturation but also proteinases activities and associated that with the richness of their species in flavonoids, tannins and alkaloids compounds. Noting that, these classes of secondary metabolites are very important at both nutritional and pharmacological levels91 which were identified in previous work92. This information is very important since protein denaturation may lead to certain pathological manifestations such as rheumatic arthritis, stroke and even cancer93 due to abnormal and excessive production of autoantigens. Another study showed that the actual bio compounds of plants, especially flavonoids can maintain the dynamic structure of proteins by limiting the oxidative stress exerted on these molecules through reactive oxygen species (ROS)94,95.

In the previous study14, two key compounds were identified, vanillin and rutin, respectively. A study made by Siddiqui et al.96 reported that vanillin may inhibit protein denaturation by inhibiting the activity of lipopolysaccharides (LPS), which can alter mRNA expression of bovine albumin and ovalbumin genes, using the mouse as the experimental model. Another study also revealed that vanillin could inhibit the inflammatory responses induced by LPS in BV-2 microglial cells97. This protective effect was explained by these researchers by the ability of vanillin to regulate changes in the early glycation process and amyloid-like aggregation of albumin, thus preventing any change in its spatial conformation.

Rutin may also be a good anti-inflammatory candidate by slowing down the secretion of Interleukin 6 (IL-6) induced by lipopolysaccharides (LPS) as well as the expression of NF-κB98 since the inhibition of these two pro-inflammatory mediators can alter the structure of proteins.

CONCLUSION

In conclusion, the anti-inflammatory assay by bovine serum albumin (BSA) denaturation test revealed that both extracts have a moderate inhibition effect on protein denaturation with almost the same effect. The various in silico tests made in the present study, also showed that some phyto-compounds of this plant could be potent candidate to treat metabolic disorders linked to diabetes and obesity, but also inflammation and skin pathologies. These compounds could also modulate the expression of some genes which could be helpful for the prevention and treatment of some illnesses linked to immunity, metabolism and the nervous system. These compounds could also exert significant cytotoxicity against various human tumor cell lines. In addition, the absorption, distribution, metabolism, excretion and toxicity of these tested compounds were also investigated as well as their physicochemical properties.

SIGNIFICANCE STATEMENT

This preliminary work brings novel information about the pharmacological potential of new phytocompounds that could be very useful for the scientific community but also for pharmaceutical industries to develop new effective drugs, more natural and respectful of human health.

ACKNOWLEDGMENTS

The authors wish to express thank the Algerian ministry of higher education and scientific research (MESRS, DGRSDT). Princess Nourah bint Abdulrahman University Researchers Supporting Project Number (PNURSP2022R43), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. Also, the authors thank Prince Sattam Bin Abdulaziz University, Al-Kharj for their scientific contributions.

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