INTRODUCTION:

Immune system is the most valuable defense system that protects its host from different kinds of antigens¹. Immune response can either be stimulated or suppressed based on the requirement. Substance that can modify the nature of immune response are known commonly as immunomodulators².

In recent days, even modern drugs are manufactured by using indigenous plant resources as raw material enriching our traditional herbal based therapy³, thus development of immunomodulatory drugs from natural compounds has attracted considerable interest.

Many researchers have reported that medicinal plant being the source for exhibiting a number of therapeutic activities, is also found to possess immunomodulatory properties that can control the outcome of certain immune responses⁴. Plant derived phytoconstituents including certain Alkaloids, flavonoids, lectin, glycoproteins, polysaccharides, sterols, and sterolins can be used as immunomodulators⁵. S. alba, commonly called as white mustard belongs to the family Brassicaceae is reported to possess cure to various ailments in folklore medicines⁶. S. alba is used in the ayurvedic system of medicine to treat migraine, cholera, cough, whooping cough, cold, flu, swollen joints and it is also used as an appetizer⁷. Despite extensive review on previous literature and keeping in view the immense importance of the members of family Brassicaceae, there is only a few scientific data available on immunomodulation study of this plant. Hence it is necessary to validate the safe and traditional use of this plant to find out new potential natural immunomodulators.

In recent years, in silico analysis has emerged as a powerful tool in the study of immunomodulatory properties of medicinal plant extracts. The computational approach leverages advanced techniques such as ADME (Absorption, Distribution, Metabolism, and Excretion) and molecular docking predictions to predict and analyze the interactions between bioactive compounds and immune system components. By simulating these interactions, researchers can identify potential immunomodulatory compounds, understand their mechanisms of action, and predict their efficacy and safety profiles. Since the integration of in vitro methods with in silico approaches offers a comprehensive framework for the discovery of novel immunomodulatory agents from medicinal plants. This study aims to synergize these two techniques to explore the immunomodulatory activity of ethanol seed extract of S. alba.

MATERIALS AND METHODS:

Collection of plant material:

The seeds of S. alba were procured during the month of April, 2014, from Herbal medicine market in Chennai city, Tamil Nadu, India. The seeds were authenticated in the Department of Medicinal Botany, National institute of Siddha, Chennai and a voucher specimen [voucher number - NISMB2052015] was submitted.

Preparation of extract:

Plant seeds were washed using distilled water thoroughly to avoid any external remains and shade dried at room temperature for a week. Dried seeds were ground to fine powder using a blender. 30gram of powdered sample was extracted with 300ml of ethanol solvent at 78^oC for 9hours⁸. The extract was concentrated using rotary evaporator at reduced pressure. Obtained seed extract was filtered and stored at 4°C for further use.

Gas Chromatography and Mass Spectrum analysis:

The composition of the ethanol extract of S. alba seed was established by GC-MS analysis. The analysis was performed on a JEOL GCMATE II GC-MS system in EI/CI mode equipped with a split/split less injector (220°C), at a split ratio of 1/10, using a VF-1MS fused-silica capillary column (30m × 0.25mm i.d.; film thickness: 0.25mm). The oven temperature was programmed from 60°C (5min) to 280°C at a rate of 4C/min and held at the temperature for 10min. Helium was used as a carrier gas at a flow rate of 0.8ml/min. Mass spectra were taken at 70 eV; a scan interval of 0.5 s and fragments from 40 to 550 Da. The spectrums of the components were compared with the database of known spectrum components stored in the NIST library⁹.

Cell culture:

Raw 264.7, a murine macrophage cell line obtained from National Centre for Cell Science (NCCS; Job no.-2121), Pune, India, was maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% heat-inactivated fetal bovine serum, 2mM L-glutamine, 100μg⁄ml penicillin and 100μg⁄ml streptomycin in a humidified incubator, at 37°C and 5% CO₂ atmosphere. For the treatment with cell, seed extract was adjusted to the initial concentration and serially diluted with DMEM to the desired concentrations (100μg/ml, 50μg/ml, 25μg/ml, 12.5μg/ml, 6.25μg/ml, 3.12μg/ml and 1.56μg/ml).

In vitro Nitric Oxide assay:

The presence of Nitric oxide was determined in cell culture media using Griess reagent¹⁰. Raw 264.7 Cells were seeded (1x10⁶ cells/ml) to flat bottom 96-well microtiter plates (100μl/well). Medium alone and the medium containing various concentrations of ethanol extract of the seeds (100μl/well) were added to each well. After 24hr of incubation, 100μl of culture supernatants were collected in a separate 96 well plates without any merge. Equal volume i.e., 100μl of Griess reagent was mixed with culture supernatants and incubated at room temperature for 10min. The absorbance values were measured at 540nm using a microplate reader. The amount of nitric oxide released was then quantified.

In vitro phagocytic assay:

a) Preparation of Candida albicans suspension:

Candida albicans procured from King Institute of Preventive Medicine and Research, Guindy, Chennai was maintained on Sabouraud Dextrose Agar medium in the form of slant culture. C. albicans of density 5x10⁷ cells/ml adjusted using DMEM was used as the test microorganism in the bioassay.

b) Nitro Blue Tetrazolium (NBT) assay:

Macrophages cell lines Raw 264.7 was used for the assay. 20μl of cell suspension with 1x10⁶cells/ml concentration along with 40μl of DMEM was seeded to flat bottom 96-well cell culture plate. 20μl of the seed extracts prepared in concentrations of 100μg/ml, 50 μg/ml, 25μg/ml, 12.5μg/ml, 6.25μg/ml, 3.12μg/ml and 1.56μg/ml was added to each well and incubated for 24h in 5% CO₂at 37°C. then 20μl of yeast cell suspension (5×10⁷ particles/ml) and 20μl of NBT were added to the wells containing Raw 264.7 cells and incubated at the same condition. After 1h of incubation DMEM was used to rinse the adherent macrophages and washed with 200μl methanol for four times. Culture plates was left to dry. 120μl of 2M Potassium hydroxide and 140μl of Dimethyl sulfoxide were added to the dried culture plates and the absorbance was measured at 540nm using microplate reader¹¹. Stimulation index (SI) was calculated the ratio of the treated well and control well absorbence.

In silico analyses:

a) Ligand preparation:

The 3D chemical structures of components identified in the seed extracts of plants were retrieved through the PubChem compound database at NCBI (The National Center for Biotechnology Information). SDF format of these compounds were used as ligands.

b) Druglikness and ADMET properties of ligands:

Drug like and non-drug like properties of any natural or artificial molecules can be distinguished with the help of Lipinski's rule of 5. Molecules that comply with 2 or more of the following rules could be predicted to possess drug likeness properties. ADME analysis includes the molecular properties of ligands for absorption, distribution, metabolism, and excretion (ADME) for drug prediction¹².

c) Target protein preparation:

The structure of target protein Macrophage colony stimulating factor protein was retrieved from Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) with PDB ID: 4NKQ serves as docking receptor. Water molecules bound to the ligands were removed from the active site of the protein receptor¹³.

d) Molecular docking:

Selected ligands that qualify ADME and Druglikness were filtered and used for the molecular docking analysis. Docking was performed using Accelry's Discovery studio 4.0¹⁴ ligand fit protocols by applying appropriate force field on the target and the drugs. Ligand with highest dock score that binds to the active binding site of the target protein was selected and finally the amino acid residues to which the ligand binds was revealed. The docking scores, internal energy of ligands, and potential mean force (PMF) values are estimated. Root-mean-square distance (RMSD) between the docked structure and the original conformation of the inhibitor in each complex was calculated.

e) Statistical analysis:

The results were expressed as mean±standard deviation. The effects of treatments were determined by analyzing the data using one-way ANOVA using the Graph Pad Prism 6.0 software package. P values <0.05 or <0.001 were considered as statistically significant.

RESULTS AND DISCUSSION:

Medicinal plants as immunomodulator are an effective alternate to conventional chemotherapy for a number of ailments¹⁵. Medicinally valuable plant namely S. alba was assessed for immunomodulatory efficacy. Nitric oxide production by macrophage cell Raw 264.7 assessed using Griess assay showed that ethanol seed extract had potential modulatory effect on NO production in vitro compared to basal control. Elevated production of NO was recorded with 40.18% at 12.5 μg/ml though the rate of NO production declined at doses above and below 12.5μg/ml in Raw 264.7 cells. NO production in Raw 264.7 was shown in figure 1 and table 1.

Production of NO by macrophages is a proinflammatory response to eradicate invading microorganisms. The principal effector molecule Nitric oxide produced in macrophages can be used as a quantitative index to evaluate macrophage activation¹⁶^,¹⁷. Substances that modify the activity of NO may possess considerable therapeutic value¹⁸. In the present study, ethanol extract of S. alba assessed for the potential ability to modulate NO production by macrophages. The seed extracts were observed to induce and inhibit NO production at the appropriate concentration. Similar to current investigation, many plant extracts have been reported to modulate NO production by murine macrophages^19,20,21^,²². NO production in the immune system acts as a major cytotoxic mediator and plays a vital role in eliminating the invading microorganisms and tumor cells. However, production of excessive NO has been associated with a range of inflammatory diseases including arteriosclerosis, ischemic reperfusion, hypertension and septic shock²³^,²⁴. Since NO is a multifunctional cell signaling molecule, the compounds that has impact on its production could also affect the signaling pathways in many related cell types²⁵. Nevertheless, such findings have tremendous therapeutic applications for neural degenerative diseases²⁶, inflammatory diseases²⁷ and many more, where macrophages are responsible for aggravating the medical conditions.

Figure 1: Effect of Sinapis alba seed extracts on NO production by Raw 264.7 macrophage cells.

Data represent the mean ± S.D. of independent experiments performed in triplicates (n=3).

Statistical significance is indicated as determined by one-way ANOVA followed by Dunnett's multiple comparison tests.

**P<0.01, ***P<0.001, ****P<0.0001.

Table 1: Percentage of NO production by Raw 264.7 cells treated with S. alba seed extract.

S. No.

Concentration

(µg/ml)

Percentage of No production

100

12.5

6.25

3.12

1.56

4.67

11.21

13.08

40.18

31.77

16.82

9.34

(-) - Reduction in NO production.

Phagocytosis is the process of removing invaded microorganisms from the host blood and other tissue fluids by macrophages²⁸. Immunomodulators are considered to enhance phagocytic activity in vitro. The increased functional ability of macrophages was evident from increased expression of NBT dye reduction. NBT assay with S. alba indicated that ethanol seed extract expressed maximum phagocytic stimulation with 83.08% being the utmost stimuli at 12.5μg/ml. However, stimulation decreased as with higher and lower concentrations. (figure 2 and Table 2). The result observed in the present study was in accord to the NBT assay reported in earlier studies²⁹^,³⁰, where various concentrations of plant extracts assayed for phagocytosis, modulated with suppression at high concentration and stimulation at lower concentration. This dual effect of plants on NBT dye reduction suggested concentration dependent activity of the plant extracts.

Figure 2: Effect of S. alba seed extract on phagocytic activity in Raw 264.7 macrophage cells.

Data represent the mean±S.D. of independent experiments performed in triplicates (n=3).

Statistical significance is indicated as determined by one-way ANOVA.

**P<0.01, ***P<0.001, ****P<0.0001.

Table 2: Percentage of stimulation of phagocytosis in Raw 264.7 cells treated with Sinapis alba seed extracts.

S. No.

Concentration (µg/ml)

Percentage of No production

100

12.5

6.25

3.12

1.56

-17.30

1.50

41.72

83.08

72.55

41.72

19.92

(-) - Reduction in phagocytosis stimulation.

Gas Chromatography-Mass Spectrometry analysis of ethanol solvent extracts of S. alba plant seed led to the identification of different organic compounds. The chemical compositions, peak area percentage, retention time, molecular weight and chemical formula of each seed extracts analyzed and identified through the NIST database were presented in figure 3 and table 3.

Ligands were made out of thirteen compounds present in the ethanol seed extract of S. alba identified using GC-MS analysis and their molecular and physicochemical properties was analyzed using Lipinski's Rule of five. Blood Brain Barrier Penetration (BBB), Human Intestinal Absorption (HIA), Caco2 cell permeability, Plasma Protein Binding (PPB), and Skin Permeability and were the parameters employed to analyze the ADME properties of the prepared ligand. Results of ADME analysis was set forth in the table 4. 3D Ligand structure of the compounds positive for Druglikness and ADME were given in the figure - 4. Sorted out ligands qualified by Lipinski's rule of five and did not show any mutagenicity were docked with target protein 4NKQ.

Figure 3: GC-MS chromatogram of the ethanol extract of Sinapis alba seed.

Table 3: Chemical compositions of ethanol extract of Sinapis alba seed.

S. No.

Name of the compound

Peak %

MW g/mol

Chemical formula

01.

02.

03.

04.

05.

06.

07.

08.

09.

10.

11.

12.

13.

5.6

11.18

13.32

17.03

17.78

18.75

19.52

19.75

20.63

21.25

22.58

23.57

26.48

Cyclohexane, 1-methyl-5-(1-methylethenyl),(R)-

Caryophyllene

Decanoic acid, 2,6,8-trimethyl-, methyl ester

Hexadecanoic acid, methyl ester

Hexadecanoic acid, ethyl ester

10,13-Octadecadienoic acid, methyl ester

1-Hexadecanone, 1-cyclophentyl-

Eicosanoic acid

11-Eicosenoic acid, methyl ester

Ethyl 3,7,11,15-tetramethyl-2-hexadecenoate

13-Docosenoic acid, methyl ester, (Z)-

Ethyl 13-docosenoate(ethyl erucate)

Neronine, 4a,5-dihydro-

0.30

1.55

0.60

0.44

7.68

2.70

28.66

31.42

0.23

7.16

1.27

17.58

0.35

136.23

204.35

228.37

270.45

284.47

294.47

308.54

312.53

324.54

296.53

352.59

347.36

C₁₀H₁₆

C₁₅H₂₄

C₁₄H₂₈O₂

C₁₇H₃₄O₂

C₁₈H₃₆O₂

C₁₉H₃₄O₂

C₂₁H₄₀O

C₂₀H₄₀O₂

C₂₁H₄₀O₂

C₂₀H₄₀O

C₂₃H₄₄O₂

C₁₈H₂₁NO₆

*Note: RT- Retention time, MW- Molecular weight

Table 4: Compounds following parameters of Lipinski’s rule and ADME properties.

S. No.	Compounds	BBB	HIA	Caco2 cell permeability	MD CK	PPB	Skin permea bility	Rule of five	Toxic ity
1.	Hexadecanoic acid, ethyl ester	14.69	100	56.86	66.31	100	-0.571	Suitable	NM
2.	Decanoic acid,2,6,8- trimethyl-, methyl ester	8.04	100	33.87	25.52	100	-0.762	Suitable	NM
3.	10,13-Octadecadienoic acid, methyl ester	13.72	100	47.11	67.08	100	-0.539	Suitable	NM
4.	11-Eicosenoic acid, methyl ester	17.55	100	48.32	67.86	100	-0.516	Suitable	NM
5.	13-Docosenoic acid, methyl ester, (Z)-	17.83	100	49.23	67.99	100	-0.500	Suitable	NM
6.	Ethyl 13-Docosenoate (ethyl erucate)	18.13	100	57.39	68.00	100	-0.498	Suitable	NM
7.	Ethyl 3,7,11,15 Tetra methyl-2-hexadecenoate	16.39	100	55.92	65.92	100	-0.540	Suitable	NM
8.	1- Hexadecanone, 1- cyclophentyl-	19.26	100	57.30	65.77	100	-0.534	Suitable	NM

Figure 4: Structure of ligands following parameters for Lipinski’s rule and ADME properties.

CONCLUSION:

Immunomodulatory activity in the present study was indicated by the stimulation of production of Nitric Oxide and phagocytosis activity. The study revealed that S. alba was capable to strengthen the immune system. Ethanol seed extract modulate immune responses significantly by enhancing above stated parameters. The in vitro test results were validated using in silico analysis resulted with the similar outcome, concluding that S. alba possess immunomodulatory efficacy. Though activity of the plant molecule can be predicted using molecular docking study, in vivo examination on animal models is essential to confirm the immunomodulatory potential of these compounds.

ACKNOWLEDGMENTS:

The authors are thankful to The Secretary and Principal, Ramakrishna Mission Vivekananda College, Mylapore, Chennai, India for providing necessary facilities. We specially thank Sophisticated Analytical Instrument Facility (SAIF), Indian Institute of Technology, Chennai, India for carrying out GC-MS studies and validation of the results. The authors also acknowledge National Centre for Cell Science (NCCS), Pune, India for supplying cell lines. We express our special gratitude to Dr. P. Kumarasamy, Professor and Head, Bioinformatics Centre and Aris cell, Tamil Nadu Veterinary and Animal Sciences University for aiding in silico studies.

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Received on 26.10.2024 Revised on 24.02.2025

Accepted on 02.05.2025 Published on 01.12.2025

Available online from December 06, 2025

Research J. Pharmacy and Technology. 2025;18(12):6021-6027.

DOI: 10.52711/0974-360X.2025.00870

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

S. No.	Compound Name	PubChem ID	Binding site	Relative energy	Dock score	RMSD (Å)
1.	11-Eicosenoic acid, methyl ester	5319603	2	17.75	121.36	0.000
2.	Ethyl 13-docosenoate (ethyl erucate)	5364508	2	14.65	142.43	0.000
3.	Hexadecanoic acid, ethyl ester	12366	2	13.83	128.78	0.000
4.	1-Hexadecanone, 1- cyclophentyl-	549696	2	15.06	125.42	0.000
5.	Levamisole (Standard)	26879	2	4.95	69.65	0.000