Elucidating the role of Shatapushpa (Anethum graveolens L.) in Rheumatoid Arthritis through In vitro and In silico Analysis

INTRODUCTION

Plants are used as medicine since centuries in various traditional medicinal system. They have helped us in curing and managing various illness. Panchakarma (five therapeutics) in Ayurveda is majorly used in managing almost 70-80% of the disease, one among the panchakarma (five therapeutics) is basti (enema). This basti (enema) helps in normalizing the vitiated vata dosha (body constituency according to Ayurveda). The basti is classified into 2 types: 1. Niruha basti (Enema based on mixture of 5 products) and 2. Anuvasana basti (Enema based on Oil).

The Niruha basti is a mixture of 5 components namely, 1. Madhu (Honey) 2. Saindhava (Salt) 3. Kalka (Paste) 4. Kwatha (Decoction) 5. Awapa (Adjuvant). The Niruha basti is prepared by adding dravyas into the kalwa yantra (Mortar and pestle) and triturating them for 45 - 60 min. In this the Kalka Dravya (paste like substance) is very important because of its action in increasing the bioavailability, boosting potency, promoting colonic action and guaranteeing therapeutic efficiency. To get the best results, the right quantity and mixing order must be maintained (Mantha et al., 2023). Erandamoola niruha basti (Enema prepared with decoction based on Ricinus communis L. roots) is one of the commonly used enema therapies in the clinical practice, in various diseases such as Rheumatoid arthritis, Osteo-arthritis and Sciatica. In this Kalka Dravya (paste like substance), Shatapushpa (Anethum graveolens L.) is used. Its role in increasing the bio-availability, boosting the potency and increasing the effect of the enema is mentioned in various classical texts.

As of 2020, rheumatoid arthritis affects 17.6 million individuals worldwide, and its prevalence has increased by 14.1% since 1990. Despite a 23.8% decrease in mortality, disability is still high. Smoking is a risk factor, and the effects are greater in women. Globally, the condition is expected to affect 31.7 million people by 2050 (Deane et al., 2021). NSAIDs, corticosteroids, DMARDs, TNF inhibitors, Janus Kinase (JAK) inhibitors, and interleukin 6 inhibitors are examples of current therapy methods that have demonstrated efficacy in treating rheumatoid arthritis. Though there are so many medications they also give possible side-effects such as Renal failure, heart failure, Gastro-intestinal ulceration, weight gain, muscle weakness, diabetes, etc., (Radu et al., 2021).

Shatapushpa (Anethum graveolens L.) which is having various properties such as Anti-inflammatory, Analgesic, Anti-microbial, Anti-cancer, Hypolipidemic, Anti-diabetic and Anti-ulcer activity (Talreja et al., 2025). With these pharmacological actions it is used in the management of Rheumatoid arthritis. Though its usage is mentioned in various classical literatures, the real mechanism of action behind it is still unknown due to presence of numerous phytochemicals. Thus, by assessing the basic phytochemical activity, thin layer chromatography, flavonoid quantification, computational pharmacology and assessing its molecular activity on the selected target will give us a clear picture why it is used in treatment of Rheumatoid arthritis in both single drug preparation and in compound formulation i.e. Erandamooladi niruha basti.

MATERIALS AND METHODS

First, raw ingredients were obtained, then an in vitro and in silico evaluation of the Shatapushpa churna was performed, and the process layout is displayed in Figure 1.

Click to enlarge

Ethical statement

Since the preparation, in vitro and in silico investigation doesn’t involve any human or animal participants the permission from Institutional Ethical Committee and Institutional Animal Ethical Committee was not required.

Statistical analysis

As the research doesn’t require any statistical analysis as it doesn’t involve any comparison of the groups, the statistical analysis wasn’t performed. For the in silico analysis the p-value in the KEGG pathway was performed in the database itself and it didn’t require any Statistical software.

Procuring Shatapushpa, Physico-chemical, Phytochemical, TLC and Total Flavonoid analysis

The drug Shatapushpa was procured from KLE Ayurveda GMP certified pharmacy and followed by that phytochemical activity, thin layer chromatography, flavonoid quantification and physico-chemical activity was performed as per API volume 3.

Gathering Phyto-chemicals

Two databases were searched for phytochemicals using the term “Apium graveolens”: Dr. Duke’s Phytochemical and Ethnobotanical Databases (Lans et al., 2020) and IMPPAT 2.0 (Mohanraj et al., 2018) (Indian Medicinal Plants, Phytochemistry and Therapeutics). The phytochemicals that were discovered were screened mostly based on oral bioavailability and pharmacological similarity. We first gathered information on the canonical smiles of each phytochemical using the PubChem database (Kim et al., 2016). Afterwards, the SwissADME database (Diana et al., 2017) was used to forecast a number of ADME variables. The web tool BindingDB (Liu et al., 2007), which is based on similarities, revealed that phytochemicals have a target with a similarity score of ≥0.85. Using the UniProtKB database, (Apweiler et al., 2004), the target names were standardized after the disease targets linked to each phytochemical were identified.

Gathering disease target and Overlapping targets

Human Protein Atlas (HPA) and Gene cards were the two databases used in collecting the disease targets related to Rheumatoid arthritis (Thul et al., 2018; Sterlzer et al., 2016). The term “Rheumatoid arthritis” was used in both the databases and the targets were collected. Overlapping targets related to Rheumatoid arthritis from the two databases and Shatapushpa (Anethum graveolens L.) related targets from Uniprot KB was put into Venny 2.1 to find out the common targets related to both disease and the drug (Collazos JCO).

Protein Interaction

The common targets were uploaded in STRING database and the construction of Protein-Protein Interaction (PPI) of the Phytochemicals of Shatapushpa (Anethum graveolens L.) and Rheumatoid arthritis targets was made (Szklarczky et al., 2023). We chose “Homo sapiens,” and a medium degree of confidence was set at 0.50 FDR to confirm reliability.

Pathway analysis

After the PPI, the KEGG (Kyoto Encyclopaedia of Genes and Genomes) pathway analysis of the targets was analysed. The selection of “Homo sapiens” as the species that could serve as the pathway of Shatapushpa (Anethum graveolens L.) and Rheumatoid arthritis.

Network creation

Network of the Phytochemicals, targets and the pathway was constructed using Cytoscape 3.7.2 (Shannon et al., 2023). The network analyser was used to do the topological evaluation. To ascertain the function and connection between the active phytochemicals and the overlapping targets of rheumatoid arthritis, the network was analysed in terms of degree strength.

Molecular docking

The Phytochemicals and targets which had higher degree in the topological evaluation was chosen for its molecular mechanism. The binding affinity of the particular chemical to the particular target was assessed. For this the chemical 3-dimensional structure was retrieved from Pub Chem database and the target compound was retrieved from RCS PDB (Berman et al., 2000). The targets were analysed and the charges were added using Auto dock 1.5.7 (Morris et al., 2009). After this the targets and the Phytochemicals were binding was done using PyRx. (Dallakyan et al., 2015). Later these was assessed on binding affinity and visualisation was done using Biovia tool (Dassault systems, 2021).

RESULTS

Physico-chemical, Phytochemical, Thin Layer Chromatography and Flavonoids

The Shatapushpa that was procured from GMP certified pharmacy was subjected for physico-chemical, phytochemical, TLC and flavonoid quantification. Shatapushpa Churna’s organoleptic characteristics, which indicate identification, purity, and lack of adulteration, were found to be consistent with standard descriptions. Low microbiological risk and a good shelf life are indicated by moisture content within certain bounds. Low siliceous and inorganic contamination is confirmed by ash tests. High extractive values support therapeutic potency by indicating a rich abundance of active components that are soluble in water and alcohol. The antioxidant, anti-inflammatory, and digestive properties can be understood by the presence of flavonoids and tannins. The presence of cardiac glycosides indicates possible impacts on metabolism and circulation. Alkaloids in alcohol extract show that some bio actives are more soluble in organic solvents. A complex phytochemical profile is shown by the several unique Rf values under UV and daylight, which provide a trustworthy fingerprint for Shatapushpa Churna authentication and quality control. At a total flavonoid content of 4.54±0.47 mg QE/g of extract, the alcoholic extract of Shatapushpa Churna exhibited a moderate to noticeable flavonoid concentration. Shatapushpa Churna’s medicinal relevance is strengthened by the fact that flavonoids are recognized to play a major role in protecting against oxidative stress, enhancing pharmacological efficacy, and scavenging free radicals. Table 1 gives the macroscopic, physicochemical results of Shatapushpa chiurna, while the Table 2 reveals the phytochemical values and flavonoid quantification. Tables 1 and 2 gives a detailed result of the Shatapushpa (Anethum graveolens) chruna in vitro study.

Analysis of the obtained Phytochemicals

A total of 815 phytochemicals related to Shatapushpa (Anethum graveolens L.) was obtained from the plant databases. After selecting them on the basis of part used “seed” and removing the duplicated from the phytochemicals we got 174 Phytochemicals. The Pub Chem ID related to these Phytochemicals were collected. These were uploaded and on the basis of “drug likeliness” and “absorption”, a total of 102 phytochemicals were obtained from Swiss ADME. After this the canonical similes obtained were uploaded into Binding DB followed by that retrieving the Gene ID of the target, we found 333 Phyto-targets related to 102 Phytochemicals.

Analysis of the disease targets and Overlapping targets

There were 6629 targets related to Rheumatoid arthritis in both the databases. These disease targets and the Phyto-targets were put into Venny 2.1.0 and the overlapping targets were recovered. There were a total of 200 common targets for both disease and the plants Figure 2 depicts the common targets between the plant and the disease.

Click to enlarge

Protein Interaction Network

A PPI network of common targets in both Shatapushpa (Anethum graveolens L.) related Phyto-targets and Rheumatoid arthritis targets were uploaded in STRING database. Figure 3 illustrates the PPI Network.

Click to enlarge

KEGG Pathway enrichment

The top 10 KEGG pathways through which the common targets help in management of Rheumatoid arthritis is depicted in Figure 4. These pathways play a potential role in the disease.

Click to enlarge

Network creation

The top 10 pathways, 200 common targets and 60 Phytochemical that are obtained by following these processes were used construction of the target-pathway-compound network. The network that is created is portrayed in Figure 5. The targets are in the big circle out-side with red circular shape, inside those targets are the phytochemicals with blue color circular layout and the pathways are in downward triangular shape in the right side.

Click to enlarge

Analysis of the Molecular docking

With the network constructed the top 4 phytochemicals (Apiole, Geranyl acetate, Scoplotein and 4-vinylguaiacol) and targets were downloaded and with this the modern medication that is Methotrexate was taken as control drug and with that the docking was done using the tools. Followed by this the binding affinity of the Phytochemicals, targets and the control drug was assessed. Scoplotein has shown the same binding affinity like the control drug methotrexate. Scoplotein has shown highest binding affinity towards PIK3CA and even methotrexate has shown good binding affinity towards that target. The Figure 6. shows the docking visualization of the control drug methotrexate and scoplotein against the selected targets. Table 3 depicts the binding affinity of the chemical constituents and the targets.

Click to enlarge

DISCUSSION

The Shatapuspha kalka is used in the preparation of Erandamoola niruha basti, which gives potency to the whole combination. Kalka Dravya may aid in the formation of a homogeneous colloid of Sneha Dravya and Kashaya Dravya by binding the hydrophilic and lipophilic ends of Basti Dravya. Shatapushpa possesses unique Shoolaghna and Vatanuloman properties. Additionally, because of its antibacterial, anti-inflammatory, analgesic, antiemetic, anti-convulsive, gastric, and mucosal protecting qualities, it soothes painful ulcers, abdominal pain, eye disorders, and uterine pain. Since the 1980s, Methotrexate has been used to treat Rheumatoid Arthritis (RA), and it is still frequently the first drug prescribed for RA. (Friedman B et al., 2019) The results of the binding energy indicate that the phytochemical binds with the protein's active site with a comparable affinity to the conventional medication.

The churna authenticity and acceptability were confirmed by its organoleptic characteristics, which included form, color, taste, and odor, all of which met conventional requirements. Because the moisture content (4.389%) was within allowable bounds, there was less chance of microbiological growth and acceptable stability. The values of acid-insoluble ash (1.812%) and total ash (8.913%) were within acceptable bounds, indicating low levels of siliceous and inorganic contaminants. Higher alcohol-soluble (20.999%) and water-soluble (30.035%) extractive values show that the active ingredients are easily extracted, demonstrating the formulation's adequate quality and purity. In a study conducted on seeds of Anethum graveolens L. It revealed that the ash value was more than 10%. When compared to the current study the values are more and further more in the same study the flavonoid content is more when compared to this with a value of 13.79 + 0.69 QE/ g of extract while the current study has given only 4.54±0.47 mg QE/g of extract this maybe because the study we have performed with hydro-alcoholic extract while in the study done in the past it was with aqueous and ethanolic extract (Hadi et al., 2024). TLC profiling exhibited multiple well-resolved spots under UV and daylight conditions, reflecting chemical complexity and providing a reproducible fingerprint for standardization. Collectively, these findings validate the formulation’s quality and support its suitability for further pharmacological and clinical investigations.

Scopoletin's anti-arthritic properties in rats with adjuvant-induced arthritis were investigated. When administered intraperitoneally at doses of 50 and 100 mg/kg, scopoletin decreased articular index scores and paw swelling in both infected and non-inoculated animals. By lowering the number of new blood vessels in the synovium and the synthesis of significant endogenous angiogenic inducers, scopoletin can reduce the clinical symptoms of rat adjuvant-induced arthritis (Pan, 2010). Scopoletin can prevent RAW 264.7 cells from producing proinflammatory cytokines, particularly IL-6, when exposed to lipopolysaccharide in vitro, most likely by preventing MAPK pathway activation (Yao et al., 2012). Scopolettin may have an anti-RA effect by inhibiting the synthesis of pro-inflammatory cytokines. Molecular docking investigations in this study showed that the chosen phytochemical component Scopoletion and the control medication methotrexate had similar binding affinities -9.3kcal/mol towards target PIK3CA.

Geranyl acetate has been demonstrated that geranyl acetate inhibits pro-inflammatory cytokines, such as TNF-α and IL-1β, which are important mediators in the pathophysiology of RA. In a study using a Complete Freund's Adjuvant (CFA) mouse model, an extract from Zanthoxylum (andaliman), which is rich in monoterpenoids such geranyl acetate and geraniol, shown anti-arthritic benefits. The extract relieved joint inflammation and cartilage damage, lowered COX-2, IL-1β, iNOS, and MMP-1 expression, and considerably decreased arthritic assessment scores (Setiadi et al., 2022).

HRAS by acting upstream of important inflammatory pathways such PI3K-AKT, NF-κB, and MAPK/ERK, which are frequently activated in RA synovial Fibroblasts (FLS), HRAS promotes angiogenesis, cytokine secretion, cell proliferation, and MMP expression (Sadeghi Shaker et al., 2023). According to this study, RA synovial tissues, particularly the intimal lining, have markedly elevated levels of H-Ras mRNA and protein. The increase is associated with synovial hyperplasia, indicating a function in tissue invasion and chronic inflammation (de Launay et al., 2010).

PIK3CB the catalytic subunit of class I PI3K enzymes, which are crucial for inflammation, immune cell signalling, and survival, is encoded by PIK3CB. Through Fcγ receptors, PI3Kβ plays a crucial role in neutrophil activation, which results in Reactive Oxygen Species (ROS) production, Chemotaxis, Degranulation (Qu et al., 2016). In RA, Fibroblast-Like Synoviocytes (FLS) are hyperproliferating and resistant to apoptosis.

MicroRNAs are short, non-coding RNAs that post-transcriptionally control gene expression. They have been linked to the aetiology of autoimmune illnesses like Rheumatoid Arthritis (RA) and cancer (Kim et al., 2002). Dysregulated miRNA expression is a contributing factor to joint degeneration, synovial hyperplasia, and persistent inflammation in RA. For instance, the immune response and inflammatory signalling pathways are regulated by miR-155 and miR-146a, which are elevated in RA (Peng et al., 2015). The disease process in RA is also significantly influenced by the miRNA-mediated interactions between various cell lines. Interestingly, the intricate relationships and integration of multilayer signalling between miRNAs and other epigenetic variables are essential for the pathophysiology of RA but are still mostly unclear (Stanczyk et al., 2008). Still, there is not enough in vivo experimental data to discuss the pleiotropic function of miRNAs in RA clinical conditions.

With these above phytochemicals present in Shatapushpa (Anethum graveolens L.) we came to know the mechanism of the drug in Amavata (Rheumatoid arthritis). The phytochemicals present in the plant has major role on the disease and the targets. Especially molecular docking visualisation has shown that where the control drug Methotrexate has docked, in the same binding site Scoplotein has also shown its docking. Additionally, methotrexate and scopoletein have a close binding affinity. All this result conclusively suggests the efficacy and mechanism of Shatapushpa (Anethum graveolens L.) in RA.

Although the study has given satisfying results, still there are some limitations for this study. The whole process is solely depended on computational method. Especially the phytochemicals which the main mode of action is substantiated is majorly procured from the database. Thus, this may bring bias for this study due to the continuous update in the databases. Nevertheless, further study on in vivo and followed by that clinical research on human samples are needed to evaluate the safety and efficacy of the drug.

CONCLUSION

The current research suggests that the paste made from Anethum graveolens L. Known as Shatapushpa, has therapeutic potential for the treatment of RA. The main research question the mechanism of action of Shatapushpa (Anethum graveolens L.) is clear by the network pharmacology and followed by the docking. In order to optimize treatment and meet unmet clinical requirements, a deeper understanding of the underlying mechanisms of RA is necessary (Peng et al., 2023).

Bioinformatics research support this, showing that Shatapushpa's active phytoconstituents such as scopoletin, geranyl acetate, apiole. HRAS and PIK3CB targets and important molecular pathways linked to RA, including as micro RNAs in cancer and pathway in cancer. Shatapushpa has demonstrated anti-inflammatory and immunomodulatory effects similar to those of methotrexate, suggesting its potential as a natural alternative or complementary agent in the management of rheumatoid arthritis. Strong computational evidence for its anti-inflammatory and immunomodulatory actions is provided by the combination of gene interaction network research, pathway enrichment, and molecular docking. As a result, Shatapushpa Kalka shows promise as an adjuvant or alternative treatment for RA, deserving of additional experimental support and clinical studies.

ACKNOWLEDGEMENT

We co-ordinally thank our college KAHER’s Shri B.M.K Ayurveda Mahavidyalaya, Shahapur, Belagavi, for their extensive support in this study.

ABBREVIATIONS

RA

Rheumatoid Arthritis

IMPPAT

Indian Medicinal Plant, Phytochemistry and Therapeutics

KEGG

Kyoto Encyclopaedia of Genes and Genomes

NSAIDs

Non- Steroidal Anti-inflammatory Drugs

DMARDs

Disease Modifying Anti-Rheumatic Drugs

ADME

Absorption, Distribution, Metabolism and Excretion

CFA

Complete Freund's Adjuvant

PPI

Protein - Protein Interaction

TLC

Thin Layer Chromatography

SD

Standard Deviation

ROS

Reactive Oxygen Species

GMP

Good Manufacturing Practices

PIK3CA

Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha

PIK3CB

Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Beta

HRAS

Harvey Rat sarcoma viral oncogene homolog

QE

Quercetin Equivalent

API

Ayurvedic Pharmacopoeia of India

HPA

Human Protein Atlas

RCS PDB

Research Collaboratory for Structural Bioinformatics Protein Data Bank

R_f

Retention factor

UV

Ultraviolet

FDR

False Discovery Rate

iNOS

inducible Nitric Oxide Synthase

COX-2

Cyclooxygenase-2

MMP-1

Matrix metalloproteinase-1

FLS

Fibroblast-like synoviocytes

TNF-α

Tumor Necrosis Factor-alpha

IL-1β

Interleukin-1 beta

IL-6

Interleukin-6

MAPK

Mitogen-activated protein kinase.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

AUTHORS CONTRIBUTION

AK: Research concept and design, Critical revision of the article; RM: Research concept and design, Data analysis and interpretation, Writing the article; KM: Collection and/or assembly of data; VSVK: Research concept and design, Data analysis and interpretation, Writing the article.