Sida Rhombifolia linn: an empirical analysis on phytochemicals and an in vitro outcome on anti-inflammatory and antioxidant perspective

Abstract

Sida Rhombifolia Linn (SR) is a drug of importance in Ayurveda, used for the multiple disease therapy. In Ayurvedic Pharmacopeia, it is purported as source of Bala in India. It is reported as a rejuvenator of muscles with therapeutic effects in joint pains, rheumatism and neurological deficits. The study objective was to strengthen the chemosystemics and pharmacology of the genera via detailed phytochemical evaluation and invitro assessment of anti-inflammatory and cytotoxic properties. Preliminary analysis demonstrated presence of compounds like alkaloids, phenolics, flavonoids, etc. The invitro assessment revealed anti-inflammatory effect in real time PCR through gene panel expression of PERK, GRP78, ATF6, IRE1A and CHOP. The cell viability was tuned to concentrations up to 120 µg/ml. Through the phytochemical evaluation studies, the ubiquity of bioactive secondary metabolites with therapeutic functions were confirmed. This propagates the potential to feature as a primary constituent in drug development after further research.

Introduction

Sida Rhombifolia Linn (SR) hails from the plant family Malvaceae. One among the known 200 of its species, it grows primarily in tropical and warm regions. It thrives effortlessly in India and Malaysia. The peak of interest arises from its wide range of medicinal uses reported through folklore, across continents¹. Its culturally familiar names are Bala, Atibala, Hastibala (Sanskrit); Wild mallow (English); Aanakurunthotti (Malayalam) and Chithamutti (Tamil)². In Ayurvedic form of medicine, it is reported as a rejuvenator of muscles with therapeutic effects in joint pains, rheumatism and neurological deficits^3,4.

Recognised by World Health Organization, this plant is listed for its use in alternative medicine health sector⁵. In Tibet, this plant extract features as an ingredient in medications of inflammatory and autoimmune disorders whereas in USA, it has been patented for reduction in sympathomimetic induced side effects¹. Similar to other plant extracts, the geographical factors attribute to the constituency of plant secondary metabolite production. Disparity in temperature, humidity, altitude, and soil composition causes distinct adaptations in plant features, thereby altering the bioactive compounds synthesis⁶.

A research study reported the anti-inflammatory component of the plant extract root by inhibiting the enzyme cyclooxygenase (COX-1 and COX-2). In the said article, pertaining to an animal study, the extract demonstrated a dose- dependent ability to reduce the oedema and induce analgesia⁷.

This study was undertaken to strengthen pharmacokinetics of SR and to justify the suitable medicinal use of this plant in pharmacognosy. The study resulted in sequestration of secondary metabolites. Additionally, the anti-inflammatory and antioxidant activity of the root extract was derived, amplifying the possibility of its use in inflammatory conditions. Further, the cytotoxicity and anti-inflammatory impact on cell lines were also derived. Thus, the drug safety across epithelial cell lines were analysed. The authors would like to underline on the singularity of this research for the geographical location of the raw material, plant part used and the effect of extract across the cell lines. Considering the variation of phytochemicals as per the environmental factors the first research characterization of the plant sourced from Kerala, India.

Materials and methods

Sample preparation

The blossoming plant of Sida rhombifolia plants were gathered from Kannur, Kerala (12°04′28″N 75°17′18″E); away from any use of pesticide or waste disposal in June 2024. The permission for the same was obtained from the Government Ayurveda College, Kannur. For quality assurance, the collected raw materials underwent physio chemical evaluation. The root identification and authentication were carried out by Dr KN Sunil Kumar, Department of Pharmacognosy, Siddha Central Research Institute, Chennai in July 2024.It has been submitted at the government institute as a voucher specimen. The authors state that the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora, were adhered to as collection protocol. For the in vitro analysis, cell lines were obtained from the National Centre for Cell Science, Pune. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Sequential extraction

SR roots were further air dried and grounded to coarse powder. The root powder (100gm) was immersed in 300 ml of each solvent, in the order of increasing polarity in a Soxhlet apparatus. The solvents were hexane, ethyl acetate, ethanol and aqueous mediums over a time span of 24 h to dissolve the phytochemicals. Subsequently, each of the filtrates were individually derived using Whatmann’s No 1 filter paper. Using ethanol provide the greatest yield at 2.0% w/w. 1.5%, 1.0%, 1.83% w/w was obtained in hexane, ethyl acetate and aqueous mediums respectively. They were then cooled to room temperature and concentrated to dryness under vacuum. This regulated drying protocol ensured prevention of microbial contamination and conservation of bioactive compounds integrity for subsequent analysis These residues were weighed, bottled and refrigerated at 4 °C for further phytochemical and biological screening methods. The process was replicated three times to confirm the original findings. Each of these underwent qualitative analysis for determination of superlative extraction medium.

Results

Qualitative analysis (Fig. 1)

Fig. 1.

Qualitative phytochemical analysis of SR root extract in different mediums (aqueous, hexane, ethanol and ethyl acetate).

Test for phenol: Extract of 1 ml of SR was mixed with phenol ciocalteacus reagent drops and further with 15% sodium carbonate drops⁸. A positive response was recorded with blue-green precipitate.

Test for alkaloids: To extract (2 ml), concentrated hydrochloric acid (2 ml) and Mayer’s reagent drops were added⁸. A positive response was observed with green-white precipitate. It was further confirmed using Dragendroff’s test and Picric acid test, where in positive responses were recorded.

Test for flavonoids: 2 N sodium hydroxide in 1 ml quantity was added to 2 ml of extract⁸. The yellow precipitate appearance denoted a positive response. Further confirmation was done using Shinoda test and ammonia test.

Test for carbohydrate: Root extract (2 ml), Molish reagent (1 ml) and concentrated sulphuric acid drops was mixed⁸. The appearance of purple-reddish colour demonstrated a positive response.

Test for saponins: 2 ml of distilled water and 2 ml root extract were mixed for 15 minutes⁸. A positive response was garnered with 1 cm layer of a lather.

Test for reducing sugars: Confirmation was derived from boiling Fehling A 1 ml and Fehling B 1 ml with extract for 10 minutes⁸, and the appearance of brown precipitate.

Test for steroid: Root extract (1 ml), chloroform (1 ml) with concentrated sulphuric acid drops were mixed⁸. It resulted in brown ring formation, deemed as a positive reaction.

Test for terpenes: Extract filtrate (1 ml) and Barfoed’s reagent (1 ml) are heated for 2 min⁸. The subsequent mahogany red color in the solution was confirmatory.

Test for cardiac glycosides: Glacial acetic acid, 5% ferric chloride, concentrated sulphuric acid are added to the root extract⁸ whereby blue colour at interface, dictated the positive response.

Test for tannins: Root extract (1 ml) and 2% of hydrochloric acid drops were mixed, leading to the appearance of red colour precipitate⁸, indicating positive response.

The phytochemical content concentration was dependant on the extraction solvent used. It was maximum in ethanol medium and minimum in hexane medium. The results attested the importance of solvent selection in the extraction process, as this significantly decides the yield percentage of phytochemicals.

Antioxidant properties

In this study, the root extract antioxidant properties assessment was done using ABTS, DPPH, superoxide radical scavenging activity, nitric oxide radical scavenging activity, metal chelating activity and reducing power of extract against standard.

ABTS (2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) assay

A spectrophotometric method, ABTS assay utilizes the oxidized ABTS radical cation reaction to antioxidants, thereby reducing the ABTS radical and losing its bluish green colour⁹. A 7 mM ABTS solution is combined with 2.45 mM potassium persulfate in a 1:1 ratio and incubated in the dark for 12–16 h to form ABTS radicals. Further, the solution was diluted with ethanol to reach an absorbance of 0.70 ± 0.02 at 734 nm. Then, 1 mL of diluted ABTS solution was mixed with varying concentrations (10–100 µL) of each root extract, adjusting the volume to 1 ml. After a 6 to10-minute incubation in the dark, absorbance is measured at 734 nm. As seen Fig. 2, in a dose reliant method; the compounds distilled from SR extract scavenged ABTS free radicals. The ABTS assay (Fig. 2) confirms the ethanol fractions of extract contain most effective radical scavenging compounds. At 500 µg, the inhibition levels reaching about 85–90% in ethanol extract which was comparable to the standard (BHT).

Fig. 2.

ABTS scavenging activity of at various root extract concentrations.

DPPH (2,2-diphenyl-1-picrylhydrazyl) assay

DPPH in methanol (33 mg in 1 L) stock solution was made with an initial absorbance of 0.8. The stock solution (5 ml) and extract solution (1 ml) were mixed to achieve different concentrations. After 30 min, absorbance was noted at 517 nm. The antiradical action was measured as percentage inhibition¹⁰. Figure 3 demonstrated the DPPH radicals percentage inhibition by the solvent extracts and concentrations of a plant sample in comparison to ascorbic acid. The ethanol and water extracts were observed to hold the highest scavenging activity. At 500 µg, the inhibition levels ranged from 75 to 80% in ethanol extract which was comparable to the standard (Ascorbic acid). All extracts demonstrated dose-dependent scavenging, wherein inhibition decreased as concentration reduced.

Fig. 3.

DPPH scavenging activity of root extract at various concentrations.

Superoxide radical scavenging

Superoxide radical scavenging was appraised by the NBT reduction method¹¹. The reaction mixture composition entailed of NBT solution (1 ml,156 M), NADH solution (1 ml, 468 M), and a sample solution (1 ml) in concentrations varying across 250 to 2500 g/ml. The reaction was instituted by adding 100 L of PMS solution (60 M PMS in phosphate buffer, pH 7.4) to the prepared mixture. On incubation, at 25 °C for 5 min, the absorbance was detected as 560 nm against a blank. It was dictated that a decrease in absorbance of the reaction mixture translated as an elevated superoxide anion scavenging activity. Figure 4 established the capacity of the plant extract among concentrations ranging from 1000 to 1 µg/mL, compared to Vitamin C (standard antioxidant). At 1000 µg/mL, the extract denoted about 85–90% scavenging activity. Thus, it has a strong, concentration-dependent superoxide scavenging activity.

Fig. 4.

Superoxide radical scavenging in percentages as per dose concentration.

Nitric oxide scavenging (NO) activity

The procedure principle evidenced that sodium nitroprusside aqueous solution at physiological pH immediately developed nitric oxide. Nitric oxide combines with oxygen and generates nitrite ions, which is then calculated utilizing the Griess reaction. Nitric oxide scavengers contend with oxygen, resulting in decreased nitrite ions production. The assay was concluded according to the method as described by Sreejayan and Rao¹². The bar diagram (Fig. 5) displays the NO scavenging activity (%) of the plant extract as compared against a standard antioxidant (Catechin) across different concentrations (100–1000 µg/mL). At 1000 µg/mL, the extract shows ~ 70% scavenging activity. The data divulged that the plant extract exhibited a dose-dependent nitric oxide scavenging effect, which is comparable to catechin, though slightly lower across all concentrations, highlighting its potential as a antioxidant agent.

Fig. 5.

Nitric oxide scavenging in percentages as per dose concentration.

Reducing power capacity

The reducing power was measured according to the reported method¹². Distinct concentrations of extracts in 1 ml of ethanol, phosphate buffer (2.5 ml, 0.2 M, pH6.6) and potassium ferrocyanide (2.5 ml, 1%) were blended. The mixture was nurtured at 50 C for 20 min. Trichloroacetic acid (2.5 ml,10%) was added and followed up with centrifugation at 3000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl₃ (0.5 ml, 0.1%). Further, the absorbance was determined at 700 nm. Elevated absorbance of the reaction mixture advocated an increased reducing power. The bar diagram (Fig. 6) contrasts the plant extract reducing power with that of BHT (Butylated Hydroxytoluene), an accepted antioxidant, at divergent concentrations (25, 50, 75, and 100 µg/mL). At 100 µg/mL, the extract approaches ~ 1.0, while BHT slightly exceeds it, nearing 1.1–1.2. Thus, the plant extract exhibits a concentration-dependent increase in reducing power and a comparable antioxidant potential, particularly at higher doses. Among the solvents tested, ethanol extract stands out as the most potent, making it a promising candidate for further isolation and purification of active antioxidant components.

Fig. 6.

Reducing power capacity as per dose per concentrations.

Metal chelating activity

Metal chelating activity was assessed through the coupling of 0.1 mM FeSO₄ (0.2 mL) and 0.25 mM ferrozine (0.4 mL) with the extract (0.2 ml). On incubation within the room temperature confines for 10 min, the mixture absorbance was decoded at 562 nm¹³. The Fig. 7 illustrates the dose reliant rise in plant extract metal chelating activity amid a concentration range of 0–500 µg/mL. As the extract consolidation dominates, the percentage inhibition (a measure of its chelating ability) rises in proportion. At 50 µg/mL, the inhibition is approximately 10%. At 100 µg/mL, it increases to around 20%. This trend continues, reaching nearly 55–60% inhibition at 500 µg/mL.

Fig. 7.

Metal chelating activity as per dose per concentrations.

This result extrapolates that the plant extract exhibits strong and concentration-dependent chelation of metal ions (likely Fe²⁺ in this assay), as indicated by increased absorbance inhibition at 562 nm. Such activity is crucial because metal chelation reduces the availability of pro-oxidant metal ions, thereby minimizing oxidative stress. Hence, the extract demonstrates promising antioxidant potential, especially at higher concentrations.

Cytoprotective effect of extract in fibroblast cell line using Alamar blue assay

The assessment of cytotoxicity and potential protective role of the root ethanolic extract, Alamar Blue assay was performed on endothelial cell lines¹⁴. The cells were doctored with varying extract concentrations (30–1000 µg/mL) over 24 h, following which the cell activity was quantified with colorimetric measurements. In this study, the assay revealed a concentration-reliant drop in cell activity. While lower concentrations (30–120 µg/mL) maintained > 90% viability, higher concentrations (≥ 250 µg/mL) significantly reduced cell survival, with viability dropping to approximately 65% at 1000 µg/ml. The outcome alludes that the extract is non-toxic at concentrations up to 120 µg/ml and thus suitable for therapeutic applications within this range.

Anti-inflammatory effect of root extract using gene expression

The anti-inflammatory component in cell lines was evaluated using real-time PCR in gene expression. The stress markers assessed were PERK, GRP78, ATF6, IRE1A and CHOP for assessing anti-inflammatory activity of the root sample. This catered to the findings in the research by Jerry Chiang WC et al.¹⁵.

Effect of root ethanol extract on PERK gene expression

PERK (PKR-like ER kinase) is a transmembrane protein and a key stress marker within the endoplasmic reticulum (ER)¹⁶. The endothelial cell lines were treated with 2 µM thapsigargin (TGP). Further two concentrations (30 and 60 µg/ml) of root ethanol extract were used over the treated cells. A dose-dependent increase in PERK expression was observed in the presence of root extract (Fig. 8a), with a nearly 1-fold increase (***p < 0.001) compared to thapsigargin-only treated cells. This upregulation suggested enhanced ER protein folding activity in response to the root extract.

Fig. 8.

Gene expression (PERK, GRP78, ATF6, IRE1A, CHOP) assessment for anti-inflammatory response of root ethanol extract where x ais denotes the mRNA expression and y axis denotes the cell lines as untreated control cells (C), Thapsigargin treated cells (TPG), cells treated at 30 and 60 µg/ml respectively.

Effect of root ethanol extract on GRP78 gene expression

Glucose-Regulated Protein 78 (GRP78) or immunoglobulin heavy chain binding protein (BiP) is a part of the Heat Shock Protein 70¹⁷. GRP78 expression was assessed in endothelial cell lines exposed to 2 µM TGP. Against the controls (C), 30 and 60 µg/ml of root ethanol extract was inculcated into the TPG treated cells. The treatment led to a 0.3-fold increase in GRP78 expression (**p < 0.01) relative to thapsigargin (Fig. 8b). This indicated a potential stress alleviation, as indicated by elevated GRP78.

Effect of root ethanol extract on ATF6 gene expression

At times of ER stress, ATF6 gene codes a transcription factor which act on target genes for the unfolded protein response¹⁸. In the study, a dose-dependent downregulation of ATF6 of up to a 2.5-fold was observed in cells treated with the root extract (30 and 60 µg/ml) compared to thapsigargin-only treated cells (Fig. 8c). The increased ATF6 expression (p < 0.001) in thapsigargin-treated cells reflects ER stress induction, which was mitigated by root extract treatment.

Effect of root ethanol extract on IRE1A gene expression

Inositol-requiring transmembrane kinase endoribonuclease-1α (IRE1α) is an unfolded protein response signal transducer during ER stress¹⁹. Gene expression analysis of the ER transmembrane protein IRE1A revealed a dose-dependent decrease of up to 0.3-fold (***p < 0.001) in response to root ethanol extract treatment against two concentrations of 30 and 60 µg/ml (Fig. 8d). Compared to thapsigargin-only cells, the extract significantly reduced IRE1A expression, indicating attenuation of ER stress initiated by thapsigargin.

Effect of root ethanol extract on CHOP gene expression

CHOP is tiered to the family of binding proteins that act as enhancers and encodes proteins for proliferation, differentiation and expression, as well as energy metabolism²⁰. CHOP expression was significantly upregulated (nearly 2-fold, ***p < 0.001) in thapsigargin-treated cells, indicating ER stress-induced apoptosis (Fig. 8e). However, on treatment with root ethanol extract (30 and 60 µg/ml), markedly reduced CHOP levels, highlighting its potential protective role against ER stress-mediated cell non-viability.

Discussion

In current study pertaining to the SR extract, the phytochemical composition was determined. The objective was to determine the presence of essential phytochemicals. Secondly, to rule out composition changes arising from difference in the plant collection geography. This was followed up with the highly sensitive Alamar Blue assay (ABA) pertaining to cytotoxicity and anti-inflammatory gene expression of the root extract in endothelial cell lines and antioxidant potential assessment. The root extract was prepared across four mediums; aqueous, ethyl acetate, ethanol and hexane and in varying concentrations as well.

The analysis was conclusive for the presence of phenols, alkaloids, saponins, carbohydrates, steroids and cardiac glycosides. This was corroborative with the reports of Dinda et al.²¹. The results in terms of anti-inflammatory action of the SR extract indicated the ethanol extract to be most efficacious in anti-inflammatory and antioxidant potential in comparison to the other mediums. This was in tandem with study by A Kumar et al., wherein the anti- inflammatory component was reported to be maximum in ethanol extract²¹. However, a contradiction was reported in the study by Mah SH et al. This study convened hexane extract to be more effective²². In another study by Chaves OS et al., the vasorelaxant property of the extract was reportedly recorded thereby contributing to the anti-inflammatory spectrum²³.

The results attributed the ethanol root derivative of S. rhombifolia has more inhibitory effects in the anti-inflammatory and antioxidant aspect on the cell line. The results regarding the ABA assay, SR extracts retain the cell viability at concentrations below 120 µg/ml. However the study by Mah et al. stated a concentration below 100 µg/ml to retain cell viability²². Also, treatment with the extract at 30 and 60 µg/ml concentrations on TPG treated cells significantly improved the anti-inflammatory gene expression. The effect was dose dependant, demonstrating its ability to mitigate ER stress-induced cytotoxicity. Assam AJ et al. stated that the anti-inflammatory component of SR extract was similarly reported in the context of antibacterial and acute toxicity aspect²⁴.

Conclusion

Phytochemical studies contribute to standardization of herbal preparation and the evolution of understanding phytoconstituents for their medical uses. The reported SR root is extensively used in government approved alternative medicine forms in India. Through this research study, the locally sourced plant and the substantiation of use was reported. With promising anti-inflammatory, antioxidant properties and concentration dependant cytotoxicity, the authors suggest in vivo trials at the reported concentrations in inflammatory clinical conditions to substantiate further drug development in future research.

Author contributions

Y.A.K: Conceptualisation, Methodology, Data analysis, manuscript writing, review. A.G: Supervision, manuscript review. K.C.L: Data curation, data analysis. D.U: Laboratory investigation.

Funding

This was a self-funded study.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.