Bridging in vitro bioactivities and in silico insights of Rubia cordifolia L. (Rubiaceae) leaf extracts for therapeutic applications

Abstract

Medicinal plants are important sources of bioactive compounds with significant therapeutic potential. This study evaluated the phenolic profile, biological activities, and molecular docking-based antiproliferative potential of the Rubia cordifolia leaf extracts in methanolic (RCLM), chloroform (RCLC), and distilled water (RCLD). Leaf extract was selected because of its richness in secondary metabolites and its roles in photosynthesis, defense mechanisms, and environmental interactions. Phytochemical screening was performed using High-Performance Liquid Chromatography (HPLC), while antioxidant activity was determined through DPPH scavenging, total antioxidant capacity (TAC), and total reducing power (TRP). Antibacterial activity was evaluated against Staphylococcus aureus, Listeria monocytogenes, and Bacillus subtilis. Cytotoxicity was investigated using the brine shrimp lethality assay and PC3 and 3T3 cell lines. Among the extracts, RCLM exhibited the strongest antioxidant activity with a DPPH IC50 of 54.41 ± 0.53 µg/mL, a TAC of 102.08 ± 0.89 µg/mL, and a TRP of 201.56 ± 0.61 µg/mL. The highest antibacterial activity was observed against Bacillus subtilis with an inhibition zone of 26 ± 0.7 mm. The methanolic extract also showed pronounced brine shrimp cytotoxic activity. Furthermore, RCLM showed > 50% inhibition against 3T3 cell lines and < 50% inhibition against PC3 cell lines. HPLC analysis revealed chlorogenic acid as the major phenolic compound (392.44 ppm), while salicylic acid was detected at the lowest concentration (3.408 ppm). Molecular docking demonstrated strong binding affinities for chlorogenic acid (− 7.7 kcal/mol) and HB acid (− 10.5 kcal/mol) to target proteins implicated in oxidative stress and cancer progression. These findings suggest that Rubia cordifolia leaf extract possesses promising pharmacological applications.

Introduction

The side effects associated with conventional chemotherapeutic agents have increased interest in plant-derived bioactive compounds as safer therapeutic alternatives¹. Medicinal plants are recognized as rich sources of bioactive phytometabolites possessing diverse biological and pharmacological activities. According to recent global projections, nearly 80% of the population in developing countries relies on herbal medicine for primary healthcare, while the global herbal medicine market is expected to exceed USD 328 billion by 2030 due to increasing demand for natural and preventive therapies². The family Rubiaceae, one of the largest angiosperm families, comprises approximately 450 genera and 6,500 species³. Among these, the genus Rubia includes nearly 70 species. Rubia cordifolia Linn. is a perennial climbing herb characterized by long cylindrical roots with thin red bark^4,5. The species is widely distributed across China, India, Pakistan, Japan, Afghanistan, Malaysia, Australia, and Nepal⁶.

Rubia cordifolia has long been used in traditional medicinal systems, including Ayurveda, Traditional Chinese Medicine, Korean medicine, and Tib-e-Unani practices, for the treatment of hemorrhage, bronchitis, rheumatism, dysentery, urinary disorders, menstrual complications, jaundice, inflammatory diseases, and wound healing^7–10. Pharmacological investigations have reported that the plant possesses anti-inflammatory, anticancer, hepatoprotective, neuroprotective, antioxidant, and antimicrobial activities¹¹. More than 80 phytoconstituents have been identified from the plant, including anthraquinones such as alizarin and purpurin, triterpenoids, glycosides, and phenolic acids, which are considered responsible for its broad pharmacological potential¹². Oxidative stress caused by excessive production of reactive oxygen species contributes significantly to the development of chronic diseases, whereas plant-derived antioxidants help neutralize free radicals and reduce cellular damage through redox-based mechanisms¹³. Similarly, medicinal plants provide important antimicrobial agents that inhibit microbial growth by disrupting cell membranes, interfering with metabolic enzymes, and inhibiting nucleic acid synthesis, thereby offering promising alternatives to synthetic antibiotics^11,12. In addition, antioxidant, antimicrobial, and cytotoxic activities are often interconnected through redox-mediated mechanisms regulated by phenolic compounds.

The biological efficacy of plant extracts is strongly influenced by the extraction solvent, as solvent polarity determines the recovery of different classes of bioactive compounds. Therefore, methanolic, chloroform, and aqueous solvents are commonly employed to obtain a broad spectrum of phytochemicals¹⁴. Leaf extract generally contains a high diversity and concentration of secondary metabolites, including phenolics, flavonoids, and alkaloids, due to their involvement in photosynthesis, plant defense, and environmental interactions¹⁵. High-performance liquid chromatography (HPLC) is widely used for the accurate identification and quantification of phenolic constituents, whereas molecular docking provides mechanistic insights into the interactions between phytochemicals and molecular targets associated with oxidative stress, microbial viability, and cellular proliferation. These integrated approaches facilitate the establishment of correlations between phytochemical composition and biological activities. Although the therapeutic significance of Rubia cordifolia has been extensively documented, most previous studies have primarily focused on root extracts, while systematic investigations on leaf extracts remain limited. Moreover, comprehensive studies integrating solvent-dependent phytochemical profiling, multi-assay biological evaluation, and in silico molecular interaction analysis of leaf extracts are still scarce. It also remains unclear whether phenolic-rich leaf extracts of Rubia cordifolia can exert coordinated biological effects through redox-mediated mechanisms influencing oxidative stress, microbial viability, and cellular proliferation.

Therefore, previous studies on Rubia cordifolia have focused predominantly on root extracts because of their well-documented traditional and pharmacological relevance, whereas investigations on leaf extracts remain comparatively limited. Despite the recognized medicinal importance of the plant, comprehensive studies integrating solvent-dependent phytochemical profiling, multi-assay biological evaluation, and in silico molecular interaction analysis of leaf extracts are still scarce. Furthermore, the coordinated redox-mediated mechanisms through which phenolic-rich leaf extracts influence oxidative stress, microbial viability, and cellular proliferation remain poorly understood. Hence, the present study was designed to evaluate the antioxidant, antimicrobial, cytotoxic, and anticancer potential of methanolic, chloroform, and aqueous leaf extracts of Rubia cordifolia. In addition, HPLC quantification and molecular docking analyses of five major phenolic acids, namely gallic acid, caffeic acid, chlorogenic acid, ferulic acid, and p-coumaric acid, were performed to establish correlations between phytochemical composition and the observed biological activities. Importantly, this study represents one of the few comprehensive investigations specifically focused on solvent-dependent leaf extracts of Rubia cordifolia through the integration of phytochemical characterization, multi-target biological screening, HPLC-based phenolic quantification, and molecular docking analysis to better elucidate the mechanistic basis of its biological activities.

Methodology

Collection and extract preparation

The leaves were taken fresh from Rubia cordifolia plants during June-November and then identified by a Taxonomist, Dr. Waseem Akhtar, from the Azad Jammu and Kashmir University, Muzaffarabad (34.3734° N, 73.4698° E), and submitted to the Department of Botany at the University of Azad Jammu and Kashmir, AKASH Herbarium, having voucher no. 373829-229. The plant was collected from the wild environment after taking permission from the landowner, complying with relevant institutional, national, and international guidelines and legislation. The plant was shade-dried, ground into a fine powder, and soaked in methanol, chloroform, and distilled water (20 g/200 mL) for 2 days to ensure optimal solvent–matrix interaction and maximum recovery of bioactive compounds without thermal degradation. Further, the prepared fresh extracts were filtered. Then, the filtrate was dried under reduced pressure using a rotary evaporator system (R-200 Buchi, Switzerland) to get the crude extracts. Finally, the extracts were weighed, and their percentage yield was calculated.

Qualitative phytochemical analysis

Qualitative tests were performed to observe the phytochemical composition of Rubia cordifolia extracts. The plant extract was prepared using the previous standard procedure¹⁶. For phytochemical assessment, standard procedures were adopted to confirm the existence of phenols (ferric chloride test), flavonoids (alkaline reagent test), alkaloids (Mayer’s reagent), saponins (foam assay), glycosides (Salkowski test), terpenoids, and steroids (Libermann’s test)^17,18.

Quantitative phytochemical analysis

The total phenolic content (TPC) of the extracts of Rubia cordifolia leaves was estimated by the Folin–Ciocalteu colorimetric method. Briefly, 0.5 mL of each extract solution was reacted with 2.5 mL of 10% Folin–Ciocalteu reagent and incubated for 5 min. After that, 2 mL of 7.5% sodium carbonate solution was added to the reaction mixture and incubated in the dark at room temperature for 30 min. The absorbance was measured at 765 nm using a UV–Visible spectrophotometer. The standard used for calibration was gallic acid, and the results were reported in terms of milligrams of gallic acid equivalents per gram of extract (mg GAE/g extract).

A colorimetric assay method with aluminum chloride was used to determine the total flavonoid content (TFC) of the leaves of Rubia cordifolia. In short, 0.5 mL of each extract was combined with 1.5 mL of methanol, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1 M potassium acetate, and 2.8 mL of distilled water. The reaction mixture was incubated at room temperature for 30 min, and then the absorbance measurement was made at 415 nm with a UV–visible spectrophotometer. Quercetin was selected as the reference compound, and the amount of flavonoids was calculated as milligrams of quercetin equivalent per gram of extract (mg QE/g extract).

High-performance liquid chromatograph (HPLC)

In brief, 1 mg of plant extract was dissolved in 5 mL of 10% methanol, then filtered through a 0.45 µm membrane filter. An Agilent 1260 HPLC system (PerkinElmer, USA) was used to analyze the phenolic acids with a C18 column (Sorbex RXC-8, 4.6 × 100 mm). The mobile phase was filtered through a 0.45 µm membrane filter; the filtered mobile phase was sonicated in an ultrasonic bath to remove air before use. The separation was achieved by using the gradient elution technique with 0.2% H3PO4, MeOH, and acetonitrile at a flow rate of 1 mL/min and a column temperature of 30 °C. Using isocratic conditions, the mobile phase was stepped to 5% at 5 min, 50% at 15 min, 70% at 25 min, and 100% at 30 min, and then held at 100% for 5 min during a total run of 35 min with detection at 210 nm. The standard and plant samples (5 µL) were injected using an autosampler at various retention times. The compounds detected, such as chlorogenic acid, p-coumaric acid, 1-amino-8-naphthol-3,6-disulfonic acid, caffeic acid, and salicylic acid, were quantified by preparing the calibration curve of each compound and correlating the peak area with the concentration of the compound¹⁹.

Antioxidant assays

DPPH assay

For the determination of DPPH scavenging activity of Rubia cordifolia leaf extracts, a previous standard method was followed²⁰. Briefly, plant extract (10 µL) was added to 0.004% DPPH solution (190 µL), and the final volume was set to 200 µL. Then, the reaction mixture was kept in the dark for half an hour, and the absorbance (517 nm) was measured. Using Ascorbic acid as a standard, the antioxidant activity was determined through the formula:

Total reducing power (TRP) assay

TRP was carried out following the standard method²¹. In this, 200 µL extract was added to phosphate buffer (500 µL) and potassium ferricyanide (500 µL). Afterwards, samples were kept at 50 °C (20 min), 500 µL of TCA was added, and centrifugation was then carried out for 10 min. Then 100 µL of 0.1% FeCl₃ was added to the supernatant, and the absorbance at 630 nm was noted. The reducing power of the extracts was expressed as ascorbic acid equivalents (AAE) based on a calibration curve prepared using different concentrations of ascorbic acid, rather than reporting raw absorbance values in µg/mL, to ensure standardized and reproducible reporting of antioxidant capacity.

Total antioxidant capacity (TAC) assay

TAC was performed using the previous method protocols with some modifications²². Briefly, 50 µL extract was added to 500 µL of reaction mixture (1.68 g of 28 mM sodium phosphate, 1.63 mL of sulphuric acid, and 0.25 g of 4 mM ammonium molybdate in 50 mL of distilled water) and incubated at 95 °C for 90 min. Finally, the absorbance was taken at a 630 nm wavelength. The total antioxidant capacity of the extracts was quantified using an ascorbic acid calibration curve and expressed as µg ascorbic acid equivalents per mL of extract (µg AAE/mL). This approach was adopted to provide a standardized and comparable measure of antioxidant capacity instead of reporting TAC directly in µg/mL of extract.

Antibacterial assay and minimum inhibitory concentration (MIC)

Three freshly cultured bacterial strains, namely Staphylococcus aureus, Listeria monocytogenes, and Bacillus subtilis, were incubated at 37 °C for almost 24 h. These bacterial strains were obtained from preserved culture at the Plant Pathology Laboratory, Quaid-i-Azam University, Islamabad. The disc diffusion method was adopted with a diameter of 6 mm impregnated with concentrations of 31.25 up to 1000 μg/mL of Rubia cordifolia extracts. Oxytetracycline antibiotic was used as a positive control, and DMSO as a negative control. The zone of inhibition (ZOI) was measured around the paper disc impregnated with the antibiotic or sample to be tested using a ruler²³. For MICs of antibacterial assay, two-fold serial dilution of antibacterial in 50-100µL of Mueller–Hinton broth across the wells. Test samples were adjusted to a 0.5 McFarland standard, diluted 1:100 in broth for the final inoculum, and finally added 50–100 µL to the wells. Plates were sealed and incubated for 28 h, along with a control for visible turbidity. MIC was recorded as the lowest concentration of antibacterial, which mainly inhibited microbial growth.

Brine shrimp cytotoxicity assay

The eggs of Artemia salina were hatched in trays with added sea salt water (3.8 g of sea salt per 1 L of distilled water). By incubating 24–48 h, 10 nauplii were added into each glass vial containing dried plant extract (50, 100, and 500 µg/mL) and 5 mL of seawater. Glass vials were incubated for 24 h at 32 °C, and then the number of alive nauplii was counted. Vincristine sulphate was used as a positive, and distilled water as a negative. Afterwards, the percentage mortality was calculated using the following formula²⁴, and then LC₅₀ values (50% lethal concentration of mortality) were determined:

Anti-cancer and cytotoxic assay

The anti-cancer potential was examined against PC3 (prostate) and 3T3 (mouse embryonic fibroblasts) cell lines following the MTT assay²⁵. PC3 cells are highly proliferative and invasive, while 3T3 fibroblasts are normal mouse embryonic fibroblasts (non-cancerous control) used to evaluate cytotoxicity. The PC3 (human prostate cancer) and 3T3 (mouse embryonic fibroblast) cell lines used in this study were obtained from the International Center for Chemical and Biological Sciences (ICCBS), H.E.J. Research Institute of Chemistry, University of Karachi, Pakistan, a recognized repository for authenticated cell lines. Furthermore, the experimental protocol involving these established human and mouse cell lines was conducted following approval from ICCBS (Reference No. AU7929-205). PC3 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM), 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 1.5 g/L sodium carbonate, and 1% antibiotic solutions. Similarly, 3T3 cells were cultured in DMEM, 10% FBS, 100 U/mL of each penicillin and streptomycin in flasks. Later, all cancer cells were incubated at 37 °C with 5% CO₂ and were sub-cultured once they formed a monolayer in a flask. The cells were detached by adding 0.25% trypsin containing 0.01% EDTA for 10 min.

The plant extract (30 µg/mL) prepared in 2% DMSO was added to a 96-well plate along with 100 µL of PC3 and 3T3 cells (1 × 105 cells/mL). Then the cells were exposed to 2% DMSO without extracts that were designated as a negative control, and Doxorubicin was used as a standard. After 24 h, 10 µL of MTT (0.5 mg/mL) was added and re-incubated for 4 h at 37 °C. Later on, 100 µL of DMSO was added, and the absorbance was taken at 570 nm. Finally, the inhibition percentage and IC₅₀ values of the cell population were assessed.

Molecular docking

Five compounds isolated and identified from Rubia cordifolia by HPLC were analyzed by molecular docking²⁶. The compounds’ structures were obtained in SDF format from PubChem and converted to PDB format using PyMOL. The STEAP1 protein (PDB ID: 8UCD), an anticancer target, was downloaded from the Protein Data Bank. The 8UCD protein was refined by removing water molecules, adding polar hydrogens, and assigning Kollman and Gasteiger charges. It was then energy-minimized and saved in PDBQT format. A grid box of size 100 × 100 × 100 Å with a spacing of 0.5 Å was set up, and its center coordinates (X, Y, Z) were optimized based on receptor and ligand size. Docking was performed to evaluate the binding interactions between the ligands and STEAP1. The docked complexes with the best binding affinities were visualized and analyzed using Ligplot + (v2.2.5), which generated interaction diagrams highlighting hydrophobic and polar interactions between the ligands and the target protein.

Statistical analysis

All experiments were performed using three independent biological replicates (n = 3), with each measurement conducted in technical triplicate. Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism. Normality and homogeneity of variance were assessed using the Shapiro–Wilk and Levene’s tests, respectively. Differences among groups were analyzed using one-way ANOVA followed by Tukey’s HSD post hoc test, while non-parametric data were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple-comparison test. Statistical significance was considered at p < 0.05. IC₅₀ values for the DPPH assay were determined by nonlinear regression analysis, whereas cytotoxicity IC₅₀ values were estimated using Probit regression analysis.

Results and discussion

Phytochemical analysis

Qualitative phytochemical analysis

Initially, Rubia cordifolia leaf extracts were prepared using three solvents, including methanol, chloroform, and distilled water, based on the difference in polarity. The extractive value was observed in the following order: methanolic extract (17.5%) > distilled water extract (15%) > chloroform extract (7.5%), as shown in Table 1. Qualitative phytochemical analysis revealed the highest secondary metabolite content in RCLM compared with RCLC and RCLD; thus, the methanolic extract exhibited the greatest potential for extracting bioactive compounds from Rubia cordifolia leaves (Table 2). The occurrence of various bioactive compounds in Rubia cordifolia leaf extracts can be attributed to the plant’s physiological and biosynthetic processes^27,28. However, the absence of some compounds in the chloroform and distilled water extracts could be due to the low polarity of the solvent, which impeded the extraction, indicating that the variations occur due to the incompatible polarity indices of the solvent²⁹. Amongst these, phenolics and flavonoids were detected in all extracts and are regarded as the primary antioxidants and radical scavengers³⁰.

Table 1.

Estimation of the extract yield of Rubia cordifolia leaf extracts prepared in different solvents.

Plant species	Solvent used	Weight of extract (g)	Extract yield (%)
Rubia cordifolia	Methanol	3.5	17.5 ± 0.30a
	Chloroform	1.5	7.5 ± 0.20c
	Distilled Water	3.0	15.0 ± 0.25b

Values are expressed as mean ± SD (n = 3). Different superscript letters (a–c) indicate significant differences among solvent extracts at p < 0.05.

Table 2.

Preliminary qualitative phytochemical profiling indicated the presence of secondary metabolites in Rubia cordifolia leaf extracts in various solvents.

Sr. No	Secondary metabolites	RCLM	RCLC	RCLD
1	Phenols	+	+	+
2	Flavonoids	+	+	+
3	Alkaloids	+	+	+
4	Terpenoids	+	+	+
5	Saponins	+	−	−
6	Glycosides	+	−	+
7	Steroids	+	−	−

RCLM, Rubia cordifolia leaf methanolic extract; RCLC, Rubia cordifolia leaf chloroform extract, RCLD: Rubia cordifolia leaf distilled water extract; + , Present; − , Absent.

Quantitative phytochemical analysis

TPC and TFC performed the quantitative phytochemical analysis. The quantitative estimation of TPC and TFC in Rubia cordifolia leaf extracts showed a wide range of variation across different solvent extracts (Fig. 1). Among the tested extracts, methanolic leaf extract (RCLM) had the highest value with the total phenolic content of 74.31 ± 0.16 mg GAE/g extract and the total flavonoid content of 43.34 ± 0.27 mg QE/g extract, followed by the aqueous leaf extract (RCLD), which had the TPC and TFC values of 74.31 ± 0.16 mg GAE/g extract and 35.12 ± 0.32 mg QE/g extract, respectively. The chloroform leaf extract (RCLC), on the other hand, had the lowest phenolic and flavonoid content with 26.87 ± 0.23 mg GAE/g extract and 18.45 ± 0.21 mg QE/g extract, respectively. Higher TPC and TFC in the methanolic extract could be due to the polar nature of methanol, which makes it better for the extraction of the polar phytochemicals like phenolics and flavonoids³¹. They have been identified as significant contributors to antioxidant and other bioactivities because of their hydrogen-donating and free radical-scavenging properties³². Thus, the higher phenolic and flavonoid contents in the methanolic extract of leaves might be responsible for its relatively higher biological potential in the present study.

Fig. 1.

Total phenolic content (TPC) and total flavonoid content (TFC) of Rubia cordifolia leaf extracts.

HPLC analysis

The HPLC chromatogram of Rubia cordifolia leaf extract indicates the occurrence of some compounds (Fig. 2) that were also present and already reported in other plants; they were utilized as confirmatory in this study. Rubia cordifolia methanolic extract exhibited the highest concentration of chlorogenic acid (392.44 ppm), followed by caffeic acid (17.984 ppm), HB acid (16.938 ppm), p-coumaric acid (13.959 ppm), and salicylic acid (3.408 ppm), respectively (Table 3). Flora is blessed with medicinal plants possessing varied bioactive compounds responsible for many reported biological effects^33–36. The reported compounds in Rubia cordifolia leaf via HPLC are a confirmatory approach, and no novel phenolic compound was reported.

Fig. 2.

HPLC chromatogram of Rubia cordifolia representing some selected phenolic compounds at respective retention times.

Table 3.

Concentration of five phenolic compounds observed in Rubia cordifolia using the HPLC method.

Compounds	RT when the single standard was run	RT when a mixture of standards was run	K-factor	Concentration (ppm)
Chlorogenic acid	2.330	2.880	0.00013	392.44 ± 2.15a
p-Coumaric acid	2.728	3.166	0.0000094	13.959 ± 0.42c
HB acid	6.976	6.759	0.00016	16.938 ± 0.55b
Caffeic acid	7.802	7.494	0.000059	17.984 ± 0.60b
Salicylic acid	15.248	15.296	0.000377	3.408 ± 0.18d

RT, Retention time; ppm, Parts per million.

Antioxidant assays

DPPH assay

The percentage inhibition and IC₅₀ (half maximal inhibitory concentration) values were determined using DPPH radical scavenging activity at five different concentrations (Fig. 3A). The IC₅₀ value represents the concentration of extract required to scavenge 50% of DPPH free radicals; therefore, a lower IC₅₀ value indicates stronger antioxidant activity. The methanolic extract (RCLM) exhibited the highest antioxidant activity with an IC₅₀ value of 54.41 ± 0.53 µg/mL, whereas the aqueous extract (RCLD) showed the lowest activity with an IC₅₀ value of 74.78 ± 0.83 µg/mL. Ascorbic acid was used as the reference antioxidant standard and exhibited an IC₅₀ value of 30.67 ± 0.70 µg/mL. The highest scavenging activity was observed in the methanolic extract compared with the aqueous and chloroform extracts (Table 4). This pattern suggests that the antioxidant activity may be associated with the presence of moderately polar phenolic phytochemicals in the methanolic extract.

Fig. 3.

Antioxidant potential of Rubia cordifolia leaf extracts observed at different concentrations (A) DPPH free radical scavenging activity, (B) Total antioxidant capacity, (C) Total reducing power assay.

Table 4.

Determination of IC₅₀ values of DPPH scavenging activity of Rubia cordifolia leaf extracts using different solvents.

Sr. No	Plant samples	DPPH scavenging activity (IC50, µg/mL)
1	RCLM	54.41 ± 0.53b
2	RCLC	68.78 ± 0.56c
3	RCLD	74.78 ± 0.83d
4	Ascorbic acid (standard)	30.67 ± 0.70a

Values are expressed as mean ± SD (n = 3). Different superscript letters (a–d) indicate significant differences among samples at p < 0.05.

RCLM, Rubia cordifolia leaf methanolic extract; RCLC, Rubia cordifolia leaf chloroform extract; RCLD, Rubia cordifolia leaf distilled water extract; IC₅₀, Half-maximal inhibitory concentration. Ascorbic acid was used as the standard antioxidant.

HPLC analysis revealed that the methanolic extract contained the highest concentration of chlorogenic acid (392.44 ppm), followed by caffeic acid (17.984 ppm), HB acid (16.938 ppm), p-coumaric acid (13.959 ppm), and salicylic acid (3.408 ppm). These phenolic compounds are well known for their antioxidant properties, primarily through free radical scavenging, hydrogen donation, metal ion chelation, and inhibition of oxidative stress³⁷. In particular, chlorogenic acid and caffeic acid are recognized as potent antioxidants capable of neutralizing reactive oxygen species (ROS), which may explain the comparatively lower IC₅₀ value and stronger antioxidant activity of the methanolic extract. Similarly, p-coumaric acid and salicylic acid may also contribute to the reducing and radical scavenging potential of the extract through synergistic antioxidant effects. Antioxidant assays indicate that these extracts could serve as a potential natural source of antioxidants, which may be attributed to the presence of highly polar and moderately polar phytochemicals that mediate the medicinal effects of plants³⁸. Similar findings were reported previously, who demonstrated DPPH scavenging activities of Rubia cordifolia extracts prepared using ethanol, methanol, aqueous solvents, and PBS extracts with polyvinylpyrrolidone (PVPP), with IC₅₀ values of 98.26, 89.47, 85.53, and 97.55 µg/mL, respectively^36,37. Moreover, IC₅₀ values ranging from 23.88 to 65.23 µg/mL have also been reported for ultrasonic-assisted extraction of Rubia cordifolia, which is consistent with the findings of the current study³⁸. Therefore, the present results suggest that Rubia cordifolia possesses considerable antioxidant potential due to the abundance of phenolic secondary metabolites capable of scavenging ROS^39–41.

Total antioxidant capacity

The current research showed that RCLM had greater antioxidant activity with a total antioxidant capacity of 38.39 to 102.08 µg AAE/mL with a standard deviation of 0.41 and 0.89, respectively, followed by RCLD (26.43 to 88.34 µg AAE/mL) and RCLC (22.43 to 69.64 µg AAE/mL) extracts, as indicated in Fig. 3B. The ascorbic acid calibration curve was used to compute these values to give a standard measure of antioxidant capacity. Equally, Rubia cordifolia extract has also been reported to contain 997.0 ± 3 mg Ascorbic acid equivalents per g extract (mg AAE/g) TAC at a concentration of 500 ug/mL, which is comparable to our study²⁹. The current TAC evidence suggests that methanol is a useful extraction solvent for extracting antioxidant potential in Rubia cordifolia³⁰ because solvent polarity largely determines the efficacy of extracting antioxidant phytochemicals. Moreover, it was stated that Rubia cordifolia extract can be used as a natural antioxidant with potential for the treatment of cancer and other diseases caused by oxidative stress³¹. The TAC values of the chosen extracts could be attributed to the presence of bioactive secondary metabolites, including phenolics and flavonoids^25,29. The elevated TAC of the methanolic extract may be attributed to its higher phenolic content, particularly chlorogenic, caffeic, p-coumaric, and salicylic acids, which contribute to antioxidant activity through free radical scavenging and ROS stabilization.

Total reducing powder assay

Total reducing power of Rubia cordifolia leaves was assessed using five different concentrations (62.5–1000 µg/mL) of the selected extracts. Results showed that the RCLM extract exhibited the highest reducing capacity, expressed as ascorbic acid equivalents (µg AAE/mL), ranging from 54.67 ± 0.50 µg AAE/mL to 201.56 ± 0.61 µg AAE/mL, while the lowest activity was recorded in the RCLD extract, ranging from 45.87 ± 0.32 µg AAE/mL to 175.69 ± 0.41 µg AAE/mL (Fig. 3C). The reducing power values were calculated using an ascorbic acid calibration curve to provide a standardized expression of antioxidant capacity. The current study confirmed a comparable range of reducing potential (77.62–93.72 µg AAE/mL) across different extracts of Rubia cordifolia reported previously^25,30. In another study, the methanolic extract of Rubia cordifolia decreased lipid peroxidase (LPO) contents and increased catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH) markers, ultimately indicating the gastroprotective effect of Rubia cordifolia, which can be attributed to its antioxidant capacity^40–42. Hence, natural antioxidants derived from plant sources may protect the body from ROS-induced damage and help alleviate oxidative stress through phyto-based therapeutic agents.

Antibacterial assay

The antibacterial activity of Rubia cordifolia extracts was evaluated against three ATCC bacterial strains (S. aureus, B. subtilis, and L. monocytogenes). Results revealed that the methanolic extract (RCLM) produced the maximum inhibitory effect against B. subtilis, with a zone of inhibition (ZOI) of 26 ± 0.7 mm at 1000 µg/mL, while the minimum ZOI (9 ± 0.5 mm) was observed at 62.5 µg/mL. Similarly, against L. monocytogenes, RCLM exhibited a maximum ZOI of 23 ± 0.5 mm at 1000 µg/mL and a minimum ZOI of 9 ± 0.5 mm at 62.5 µg/mL. The chloroform extract also demonstrated antibacterial activity, showing a maximum ZOI of 20 ± 0.5 mm at 1000 µg/mL and a minimum inhibition of 10 ± 0.5 mm against B. subtilis. Against S. aureus, the chloroform extract exhibited a maximum ZOI of 11 ± 0.5 mm at 1000 µg/mL and a minimum ZOI of 8 ± 0.5 mm at 62.5 µg/mL. Oxytetracycline was used as a positive control, whereas DMSO served as a negative control and showed no inhibitory activity against the tested bacterial strains. All these Results are depicted in Figs. 4A–C and 5. Overall, antibacterial activity increased in a concentration-dependent manner; however, statistical analysis demonstrated that the observed differences among the tested groups were not statistically significant (ANOVA, p = 0.585952; p > 0.05). Therefore, although numerical variations in antibacterial activity were observed, these differences should be interpreted cautiously and not considered statistically significant under the present experimental conditions. At concentrations below 1000 µg/mL, the extracts predominantly exhibited bacteriostatic effects, whereas bactericidal activity was observed at 1000 µg/mL due to the absence of bacterial growth upon subculture. The minimum inhibitory concentration (MIC) values against the tested bacterial strains are presented in Table 5, while the statistical analysis is summarized in Table 6.

Fig. 4.

Antibacterial activity of Rubia cordifolia extracts determined at different concentrations (A) Methanolic extract, (B) Chloroform extract, (C) Distilled water extract.

Fig. 5.

ZOI against S. aureus, L. monocytogenes, and B. subtilis. (A–C) Indicate methanol extracts, (D–F) present chloroform extracts, and (G–I) show distilled water extracts of Rubia cordifolia.

Table 5.

MICs of bacterial strains treated with different solvent extracts.

Solvent extract	Staphylococcus aureus	Listeria monocytogenes	Bacillus subtilis
Methanolic	62.3 ± 1.2a µg/mL	60.7 ± 1.5a µg/mL	60.4 ± 1.1a µg/mL
Chloroform	61.2 ± 1.0a µg/mL	62.1 ± 1.3a µg/mL	60.8 ± 1.4a µg/mL
Distilled Water	62.0 ± 1.4a µg/mL	60.2 ± 1.6a µg/mL	62.4 ± 1.2a µg/mL

Values are expressed as mean ± standard deviation (SD) of three independent experiments. MIC represents the lowest concentration of extract required to inhibit visible bacterial growth and is expressed in µg/mL. Different superscript letters within a column indicate significant differences among solvent extracts (p < 0.05) according to one-way ANOVA followed by Tukey’s post hoc test. Identical superscript letters indicate no significant difference (p > 0.05).

Table 6.

Single-factor ANOVA comparing the means of three experimental groups, showing sum of squares (SS), degrees of freedom (df), mean squares (MS), F-statistic, p-value, and critical F-value.

Groups	Count	Sum	Average	Variance
Anova: single factor
Column 1	3	185.5	61.83333	0.583333
Column 2	3	182.99	60.99667	1.760033
Column 3	3	182.5	60.83333	2.083333

Source of variation	SS	df	MS	F	p-value	F crit
ANOVA
Between groups	1.726689	2	0.863344	0.585093	0.585952	5.143253
Within groups	8.8534	6	1.475567
Total	10.58009	8

One-way ANOVA analysis revealed that there was no statistically significant difference among the tested plant extracts in DPPH radical scavenging activity (F = 0.585, p = 0.586 > 0.05). Therefore, although minor numerical variations in mean antioxidant activity were observed, these differences were not statistically meaningful at the 95% confidence level and should be interpreted cautiously under the present experimental conditions.

Plant extracts can inhibit bacterial growth through multiple mechanisms, including disruption of cell membranes, inhibition of cell wall synthesis, interference with protein and nucleic acid biosynthesis, and induction of oxidative stress mediated by bioactive phytochemicals^42,43. The antibacterial activity observed in the methanolic extract may be associated with phenolic compounds such as chlorogenic acid, caffeic acid, HB acid, p-coumaric acid, and salicylic acid, which are known to disrupt bacterial membranes, alter cellular permeability, interfere with enzymatic pathways, and induce oxidative stress in microbial cells⁴⁴. Antimicrobial agents derived from medicinal plants remain important therapeutic candidates against infectious diseases⁴⁵. In the present study, the methanolic extract of Rubia cordifolia exhibited comparatively higher antibacterial activity than the other extracts tested; however, due to the non-significant ANOVA results, these differences should be considered preliminary observations rather than definitive evidence of superior antibacterial efficacy. The findings are generally consistent with previous reports describing the bacteriostatic potential of Rubia cordifolia extracts against various bacterial strains⁴⁶. Therefore, the plant may serve as a promising source of bioactive compounds for future antimicrobial investigations in food and pharmaceutical applications, although additional studies with larger sample sizes and detailed mechanistic validation are required^47,48.

Brine shrimp cytotoxicity assay

As methanolic extracts showed the highest antioxidant and antibacterial potential, RCLM was selected to determine cytotoxic effects in Rubia cordifolia leaves. Results revealed an 80% mortality rate at 1000 µg/mL and an IC₅₀ of 13.519 ppm, with 5.284 to 34.585 lower and upper confidence intervals, respectively (Fig. 6). The brine shrimp inhibition test was carried out to preliminarily validate the pesticidal effects of plant extracts, as used by many scientists, and also confirms general toxicity, potential bioactivity, and pesticidal activity, in correlation with mammalian cytotoxicity^49,50. The observed cytotoxicity may be associated with phenolic compounds such as chlorogenic acid, caffeic acid, HB acid, p-coumaric acid, and salicylic acid, which can induce oxidative stress imbalance, membrane disruption, and apoptosis-related cellular damage⁵¹. The current findings indicate the existence of toxic compounds in Rubia cordifolia extract. They can thus be utilized as a general, non-selective toxicity, showing that Rubia cordifolia leaf extract possesses compounds that confer general cytotoxicity but not selective anti-cancer activity.

Fig. 6.

Cytotoxic potential of Rubia cordifolia leaf methanolic extract in terms of mortality percentage of brine shrimps.

Cell toxicity and anti-cancer assay

In this assay, RCLM showed a non-cytotoxic effect against the PC3 cell line, with less than 50% inhibition, whereas 78.9% inhibition was observed against the 3T3 cell line, indicating notable non-selective toxicity against the 3T3 cell line. Doxorubicin (standard) showed 89.9% inhibition against the tested cancer cell lines (Table 7). These findings suggest that the methanolic leaf extract of Rubia cordifolia does not possess selective anti-prostate cancer activity under the present experimental conditions. Instead, the extract exhibited generalized cytotoxicity, particularly toward the normal 3T3 cell line, indicating possible non-specific cellular toxicity rather than targeted anticancer efficacy. An effective anti-cancer bioactive agent or drug would ideally exhibit greater cytotoxicity toward cancer cells while sparing normal cells. So, Rubia cordifolia leaf extract lacks selective anti-cancer potential under designed experimental conditions, indicating that crude extracts possess bioactive secondary metabolites with general cytotoxic efficacy rather than selective anti-cancer action⁵².

Table 7.

Percentage inhibition and IC₅₀ values indicating cytotoxicity potential of Rubia cordifolia methanolic extract against PC3 and 3T3 cell lines.

Sample	PC3 cancer cell lines
	Percentage Inhibition	IC50 ± SD	Percentage Inhibition	IC50 ± SD
RCLM (30 µg/mL)	32.6	Inactive	62.6	18.4 ± 0.5a
	48.2	Inactive	78.9	15.2 ± 1.4b
	39.7	Inactive	74.3	8.7 ± 0.5c
Doxorubicin (30 µM)	89.9	1.9 ± 0.12a	89.9	0.1 ± 0.02d

*Doxorubicin was used as a standard; RCLM: Rubia cordifolia leaf methanolic extract; IC50: Half-maximal inhibitory concentration; SD: Standard deviation.

Cancer is one of the main causes of death throughout the world, and medicinal plants possess anti-cancer properties by virtue of secondary metabolites⁵³. Therefore, the market demand for medicinal plants is rising day by day due to their potent anti-cancer potential, and there is a need to test plants and their parts (leaf, stem, root, bulb, tubers, rhizome, etc.) to confirm active anti-cancer phyto-constituents. Herbal medications against cancer are a normal practice in underdeveloped countries of the world⁵⁴. It has been reported that inhibition of human laryngeal carcinoma HEp-2 cells occurs in a concentration-dependent manner, and apoptotic cell death in Hep-2 cells was observed using Rubia cordifolia leaf methanolic extract⁵⁴. These phenolic compounds are known to exert cytotoxic and antioxidant effects through multiple mechanisms, including modulation of oxidative stress, induction of apoptosis, disruption of mitochondrial membrane potential, inhibition of cellular proliferation, and regulation of inflammatory pathways⁵⁵. Chlorogenic acid, identified as the most abundant compound (392.44 ppm), has been reported to influence ROS-mediated signaling and apoptosis-related pathways in different cancer models. Similarly, caffeic acid and p-coumaric acid may contribute to cytotoxic effects through free radical modulation and interference with cellular redox balance, whereas salicylic acid has been associated with stress signaling and growth regulation⁵⁶. However, in the present study, these compounds did not demonstrate selective toxicity toward PC3 prostate cancer cells, suggesting that their combined activity within the crude extract may have produced generalized cytotoxicity rather than cancer-specific effects.

However, the current study did not demonstrate significant cytotoxic activity against the PC3 prostate cancer cell line; therefore, definitive anticancer claims against prostate cancer cannot be concluded from the present experimental data. Similarly, although molecular docking analysis demonstrated favorable binding interactions of chlorogenic acid and HB acid with the STEAP1 protein (PDB ID: 8UCD), these findings should be considered only as predictive in silico observations and not as direct evidence of anticancer activity. The docking results provide preliminary mechanistic insight into possible ligand–protein interactions but do not confirm functional inhibition of prostate cancer progression without biological validation. Therefore, further investigations involving apoptosis assays, caspase activation studies, colony formation assays, gene expression analysis, and additional in vitro and in vivo experiments are required to validate the biological relevance of the docking predictions and to clarify the molecular mechanisms underlying the observed cytotoxic effects.

Molecular docking

Molecular docking analysis was performed as a predictive and hypothesis-generating computational approach to investigate the potential interactions of the identified phenolic compounds with the STEAP1 protein (PDB ID: 8UCD), a reported therapeutic target associated with prostate cancer progression. The three-dimensional structures of the selected compounds were retrieved from the PubChem database in SDF format and converted into PDB format using PyMOL software, while the crystal structure of STEAP1 was obtained from the Protein Data Bank (Fig. 7). The docking results were evaluated based on binding affinity values, hydrogen bonding patterns, hydrophobic interactions, and the involvement of active-site amino acid residues (Table 8 and Fig. 8). Lower binding energy values indicate stronger ligand–protein interactions and greater thermodynamic stability of the complexes. Among the tested compounds, chlorogenic acid demonstrated the strongest interaction with STEAP1, exhibiting the lowest binding energy of − 10.5 kcal/mol. The compound formed multiple hydrogen bonds with Tyr182(C), Tyr219(C), Hem402(C), and Arg215(C), along with hydrophobic interactions involving Phe272(C), Met216(C), Pro183(C), Tyr188(C), and Arg185(C). The presence of multiple hydroxyl and carboxyl functional groups in chlorogenic acid may enhance binding stability through the formation of stable hydrogen-bonding networks within the active site of the protein. Previous studies have also reported the biological significance of chlorogenic acid through modulation of the PI3K/AKT/PTEN signaling pathway, which is associated with apoptosis and suppression of cancer cell proliferation⁵⁷.

Fig. 7.

The STEAP1 protein (PDB ID: 8UCD), an anticancer target that was downloaded from the Protein Data Bank.

Table 8.

Summary of docking results (GC–MS compounds) of 5 potential hits, functional groups involved in hydrophobic interactions, and hydrogen bonds with their binding affinities against the STEAP1 (8UCD) target protein.

Compounds	Chemical Structure	Hydrophobic interactions	Hydrogen bonds with distance	Binding affinity
Chlorogenic acid		Phe272(C) Met216(C) Pro183(C) Tyr188(C) Arg185(C)	Tyr182(C) O–O9 = 3.14 Å Tyr219(C) OH–O5 = 3.17 Å OH–O6 = 2.92 Å Hem402(C) O2A–O4 = 3.04 Å O2A–O3 = 3.22 Å Arg215(C) NH1–O5 = 3.04 Å	− 10.5
HB acid		Tyr252(C) Leu67(B) His72(B) Trp71(B) Leu232(C) Lbn401(B) Ile75(B) Phe68(B)		− 10.5
P-coumaric acid		Phe164(A) Pro132(A) Trp157(A)	Leu128(A) O–O1 = 3.18 Å	− 7.2
Caffeic acid		Tyr188(B) Pro183(B) Tyr182(B)	Met184(A) O–O1 = 2.89 Å Pro183(A) O–O2 = 3.03 Å Arg185(B) NH2–O3 = 2.88 Å	− 7.1
Salicylic acid		Phe164(A) Trp157(A) Leu131(A) Leu128(A) Pro132(A)		− 6.3

Fig. 8.

Interaction plots of 5 potential hits bounded within the active site of STEAP1 (8UCD) target protein, red spikes present hydrophobic residues, green colored residues present hydrogen bonds along with their bond distance within the target of 4 Å.

HB acid also exhibited a strong binding affinity of − 10.5 kcal/mol, mainly stabilized through hydrophobic interactions with Tyr252(C), Leu67(B), His72(B), Trp71(B), Leu232(C), Ile75(B), and Phe68(B), suggesting favorable accommodation within the hydrophobic region of the STEAP1 binding pocket. In contrast, p-coumaric acid and caffeic acid showed moderate binding affinities of − 7.2 and − 7.1 kcal/mol, respectively. P-coumaric acid formed hydrogen bonds with Leu128(A), whereas caffeic acid interacted with STEAP1 through hydrogen bonds with Met184(A), Pro183(A), and Arg185(B), in addition to several hydrophobic interactions. Previous reports suggest that caffeic acid may suppress prostate cancer progression through inhibition of the NF-κB signaling pathway, thereby reducing invasion, angiogenesis, and metastasis^58,59. These findings indicate that hydroxyl-substituted phenolic acids can interact effectively with active-site residues through both polar and hydrophobic contacts. Salicylic acid exhibited the lowest binding affinity (− 6.3 kcal/mol), indicating comparatively weaker interaction with the target protein⁶⁰. Overall, the molecular docking findings provide preliminary computational insight into possible ligand–protein interactions only and should not be interpreted as direct evidence of anticancer activity or mechanistic suppression of prostate cancer progression. Furthermore, the present study did not include apoptosis assays, caspase activation studies, or other functional validation experiments; therefore, no definitive conclusions regarding anticancer mechanisms can be drawn from the docking analysis alone. Additional in vitro and in vivo investigations are necessary to validate the biological relevance of these computational predictions.

Conclusion

The present study showed that Rubia cordifolia leaves had significant antioxidant, antibacterial, and cytotoxic activity, with the methanolic extract showing comparatively stronger biological activity compared to chloroform and aqueous extracts. The phytochemical screening and HPLC analysis yielded several phenolic compounds that could be responsible for the observed biological activities, and preliminary computational knowledge of the interactions between selected phytochemicals and biologically relevant target proteins involved in oxidative stress and cancer progression was provided by molecular docking. In general, the results indicate that the leaves of Rubia cordifolia can be a valuable source of bioactive phytochemicals that have possible pharmaceutical applications. The present study is, however, mainly in vitro and in silico; thus, in vivo biological mechanisms, selectivity, and toxicity profile, as well as clinical relevance of extracts, still need to be well delineated. Moreover, no experimental evidence was found to confirm direct connections between particular chemicals and effects observed on organisms, and, consequently, the results must be interpreted carefully. Further, bioactivity-guided fractionation, mechanistic studies, apoptosis and signalling pathway studies, in vivo validation, detailed toxicity studies, optimization of formulation, and development of advanced drug delivery systems will help to validate the therapeutic potential of Rubia cordifolia and its possible use as a therapeutic plant formulation.

Author contributions

Wasim Akhtar, Rukh e Fatima Naqvi, Neelum Nasar, Sadia Zafar, Ilham Khan, and Momna Asif: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools, or data; Performed statistical analysis; Wrote the paper; Reviewed and edited the manuscript. Shaimaa A. M. Abdelmohsen, Abeer Ahmed Alghamdi, Najla Alotaibi, Manar Fahad Albarak, Dalal Almatrudi, Badriah Albarzan, Wasim Akhtar, and Rukh e Fatima Naqvi: Conceived and designed the experiments; Performed the experiments; Contributed reagents, materials, analysis tools, or data; Assisted in data curation; Wrote the paper; Reviewed and edited the manuscript. Ilham Khan, Neelum Nasar, Shaimaa A. M. Abdelmohsen, Abeer Ahmed Alghamdi, Najla Alotaibi, Manar Fahad Albarak, Dalal Almatrudi, Momna Asif, and Badriah Albarzan: Conceived and designed the experiments; Contributed reagents, materials, analysis tools, or data; Supervised the study; Validated the results; Wrote the paper; Reviewed and edited the manuscript. All authors have approved the final version of the manuscript.

Funding

This research project was funded by the Deanship of Scientific Research and Libraries, Princess Nourah bint Abdulrahman University, through the Pioneer Researcher Funding Initiative, Grant No (PRFI- 2026).

Data availability

Data is available on request from the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.

Ethical consent

The PC3 (human prostate cancer) and 3T3 (mouse embryonic fibroblast) cell lines used in this study were obtained from the International Center for Chemical and Biological Sciences (ICCBS), H.E.J. Research Institute of Chemistry, University of Karachi, Pakistan, a recognized repository for authenticated cell lines. Furthermore, the experimental protocol involving these established human and mouse cell lines was conducted following approval from ICCBS (Reference No. AU7929-205). All the experimental procedures were performed following national and international standard guidelines.