Medicinal plants remain a critical reservoir of structurally diverse bioactive compounds that support drug discovery and phytopharmaceutical development. According to the World Health Organization (WHO), a considerable percentage of people throughout the world use herbs for basic health care purposes (WHO, 2019). Odukoya et al. (2022) report that medicinal plants recommended for cardiac dysfunction are rich in secondary metabolites, including alkaloids, flavonoids and polyphenols. These plants exert cardioprotective effects by reducing inflammation and lowering lipid profile levels. Hypertension is also mitigated by these herbs, contributing to cardio-protection. Plant secondary metabolites, including phenolics, alkaloids, glycosides and amide-containing compounds, are widely documented for their anti-inflammatory, antioxidant and cardioprotective properties (Dai and Mumper, 2010; Pan et al., 2014). Contemporary phytochemical investigations increasingly employ advanced analytical platforms such as GC-MS and FTIR spectroscopy to enable precise chemical profiling and to validate traditional therapeutic claims through experimental pharmacology (Sasidharan et al., 2010; Movasaghi et al., 2008).
       
Biophytum sensitivum (L.) DC. (Oxalidaceae) is an herbaceous species that is found extensively in the tropical areas of India. The plant occupies a significant position in Ayurvedic and Siddha systems of medicine, where it is traditionally employed for the management of inflammatory disorders, metabolic disturbances, wound healing and cardiovascular conditions (Roopa et al., 2022). Ethnobotanical records and pharmacognostic documentation recognize B. sensitivum as a medicinally valuable species with reported antioxidant, immunomodulatory and hypo-glycemic activities. Experimental investigations have demonstrated glucose-lowering and lipid-modulating effects of B. sensitivum extracts, suggesting potential modulation of cardio-metabolic pathways (Sakthivel and Guruvayoorappan, 2012).
       
Cardiovascular diseases (CVDs) continue to be the primary cause of death around the world, with an estimate of more than 20 million people dying due to CVD each year, as per recent WHO estimations (WHO, 2025). From chemotherapeutic drugs, doxorubicin is widely recognized as one that causes dose-dependent cardiotoxicity through oxidative stress, lipid peroxidation and myocardial necrosis (Bhutani et al., 2025). CVD deaths were the lowest in developed nations as compared toLow/Middle Income Countries. The developing countries have a major burden of CVD risk factors among all age groups. Unfortunately, more than 80% Global deaths occurred due to CVD (Roth et al., 2015).
       
Restoration of lipid parameters,high-density lipoprotein cholesterol (HDL-C) in particular and preservation of myocardial histoarchitecture are widely accepted indicators of cardio-protection (Pandey et al., 2024).Consequently, plant-derived antioxidants and phenolic-rich extracts have been actively investigated for their protective role against doxorubicin-induced myocardial injury.Nitrogen-containing heterocycles and amide-based compounds represent pharmacologically significant structural classes due to their capacity to participate in hydrogen bonding, enzyme modulation and redox interactions. Detection of amide-associated functional groups and related derivatives within medicinal plants provides a mechanistic basis for exploring their therapeutic potential (Chernyshov et al., 2022).
       
However, comprehensive phytochemical profiling of B. sensitivum, integrating spectroscopic characterization with experimental validation of cardioprotective activity, remains limited. For this reason, the current research work was aimed at (i) quantification of important phytochemical components of B. sensitivum extracts; (ii) characterization of secondary metabolites using GC-MSand FTIR analysis, focusing on amide-related chemical structures; and (iii) determining the cardioprotective potential of the hydroalcoholic extract against doxorubicin-induced myocardial damage in albino Wistar rats.

Plant source and identification
 
The whole plant of B. sensitivum (L.) DC. was harvested from Kovilpatti, Tamil Nadu, India (lat. 9.17°N, long. 77.87° E) during August-September, 2025. Identification of the plant was conducted based on standard flora of the region and was also verified by an expert taxonomist at St. Xavier’s Research Foundation, Palayamkottai. The voucher specimen (Ref. No. XCH-40483) is preserved in the herbarium for future records.
 
Preparation of extracts
 
The plant materials were first washed in running tap water and then distilled H₂O before being dried in shade at 25± 2°C under appropriate ventilation for 14 days, after which they were milled using a REMI RAS-4 laboratory mill (REMI Elektrotechnik Ltd., Mumbai, India). About 100 g of the dried material was allowed to macerate separately in 70% ethanol, methanol and distilled H2O in a ratio of 1:10 (w/v) for 72 hours with shaking intermittently at 120 rpm in an Orbitek orbital shaker (Scigenics Biotech, India). Lyophilization of the aqueous extract was done in a Labconco Free-Zone 2.5 L freeze drier (Labconco Corp., USA) in order to prevent degradation of any of the bioactive compounds. Percentage yield was calculated gravimetrically using a Sartorius Entris analytical balance (±0.1 mg accuracy). Dried extracts were kept in amber glass bottles and stored at 4 °C until the next process (Devi et al., 2023; Harborne, 1998; WHO, 2011).
 
Qualitative phytochemical screening
 
The preliminary screening for the presence of phytochemicals was done using the qualitative tests. Proteins were determined through Biuret test (Mihalcea and Drochioiu 2025), which showed the presence of peptide bond in violet colour and was confirmed using UV spectrophotometry at 540 nm by using Shimadzu UV-1800 UV-Visible spectrophotometer (Shimadzu Corporation, Japan). The total phenolics content was determined using Folin-Ciocalteu reagent, which was later quantified using UV spectrophotometry at 765 nm (Hu et al., 2025). The cardiac glycoside was tested by Salkowski’s reagent (Ponce et al., 2025).
 
Phytochemical quantification
 
The protein content was assessed by Biuret method using bovine serum albumin as a reference and the absorbance was taken at 540 nm with Shimadzu UV-1800 spectrophotometer. Linearity (R² ≥ 0.99) was observed in calibration curves prepared. Phenolic content was assessed using the Folin-Ciocalteu method and reported in terms of mg Gallic acid equivalent per 100 g of dry weight and the absorbance was read at 765 nm. The cardiac glycosides were measured using Baljet reagent with digitoxin as reference material and absorbance was taken at 495 nm (Mihalcea and Drochioiu, 2025; Hu et al., 2025; Ponce et al., 2025).
 
FTIR spectroscopy
 
FTIR analysis was carried out using a FTIR spectrometer, Bruker Alpha II (Bruker Optik GmbH, Germany), fitted with a DTGS detector. The spectra were acquired within the region of 4000-400 cm^-1 at a spectral resolution of 4 cm^-1 using 32 scans/sample. Spectroscopic data were analyzed with the help of OPUS software version 7.5, Bruker (Patel et al., 2025).
 
GC-MS analysis
 
GCMSanalysis of the methanolic extract was conducted using the Agilent 7890B Gas Chromatograph coupled with the Agilent 5977A Mass selective detector from agilent Technologies (USA). The separation process was done using an HP-5MS column (30 m × 0.25 mm i.d. × 0.25 µm film thickness). Pure helium was used as the mobile phase in a constant flow rate of 1 mL/min. Split injection (10:1) of one microliter sample, which was filtered using a 0.22 µm PTFE syringe filter (Millipore, USA), was done at an injector temperature of 250°C. The initial temperature (60°C) was kept constant for 2 minutes and then increased to 280°C in the rate of 10°C/min, with the last stage being held for 10 minutes. Electron ionization was done to acquire the mass spectra of compounds at 70 eV ionization energy. The temperature of the ion source, quadrupole and scan range were set at 230°C, 150°C and m/z 50-600, respectively. The data acquisition and further processing were done using Agilent MassHunter Workstation Software (Version B.07.00), while identification was done based on the similarity index of the spectra compared to NIST 14 MS Library with the cut-off ≥90% (Khan et al., 2026).
 
Animal subjects and ethics clearance
 
Adult albino Wistar rats weighing between 150 to 200 g were maintained in polypropylene cages. Animals were provided with a pellet diet from Hindustan Unilever Ltd. India along with filtered water in sufficient quantity. Animals were kept in controlled environmental conditions with temperatures maintained between 25±2°C, humidity at 55 to 60% and a 12-hr light and dark period.The experimental procedures were reviewed and approved by the Institutional Animal Ethics Committee (IAEC) under approval number IAEC/R. Jasmine Juliet/MKU/20PFOS9932/Ph.D/KMCP/05/2023 (Utami et al., 2026; Thakur et al., 2022).
 
Induction of myocardial injury and treatment protocol
 
Hydro-alcoholic extract of B. sensitivum was applied. Myocardial damage was created using a single injection of doxorubicin hydrochloride (Cipla Ltd., India) [intraperitoneal (I.P)] for three consecutive days (cumulative dose)at a dose of 15 mg/kg body weight. Rats were divided into four different groups (n=4), which were namely normal control, toxic control and two other groups that were treated with hydroalcoholic extract of B. sensitivum in doses of 150 mg/kg/day (P.O) for 21 daysand 300 mg/kg/day (P.O) for 21 daysbody weight, respectively.
 
Biochemical analysis
 
Blood samples were obtained under mild anaesthesia at the conclusion of the experiment and centrifuged by a REMI C-24 BL refrigerated centrifuge at 3000 rpm for 10 min at 4°C to obtain serum. The HDL-C concentrations were analyzed using an enzymatic colorimetric diagnostic assay kit (Erba Mannheim, Germany), measuring absorbance at 600nm. The cardiac biomarker of cTnI was analysed by ELISA kit. The other Cardiac biomarkers such as CK-MB, LDH, SGOT or oxidative stress parameters analysis will be carried out based on our requirement of experiments in future research work. 
 
Histopathological examination
 
Excision of heart tissue was done immediately following sacrifice and rinsing in physiological saline. They were then fixed in 10% neutral buffered formalin (Merck, Germany). The procedure for preparation of tissue samples was conducted on Leica TP1020 tissue processor and then embedded with Leica EG1150 embedding station in paraffin blocks. The cutting of 5 micrometre thick slices was achieved through the use of Leica RM2235 rotary microtome. Haematoxylin and eosin staining techniques were used to prepare the sections. Microscopic evaluation was conducted using Olympus CX43 optical microscope with digital imaging technique.
 
Statistical analysis
 
All tests were run in triplicate or with six subjects per group. Data was presented as mean ± SEM. All statistical analyses were done with Graphpad Prism software version 9.0 (USA). Statistically significant results were taken to be those where p<0.05 Devi et al. (2023).

Plant authentication
 
The collected specimen was taxonomically authenticated as B. sensitivum (L.) DC. (Fig 1) belonging to the family Oxalidaceae and a voucher sample was deposited in the institution’s herbarium (Ref. No. XCH-40483). Morphological identification was performed based on diagnostic characteristics, including pinnate leaf arrangement, sensitive leaflet movement and yellow inflorescence. Proper taxonomic authentication and voucher deposition are essential to ensure reproducibility and accuracy in phytochemical and pharmacological investigations (Heinrich et al., 2020; de Boer et al., 2015). Accurate botanical identification forms the foundation for reliable correlation between chemical composition and biological activity.


 
Qualitative phytochemical analysis
 
The results of phytochemical qualitative analysis of the extracts of B. sensitivum (Table 1) showed that the presence of proteins, phenols and cardiac glycosides were common in all extracts with variations in intensity.T he Biuret reaction produced a prominent violet colouration in the ethanolic and methanolic extracts, indicating a higher abundance of proteinaceous or peptide-linked constituents, whereas the aqueous extract exhibited comparatively reduced intensity. The Folin-Ciocalteau assay yielded a characteristic blue chromophore in all extracts, confirming the presence of phenolic compounds, with stronger development observed in hydroalcoholic extracts. Kellar Kilani test showed an intense brown ring formation in all extracts, indicating the presence of cardiac glycosides, especially ethanol extract.

The qualitative predominance of phenolics and glycosides in hydroalcoholic extracts supports the quantitative findings presented in Table 2, where ethanolic extraction yielded significantly higher concentrations of these constituents. The efficiency of hydroalcoholic extract in the extraction of moderately polar secondary metabolites is attributed to increased solubility dynamics (Dai and Mumper, 2010).        



            

The phenols are vital in addressing oxidative stress by donating hydrogen atoms and stabilising radicals, which are critical to cardio protection. Cardiac glycosides, although traditionally associated with positive inotropic effects, also contribute to the modulation of ion homeostasis and myocardial contractility. The concurrent detection of phenolics and glycosides therefore provides an initial phytochemical rationale for the cardioprotective effects observed in vivo (Table 5).
       
The qualitative screening thus establishes a foundational chemical profile that supports subsequent spectroscopic characterisation (Table 3) and GC-MS identification of nitrogen-containing bioactive compounds (Table 4).




 
Quantitative phytochemical composition and statistical validation
 
The quantitative phytochemical composition of B. sensitivum extracts is presented in Table 2. The ethanolic extract exhibited the highest protein content (360±10.8 mg/100 g), followed by the methanolic (310±9.3 mg/100 g) and aqueous extracts (230±6.9 mg/100 g). Similar trends based on solvents were also noticed for total phenolics, where the ethanol extract had a concentration of 276±8.3 mg/100 g compared to 236±7.1 mg/100 g for methanol and 68±2.0 mg/100 g for water. The concentration of cardiac glycosides was also highest in the ethanol extract at 2.20±0.07 mg/100 g, compared to 0.70±0.02 mg/100 g for methanol and 0.15±0.005 mg/100 g for water. One-way ANOVA showed there were significant differences between different solvents regarding concentrations of protein (F2,6 = 135.2, p<0.0001), total phenolics (F2,6 = 259.8, p<0.0001) and cardiac glycosides (F2,6>1000, p<0.0001). The Tukey post hoc test revealed the presence of significant higher amounts of all phytochemicals in ethanolic extract than methanolic and aqueous extracts (p<0.05) represented by different superscript letters in Table 2.
       
The high efficiency of extraction in the case of ethanol might be due to its intermediate polarity, which makes it capable of dissolving polar as well as slightly non-polar plant compounds. Polarities play an important role in the extraction process of phenols; therefore, a higher percentage is obtained using hydroalcoholic solvents than purely aqueous solutions (Ghaffar and Perveen, 2025). The balanced polarity of ethanol allows improved extraction of phenolics and glycosidic compounds through enhanced solvent-solute interactions. Given that phenolic constituents contribute to cardioprotection via radical scavenging and inhibition of lipid peroxidation (Najjar and Feresin, 2021), the significantly higher phenolic and glycoside content in the ethanolic extract supports its biological relevance for subsequent cardioprotective evaluation.
 
FTIR spectral analysis and functional group characterisation
 
The FTIR (Fig 2) for the ethanolic extract showed clear peaks indicative of functional groups as listed in Table 3. The broad peak appearing at 3444.63 cm^-1 was due to O-H and N-H stretching vibrations showing that the extract contained phenolic hydroxyl groups and amides. The peak appearing at 1749.32 cm^-1 signified the presence of C=O stretching vibrations that are normally found in esters and amide groups, whereas the one appearing at 1625.88 cm^-1 is attributed to C=C stretching or conjugated C=O vibration in aromatic structures. The peaks found at 781.12 cm^-1 and 704.93 cm^-1 confirmed the presence of substituted aromatic rings. The spectra were in line with the occurrence of nitrogenised aromatic or phenolic structures in medicinal plants’ extracts (Movasaghi et al., 2008). The presence of amide-associated functional groups correlates with the propanamide derivative identified in GC-MS analysis (Table 4; Fig 3), providing complementary structural validation.




 
GC-MS profiling and identification of bioactive constituents
 
Multiple nitrogen heterocyclic, aromatic amides, indole compounds and phenolic acid analogues have been detected in the methanolic extract of B. sensitivum by GC-MS (Fig 3; Table 4). The chromatographic profile demonstrated a predominant peak at RT 20.355 min (15.26%), tentatively identified as a propanamide-related aromatic amide derivative. Another halogenated propanamide compound, Propanamide, 3-bromo-N-(4-bromo-2-chlorophenyl)-, was detected at RT 17.868 min with 8.93% relative abundance. The repeated occurrence of amide scaffolds at different retention times indicates structural diversity within the amide fraction of the extract. Amide-containing heterocycles are widely recognized in medicinal chemistry for their biological relevance due to hydrogen-bonding capability and enzyme interaction potential (Vitaku et al., 2014). Specifically, the aromatic amide derivatives have been observed to possess antioxidant and oxidative stress-modifying abilities, mechanisms that can play an important role in cardio protection (Ullah et al., 2024). The predominance of propanamide derivatives in the present study therefore provides a phytochemical basis for the observed improvement in serum HDL-C levels and histological preservation of myocardial tissue (Qayyum et al., 2022).
       
Apart from the propanamide class, another chemical entity known as 2-Myristynoyl-glycinamide (8.16%) was found, which belongs to the fatty acid amide group. Studies suggest that fatty acid amides help stabilize the membranes and control lipids metabolic pathways, thereby leading to lipid stability (Santos-Buelga et al., 2019). The identification of such lipid-associated amide derivatives is consistent with the HDL-restorative effect observed in vivo. An indole-related compound detected at RT 18.142 min (13.72%) further supports the antioxidant framework of the extract. Indole derivatives have been shown to be anti-inflammatory and protect against endothelial damage; they are often found in the secondary metabolic products of plants (Sravanthi and Manju, 2016). Additionally, a phenolic acid derivative, Benzoic acid, 2,4-bis[(trimethylsilyl)oxy]-, trimethylsilyl ester (6.08%), was detected, indicating the presence of phenolic constituents, which are well established for their radical scavenging capacity and cardioprotective relevance (Torres-Fuentes et al., 2022; Kifayatullah et al., 2017).
       
Collectively, the GC-MS profile demonstrates the predominance of nitrogen-containing amide derivatives and heterocyclic scaffolds, supported by phenolic and indole constituents. These structural classes are mechanistically associated with antioxidant defense, lipid regulation and membrane stabilization, thereby corroborating the biochemical and histopathological findings of the present study. However, as compound identification was based on NIST library matching, further structural confirmation through advanced spectroscopic techniques is warranted.
 
Effect of extract on serum HDL and cTnI
 
Effect of B. sensitivum extract on serum HDL-C concentration is shown in Table 5 and 6. Doxorubicin caused significant reduction in HDL-C levels (23.85 ± 0.95 mg/dL) when compared to normal control group (40.21±0.90 mg/dL). One-way ANOVA test showed that there is a significant difference between the groups (F3,20 = 63.2, p<0.0001). Tukey’s post hoc test (Table 6) showed that HDL-C levels in the toxic control group were significantly lower as compared to those in normal control group (p<0.0001). The hydroalcoholic extract significantly improved the HDL-C levels at 150 mg/kg (36.15±0.85 mg/dL) and 300 mg/kg (35.85±0.85 mg/dL) dosages when compared to the toxic control (p<0.0001). However, there was no significant difference between the two dosage groups (p>0.05). Restoration of HDL-C levels indicates improvement in lipid homeostasis and reverse cholesterol transport, mechanisms that are essential in mitigating cardiovascular risk (Rosenson et al., 2016). Plant-derived phenolics have been shown to regulate lipid metabolism and enhance antioxidant defense systems (Kumar and Goel, 2019), which may explain the observed lipid modulation.




       
Wu et al. (1998) cTn I is a very sensitive biomarker. DOX group showed marked elevation in serum cTnI content. Pre-treatment of Hydro alcoholic extract of Biophytum sensitivum (150 mg/kg/day and 30 0 mg/kg P.O) brought down the DOX induced elevation in cTn I level, reflecting the reduced membranous damage (Table 7).


 
Histopathological evaluation of myocardial tissue
 
Histopathological examination of cardiac tissues (Fig 4) demonstrated distinct structural alterations among the experimental groups. The normal control group exhibited well-organised myocardial fibres with intact architecture, centrally located nuclei and absence of inflammatory infiltration. In contrast, the doxorubicin-induced toxic control group showed pronounced myocardial degeneration characterized by cytoplasmic vacuolization, myofibrillar disruption, inflammatory cell infiltration and extensive areas of necrosis, confirming successful induction of cardiotoxicity.


       
Administration of the hydroalcoholic extract of B. sensitivum resulted in marked attenuation of myocardial damage. The 150 mg/kg treatment group exhibited moderate restoration of myocardial architecture with reduced inflammatory infiltration and limited necrotic areas. The 300 mg/kg treatment group demonstrated substantial structural recovery, with near-normal myocardial fibre arrangement, preservation of cellular integrity and minimal necrotic changes compared to the toxic control.Doxorubicin-induced cardiotoxicity is widely attributed to excessive reactive oxygen species generation, mitochondrial dysfunction and lipid peroxidation-mediated membrane damage (Octavia et al., 2012). The structural preservation observed in extract-treated groups suggests mitigation of oxidative injury and stabilization of myocardial membranes. This antioxidant protection is attributed to the high amount of phenolics present in the extract and nitrogenous bioactive compounds identified using gas chromatography-mass spectrometry analysis. These compounds possess anti-oxidant and anti-inflammatory activities.
 
Integrated chemical-biological correlation
 
The integration of statistically validated phytochemical quantification (Table 3), functional group confirmation (Table 5; Fig 2), GC-MS identification of a prominent propanamide derivative (Table 4; Fig 3), restoration of HDL-C levels with strong statistical significance (Table 6) and histopathological improvement (Fig 4) collectively substantiates the cardioprotective potential of B. sensitivum. The significant enrichment of phenolics and the presence of nitrogen-containing amide derivatives suggest a multifactorial mechanism involving oxidative stress attenuation, lipid regulation and myocardial structural preservation. These findings provide comprehensive chemical and biological evidence supporting the therapeutic relevance of B. sensitivum and warrant further mechanistic investigations at the molecular level.

The present investigation establishes Biophytum sensitivum (L.) DC. is a phytochemically enriched therapeutic plant with experimentally validated cardioprotective potential. Restoration of lipid parameters and preservation of myocardial architecture in a doxorubicin-induced model underscore its protective efficacy against oxidative cardiac injury. The identification of phenolic constituents and nitrogen-containing amide derivatives provides a plausible biochemical basis for its activity, potentially mediated through antioxidant, anti-inflammatory and membrane-stabilizing pathways. Together, these findings substantiate the traditional medicinal relevance of B. sensitivum and position it as a promising candidate for further cardiovascular pharmacological exploration. Future investigations focusing on molecular docking, target-specific assays and pathway elucidation are warranted to define its precise therapeutic mechanisms.

The authors gratefully acknowledge the instrumental support provided by ANJAC College, Sivakasi and V.V. Vanniaperumal College for Women, Virudhunagar, Tamil Nadu, India, for facilitating the GC-MS and FTIR analyses of the methanolic and ethanolic plant extracts.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Ethics Statement
 
All animal experimental procedures were reviewed and approved by the Institutional Animal Ethics Committee (IAEC) under approval number IAEC/R. Jasmine Juliet/MKU/20PFOS9932/Ph.D/KMCP/05/2023.

The authors confirm that there are no conflicts of interest associated with this study.