Exploration of Selective Cytotoxic and Anticancer Activity of Helicteres isora Against Skin Cancer Cells: A Phytochemical and Bioanalytical Approach

Cancer and diabetes are among the most serious health challenges worldwide and are responsible for significant morbidity and mortality. The rapid increase in incidence of these diseases has created growing demand for safer and more effective therapeutic agents. Although several synthetic drugs are available for the treatment of these conditions, many of them are associated with adverse effects and high treatment costs. Consequently, researchers are increasingly exploring natural products derived from medicinal plants as a potential source of new therapeutic compounds (Giovannucci et al., 2010).

       Cancer develops when a normal cell undergoes genetic alterations that lead to uncontrolled growth and loss of normal regulatory mechanisms. These abnormal cells can invade surrounding tissues and may spread to other parts of the body through metastasis (Hanahan et al., 2011). According to global health reports, cancer is one off the leading causes of death worldwide and continues to increase due to factors such as aging, lifestyle changes and environmental exposures (Klement, 2024). Conventional cancer treatments such as chemotherapy, radiation therapy and surgery have improved survival rates but these approaches are often associated with serious side effects and toxicity to normal cells (Stein et al., 2008). In recent years, natural products are plant derived compounds have gained attention as potential anticancer agents because they may selectively target cancer cells while causing comparatively fewer adverse effects (Rincón et al., 2025).

     Diabetes mellitus is a chronic metabolic disorder characterized by elevated blood glucose levels resulting from defects in insulin secretion, insulin action or both (American diabetes association., 2010). Persistent hyperglycaemia in diabetic patients can lead to long term complications affecting vital organs such as the hearts, kidneys, nerves and eyes (Antar et al., 2023).

    Enzymes such as α-amylase and α-glucosidase play an important role in carbohydrate digestion. The inhibition of these enzymes is considered as a useful strategy for controlling postprandial blood glucose levels (Febriyanti et al., 2025). Plant based therapies have attracted considerable interest in diabetes management because many medicinal plants contain bioactive compounds capable of improving glucose metabolism and reducing oxidative stress (Jacob et al., 2018).

     So, cancer and diabetes are two major non-communicable diseases that significantly affect global health. Their prevalence has increased rapidly in recent decades due to factors such as lifestyle changes, aging populations, and environmental influences (Liu et al., 2025). Biologically, the connection between cancer and diabetes is associated with metabolic disturbances such as hyperglycaemia, insulin resistance, and elevated insulin levels, which can promote abnormal cell growth and contribute to tumour development (Cignarelli et al., 2018).

      Although current treatments such as chemotherapy and antidiabetic drugs are widely used, they often cause side effects and long-term complications. Therefore, there is an increasing need to explore safer and more effective therapeutic approaches (Bigorra et al., 2021).

     Natural products derived from plants have historically played a crucial role in the discovery of therapeutic agents. Many modern drugs used in clinical practice originate from natural sources or their derivatives. Natural products contain a wide variety of bioactive secondary metabolites such as phenolics, flavonoids, alkaloids, terpenoids, and glycosides. These compounds are known to possess several pharmacological activities including antioxidant, antimicrobial, antidiabetic, and anticancer properties (Les et al., 2024).  Compared to many synthetic drugs, natural compounds are often considered safer due to their relatively lower toxicity and better biological compatibility. As a result, plant-derived compounds are increasingly explored as potential alternatives for disease prevention and treatment. Natural products continue to play a vital role in modern drug discovery and development. Advances in phytochemical and analytical techniques have facilitated the identification of novel compounds with promising therapeutic potential (Liu et al., 2024).

      Due to their diverse biological activities and long history of traditional use, medicinal plants are receiving increasing attention from researchers worldwide. Scientific investigations are being conducted to validate their therapeutic potential and identify bioactive constituents responsible for their pharmacological effects.

     Helicteres isora L. is a medicinal plant belonging to the family Sterculiaceae. It is commonly known as the Indian screw tree and is widely distributed in tropical and subtropical regions of Asia, particularly in India. Different parts of Helicteres isora, including fruits, bark, and leaves, have been traditionally used in various herbal systems of medicine for the treatment of gastrointestinal disorders, infections, inflammation, and metabolic conditions such as diabetes (Thapa., 2024).

      Phytochemical investigations have reported that Helicteres isora contains several bioactive compounds including phenolics, flavonoids, tannins, alkaloids, and terpenoids. These secondary metabolites are known to contribute to various biological activities. Previous studies have indicated that extracts of Helicteres isora exhibit multiple pharmacological properties such as antioxidant, antimicrobial, anti-inflammatory, and antidiabetic activities (Sharma et al., 2016). Phenolic and flavonoid compounds play a significant role in reducing oxidative stress by scavenging free radicals. Their ability to modulate multiple biological pathways makes them promising candidates for managing complex diseases like cancer and diabetes (Suthar et al., 2009).

    Advanced analytical techniques are essential for identifying and characterizing bioactive compounds present in plant extracts. These techniques help in understanding the chemical composition responsible for biological activities. High Performance Thin Layer Chromatography (HPTLC) is an effective method for obtaining the fingerprint profile of plant extracts. It allows the separation and identification of different phytoconstituents based on their retention factor (Bhujbal et al., 2024). Liquid Chromatography–Mass Spectrometry (LC-MS) is a powerful analytical technique used for the identification of bioactive compounds based on their molecular mass and structure. It provides detailed information about the chemical constituents present in the extract (Ramachandram et al., 2016).

        Although Helicteres isora has been reported to possess certain pharmacological activities, comprehensive studies integrating phytochemical profiling using HPTLC and LC-MS with evaluation of antioxidant, antidiabetic, and anticancer activities are still limited. Therefore, the present study was undertaken to evaluate the phytochemical profile of Helicteres isora using HPTLC and LC-MS techniques and to investigate its antioxidant, antidiabetic, and anticancer activities along with cytotoxicity assessment.

Plant Material Collection and Authentication

The plant material of Helicteres isora was collected from Aundha Nagnath, located in Hingoli district of Maharashtra, India. Aundha Nagnath is a historically and ecologically significant region located in Hingoli district of Maharashtra, India. The area is well known for the ancient Aundha Nagnath temple, one of the twelve revered Jyotirlingas dedicated to Lord Shiva. Apart from its religious importance, the region also possesses a diverse natural environment that supports a variety of plant species.

    The collection was carried out during the appropriate season to ensure the availability of healthy and mature plant parts. The selected plants were free from disease and physical damage. After collection, the plant material was thoroughly washed with water to remove soil and other contaminants. The cleaned samples were shade-dried at room temperature to preserve their phytochemical constituents. Once completely dried, the plant material was coarsely powdered using a mechanical grinder and stored in airtight containers for further pharmacognostic and phytochemical analysis.

Fig. – Collection of H. isora from Aundha Nagnath

 Preparation of Plant Extract by Soxhlet extraction method

      5 g of dried and powdered plant material was placed in a thimble and kept inside the Soxhlet apparatus. 300 mL of methanol was taken as the solvent.

         The setup was heated using a heating mantle. As the solvent heated up, it evaporated and moved towards the condenser, where it cooled and changed back into liquid form. This liquid then collected in the chamber containing the plant material.

      When the solvent level reached a certain point, it automatically flowed back into the flask, carrying the extracted compounds along with it. This cycle kept repeating and helped in proper extraction of the plant constituents.

The process was continued for about 8 hours, completing around 6–7 cycles. After extraction, the collected extract was allowed to dry in air to remove the remaining solvent. The dried extract was then stored properly for further analysis (Redfern et al., 2014; Kasiramar et al., 2019).

   Analysis such as preliminary phytochemical screening, total phenolic content (TPC), and total flavonoid content (TFC) analyses were carried out at the Botany Research Laboratory, N. E. S. Science College Nanded. Antioxidant and antidiabetic assays were performed at Infinite Biotech Sangli. HPTLC and LC-MS analyses were conducted at CSIR-National Chemical Laboratory Pune, while FTIR analysis was carried out at Annasaheb Dange College Ashta.

Preliminary Phytochemical Analysis

Preliminary phytochemical screening of the extract was performed using standard qualitative methods to detect the presence of major secondary metabolites such as alkaloids, flavonoids, phenolics, tannins, saponins, glycosides, and terpenoids.

Test for glycosides:

1. Kellar killani test: The extract was treated with glacial acetic acid containing ferric chloride, followed by careful addition of concentrated sulphuric acid along the side of the test tube. The formation of characteristic colour changes was observed.

2. Raymond’s test: The extract was mixed with dinitrobenzene in methanolic alkali and gently heated. The appearance of violet colour was noted.

3. Legal’s test: The extract was treated with pyridine and sodium nitroprusside solution, and the development of pink to red colour was observed.

Test for Alkaloids

1. Mayer’s Test: The test solution was treated with Mayer’s reagent (potassium mercuric iodide). The formation of a cream-colored precipitate was observed, indicating the presence

Test for Flavonoids

1. Ferric Chloride Test: The extract was treated with a few drops of ferric chloride solution.

2. Shinoda Test: The extract was treated with small pieces of magnesium ribbon followed by the addition of concentrated hydrochloric acid.

3. Zinc Hydrochloric Acid Reduction Test: The extract was mixed with zinc dust and a few drops of hydrochloric acid.

4. Alkaline Reagent Test: The extract was treated with sodium hydroxide solution, followed by the addition of dilute acid.

5. Lead acetate solution test: The extract was treated with a few drops of lead acetate solution

Test for steroids:

1. Chloroform test: The extract was dissolved in chloroform. Concentrated sulphuric acid was then carefully added along the side of the test tube.

2. Salkowski’s Test: The extract solution was carefully treated with concentrated sulphuric acid along the side of the test tube to form a separate lower layer.

Test for saponins:

Foam Test: The extract was mixed with water and shaken vigorously for a few minutes.

Test for carbohydrates:

 1. Molisch’s Test: The extract was treated with a few drops of Molisch’s reagent, followed   by the careful addition of concentrated sulphuric acid along the side of the test tube.

2. Benedict’s test: The extract was treated with Benedict’s reagent and heated in a water bath for a few minutes.

Test for proteins:

1. Millon’s Test: The extract was treated with Millon’s reagent and heated in a water bath.

2. Xanthoproteic Test: The extract was treated with concentrated nitric acid and heated gently.

3. Biuret Test: The extract was treated with sodium hydroxide solution followed by the addition of dilute copper sulphate solution.

4. Ninhydrin Test: The extract was treated with ninhydrin reagent and heated.

Test for starch:

1. Starch reagent test: About 1 mL of the extract was mixed with sodium chloride solution and heated. After heating, starch reagent was added to the mixture.

Test for tannins:

1. Gelatin Test: The extract was dissolved in distilled water, followed by the addition of gelatin solution and sodium chloride solution.

2. NaOH Test: The extract was treated with sodium hydroxide solution and shaken well.

(Shaikh et al., 2020; Basumatary., 2016; Kalita et al.,2017)                 

Determination of Total Phenolic Content

      The total phenolic content of the extract was evaluated using the Folin–Ciocalteu colorimetric method. The extract was prepared at different concentrations ranging from 20 to 100 µg/mL for analysis.

       For the assay, 1 mL of the extract was diluted with 9 mL of distilled water in a 25 mL volumetric flask. Subsequently, 1 mL of Folin–Ciocalteu reagent was added and the mixture was mixed thoroughly. After an incubation period of 5 minutes, 10 mL of 7% sodium carbonate solution was added, and the volume was adjusted up to 25 mL with distilled water.

        A series of gallic acid standard solutions (20–100 µg/mL) were prepared following the same procedure to generate a calibration curve. The reaction mixtures were allowed to stand at room temperature for 90 minutes.

       The absorbance of both sample and standard solutions was recorded at 550 nm against a reagent blank using a spectrophotometer. All experiments were conducted in triplicate to ensure reliability of results. The total phenolic content was expressed as micrograms of gallic acid equivalents (µg GAE/g) of extract (Chang et al.,2002; Ulloa et al., 2024; Nikolaeva et al., 2022).

5. Determination of Total Flavonoid Content

    The total flavonoid content of the extract was determined using the aluminium chloride colorimetric method. For the assay, 1 mL of the extract was mixed with 4 mL of distilled water in a 10 mL volumetric flask.

      To this mixture, 0.30 mL of 5% sodium nitrite solution was added and allowed to stand for 5 minutes. Subsequently, 0.30 mL of 10% aluminium chloride was added and the mixture was incubated for another 5 minutes. Thereafter, 2 mL of 1 M sodium hydroxide was added, and the final volume was adjusted to 10 mL using distilled water.

      Standard solutions of quercetin (200–1000 µg/mL) were prepared following the same procedure to obtain a calibration curve. The absorbance of both the sample and standard solutions was measured at 510 nm against a reagent blank using a spectrophotometer.

        All determinations were carried out in triplicate to ensure accuracy. The total flavonoid content was expressed as micrograms of quercetin equivalents (µg QE/g) per gram of extract (Muchandi et al., 2017; Shraim et al., 2021)

Antioxidant Assay

      The antioxidant activity of the extract was evaluated based on its free radical scavenging ability using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals.

      Different concentrations of the extract (20–100 µg/mL) were prepared, and 1 mL of each concentration was taken in separate test tubes. To each tube, 1.5 mL of 0.1% DPPH solution in methanol was added. The reaction mixtures were then incubated in the dark at room temperature for 30 minutes.

      Following incubation, the change in colour from deep purple to yellow was monitored, and the absorbance was measured at 510 nm using a colorimeter against an appropriate control.

      The percentage of DPPH radical scavenging activity was calculated using the following formula:

(Baliyan et al., 2002; Rumpf et al., 2023).

7. Antidiabetic Assay

     The α-amylase inhibitory activity of the extract was evaluated following the method of Bernfeld.

     Different concentrations of the extract (20–100 µg/mL) were prepared, and 100 µL of each concentration was mixed with 500 µL of 0.1 M phosphate buffer (pH 6.9) containing α-amylase enzyme. The reaction mixture was incubated at 25°C for 10 minutes.

     Subsequently, 500 µL of 1% starch solution prepared in 0.1 M phosphate buffer (pH 6.8) was added, and the mixture was further incubated at 25°C for 10 minutes.

      For the control, the enzyme solution was replaced with buffer under similar conditions. After incubation, 1 mL of dinitrosalicylic acid (DNS) reagent was added to both test and control tubes. The mixtures were then heated in a boiling water bath for 10 minutes, followed by cooling to room temperature.

     The absorbance was measured at 540 nm using a spectrophotometer. The percentage inhibition of α-amylase activity was calculated using the following equation:

(Zephy et al., 2015; Maritim et al.,2003).

HPTLC Analysis

     The Helicteres isora sample solution was prepared by dissolving 5 mg of sample in 5 mL of distilled water, followed by sonication for 5 minutes. The volume was then adjusted to 10 mL with methanol. Further dilution was carried out by taking 1 mL of this solution and making up to 10 mL using methanol and water (1:1) to obtain a final concentration of 50 ppm.

     The sample was applied onto pre-coated silica gel 60 F254 HPTLC plates using a Linomat 5 applicator. Each track was applied as bands at a fixed position with controlled dosage.

      Chromatographic development was carried out in a twin-trough chamber using a mobile phase consisting of toluene, ethyl acetate, and formic acid in the ratio of 6:3:1 (v/v/v). The chamber was saturated for about 20–25 minutes prior to development. The plate was developed up to a distance of 70 mm.

       After development, the plate was dried at room temperature and visualized under UV light at 254 nm. Densitometric scanning was performed at 329 nm using a TLC scanner to record the chromatographic profile of the sample (Chewchinda et al., 2020; Sujatha et al., 2019; Thomas et al., 2020).

 LC-MS analysis

    The phytochemical profiling of the plant extract was carried out using Liquid Chromatography–Mass Spectrometry (LC–MS) analysis. The extract was first filtered through a membrane filter to remove any particulate matter before injection into the system.

    The LC–MS analysis was performed using a suitable chromatographic column under optimized operating conditions. A compatible mobile phase system was used to achieve effective separation of the compounds present in the extract. The sample was introduced into the system, and separation of phytoconstituents was carried out based on their interaction with the stationary and mobile phases.

      The eluted compounds were subsequently detected using a mass spectrometer equipped with an appropriate ionization source. The mass spectra of the compounds were recorded over a defined mass range. The identification of compounds was carried out based on their retention time, molecular mass (m/z values), and comparison with available spectral databases.

      The obtained chromatogram provided multiple peaks corresponding to different phytoconstituents present in the extract, indicating the presence of diverse bioactive compounds (Thorsteinsdottir et al., 2021; Gandu et al., 2025; Jouaneh et al., 2022).

Cytotoxicity Assay

     L929 fibroblast cells were maintained in suitable culture medium and incubated at a density of approximately 1 × 10⁴ cells/ml for 24 hours at 37°C in a humidified atmosphere containing 5% CO₂.

       After incubation, the cells were seeded into 96-well tissue culture plates by dispensing 100 µl of cell suspension per well (approximately 1 × 10⁴ cells/well). The cells were then treated with different concentrations of the test samples (20, 40, 60, 80, and 100 µg/ml). Control wells containing cells treated with 0.2% DMSO in PBS were maintained for comparison. All treatments, including controls, were performed in triplicate to ensure accuracy and reproducibility.

     The plates were incubated for an additional 24 hours under standard culture conditions (37°C and 5% CO₂). Following the incubation period, the culture medium was carefully removed from each well, and 20 µl of MTT solution (5 mg/ml prepared in PBS) was added.

      The plates were then incubated for 4 hours to allow the formation of formazan crystals by metabolically active cells. The development of dark-coloured formazan crystals indicated the presence of viable cells, as the yellow MTT reagent is reduced only by living cells.

         After incubation, the medium was completely discarded, and 200 µl of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formed formazan crystals. The plates were kept for approximately 10 minutes at 37°C, protected from light using aluminium foil.

       Finally, the absorbance of each well was measured using a microplate reader at a wavelength of 550 nm. The experiment was conducted in triplicate, and the results were used to calculate the percentage of cell viability and inhibition (Stephen., 2005; Sigma-Aldrich. (n.d.); Mosmann.,1983; Hansen et al., 1989).

Anticancer Assay

         B16F10 melanoma cells were maintained in appropriate culture medium and incubated at a density of approximately 1 × 10⁴ cells/ml for 24 hours at 37°C in a humidified atmosphere containing 5% CO₂.

        Following incubation, the cells were seeded into 96-well tissue culture plates by adding 100 µl of cell suspension per well (approximately 1 × 10⁴ cells/well). The cells were then treated with varying concentrations of the test samples (25, 50, 75, 100, and 125 µg/ml). Control wells containing cells treated with 0.2% DMSO in PBS were included for comparison. All treatments were performed in triplicate to ensure reliability of the results.

       The plates were further incubated for 24 hours under standard conditions (37°C and 5% CO₂). After the incubation period, the culture medium was carefully removed from each well, and 20 µl of MTT solution (5 mg/ml prepared in PBS) was added.

       The plates were incubated again for 4 hours to allow viable cells to convert the yellow MTT into dark purple formazan crystals. The formation of these crystals indicates metabolically active cells.

       After incubation, the medium was completely removed, and 200 µl of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formed formazan crystals. The plates were kept for approximately 10 minutes at 37°C and protected from light by covering with aluminium foil.

     Finally, the absorbance of each well was measured using a microplate reader at a wavelength of 550 nm. All experiments were conducted in triplicate, and the data obtained were used to calculate the percentage of cell viability and inhibition (Stephen., 2005; Sigma-Aldrich. (n.d.); Mosmann.,1983; Hansen et al., 1989)

FTIR Analysis

        Fourier Transform Infrared (FTIR) spectroscopy was employed to identify the functional groups present in the plant extract. The dried extract was prepared for analysis by mixing with potassium bromide (KBr) to form a fine powder, which was then compressed into a transparent pellet using a hydraulic press.

  The prepared pellet was placed in the FTIR spectrophotometer, and the spectrum was recorded over a range of 4000 to 650 cm⁻¹.The obtained spectra were analysed to identify characteristic absorption peaks corresponding to different functional groups present in the extract.

        The observed peaks were interpreted based on standard reference values to determine the presence of functional groups such as hydroxyl, carbonyl, amine, and other chemical moieties. This analysis provided useful information regarding the chemical nature of the bioactive compounds present in the extract (Gang et al., 2024; Shen et al., 2023; Munajad et al., 2018).

The present investigation comprehensively evaluated the phytochemical composition and pharmacological potential of Helicteres isora using a combination of qualitative, quantitative, analytical, and biological approaches. Preliminary phytochemical analysis confirmed the presence of major secondary metabolites such as flavonoids, phenols, tannins, alkaloids, and other bioactive constituents, indicating its rich phytochemical profile.

Quantitative estimation revealed significant levels of total phenolic content by the Folin–Ciocalteu method and total flavonoid content by the aluminium chloride method, suggesting strong antioxidant potential. This was further supported by antioxidant assays, which demonstrated notable free radical scavenging activity of the extract. Additionally, the extract exhibited promising antidiabetic activity, indicating its potential role in managing metabolic disorders.

 Advanced analytical techniques further validated these findings. HPTLC profiling provided characteristic fingerprint patterns confirming the presence of diverse phytoconstituents, while LC–MS analysis enabled the identification of several bioactive compounds that may be responsible for the observed biological activities. FTIR analysis confirmed the presence of functional groups corresponding to these phytochemicals, thereby supporting the overall chemical characterization of the extract.

Importantly, the study demonstrated significant cytotoxic and anticancer activity of Helicteres isora against skin cancer cell lines, indicating its potential as a promising source of anticancer agents. Notably, the cytotoxic and anticancer potential of this plant remains relatively underexplored, and the present work contributes novel insights into this aspect by systematically evaluating its effects using in-vitro models.

Overall, the findings suggest that Helicteres isora possesses considerable therapeutic potential due to its rich phytochemical composition and diverse biological activities. However, despite these encouraging results, limited scientific investigations are available, particularly regarding its cytotoxic and anticancer mechanisms. Therefore, further detailed studies involving molecular mechanism elucidation, isolation of active compounds, and clinical validation are required to establish its efficacy and safety for therapeutic applications.

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