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    In vitro Evaluation of the Anti-Carcinogenic Activity of Swarnavari Bhasma: An Ayurvedic Herbo-Mineral Formulation

    Keyur Dudhat1★, Anita Wanjari1★ Corresponding author

    1. 1Department of Rasa Shastra and Bhaishajya Kalpana, Mahatma Gandhi Ayurved College, Hospital and Research Centre, Salod, Wardha, Maharashtra, INDIA.

    CORRESPONDENCE

    Keyur Dudhat

    MD Department of Rasa Shastra and Bhaishajya Kalpana, Mahatma Gandhi Ayurved College, Hospital and Research Centre, Salod, Wardha, Maharashtra, INDIA.

    drkeyur8@gmail.com

    Received: 02-11-2025; Revised: 16-02-2026; Accepted: 03-04-2026.

    Volume 18, Issue 4 · pp. 1360–1368 · PUBLISHED Oct-Dec 2026 · DOI: 10.5530/pres.20260310

    ABSTRACT

    Background Cancer remains a formidable global health challenge, and the severe side effects and high costs of conventional treatments like chemotherapy necessitate the exploration of safer, more accessible therapeutic alternatives. Ayurveda, the traditional Indian system of medicine, offers a rich repository of herbo-mineral formulations with potential anticancer properties. This study investigates Swarnavari Bhasma, a novel Ayurvedic formulation, for its anti-carcinogenic potential. Aim The primary aim of this study was to prepare and standardize Swarnavari Bhasma and to assess its in vitro cytotoxic activity against a panel of human cancer cell lines. Materials and Methods Swarnavari Bhasma was prepared using traditional Ayurvedic Rasashastra methods, including Shodhana (purification) and Marana (incineration) of its core mineral components, Swarna Makshika (Copper Pyrite) and Kapardika (Calcium carbonate). The final formulation was created by integrating these Bhasmas with Haridra (Curcuma longa) and processing them with decoctions of Triphala, Pippali (Piper longum), and a Vincristine solution. The product was characterized using classical Ayurvedic tests (Bhasma Pariksha) and modern instrumental techniques including Field Emission Scanning Electron Microscopy (FESEM), X-ray Diffraction (XRD), Particle Size Analysis and Fourier Transform Infrared Spectroscopy (FTIR). The anti-carcinogenic activity was evaluated using the MTT cytotoxicity assay on four human cancer cell lines: MCF-7 (breast), A549 (lung), Hep G2 (liver), and PC-3 (prostate). 5-Fluorouracil (5-FU) was used as the standard positive control. Results Swarnavari Bhasma demonstrated significant, dose-dependent cytotoxic activity against all four tested cell lines. The formulation showed the highest efficacy on the MCF-7 breast cancer cell line (IC₅₀ = 65.14 µL/mL) and the lowest efficacy against the PC-3 prostate cancer cell line (IC₅₀ = 121.28 µL/mL). Analytical characterization confirmed the final product as a highly crystalline, multi-phase herbo-mineral composite with a unique porous, quasi-spherical micro-aggregate morphology and a significant nanoparticle population (mean size ~131.8 nm). Conclusion The study successfully demonstrates that Swarnavari Bhasma, prepared and standardized through a combination of classical and modern methods, possesses significant in vitro anti-carcinogenic properties. Its multi-targeted nature, derived from the synergy of its diverse components, makes it a promising candidate for further development as an integrative therapy in oncology.

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    INTRODUCTION

    Cancer, a multifactorial disease characterized by uncontrolled cell growth, is a leading cause of mortality worldwide. Projections indicate a substantial rise in cancer incidence in India, underscoring the urgent need for novel therapeutic strategies (Aggarwal et al., 2025). While conventional treatments such as chemotherapy and radiation have improved survival rates, they are frequently associated with debilitating side effects, high costs, and the development of drug resistance, limiting their overall effectiveness (Anand et al., 2023; Zafar et al., 2025). This has spurred interest in exploring alternative and integrative treatment options, including those from traditional systems of medicine like Ayurveda.

    Ayurveda offers a holistic approach to health, viewing cancer, or Arbuda, as a disease arising from an imbalance of the three fundamental biological energies (Tridoshas: Vata, Pitta, and Kapha) that corrupt the body's tissues (Dhatus), particularly muscle (Mamsa) and blood (Rakta) (AYUSH Cancer Conclave, 2019 Accepted Posters Papers with Abstracts 2019; Prajapati et al., 2024). Rasashastra, the branch of Ayurvedic pharmaceutics dealing with herbo-mineral preparations, utilizes specific processing techniques to enhance the therapeutic properties of metals and minerals while minimizing toxicity (Savrikar and Ravishankar, 2011; Wijenayake et al., 2014).

    This study focuses on Swarnavari Bhasma, a novel herbo-mineral formulation designed based on Ayurvedic principles. It utilizes Swarna Makshika (Chalcopyrite) and Varatika or Kapardika (Cowrie shell) as affordable and effective substitutes (Abhav Pratinidhi Dravya) for the traditionally used but costly Swarna (Gold) and Vajra (Diamond) Bhasmas (Sri Govinda Das. Edited by Shri Bramhashankar Mishra. “Bhaishajya Ratnavali”. 1st ed. Varanasi; Chaukhambha Sanskrit Bhavan; 2006. Pp.80. n.d.; Vaidya Lakshmipati shashtri, Yogaratnakara ed. With ’Vidyotini’ Hindi commentary by Bhimashankar Shastri published by chaukhamba prakashan, Varanasi, reprint-2010- Abhavavarga-verse no.38-page no.173 n.d.) These mineral bases are combined with a synergistic blend of potent herbs. Haridra (Curcuma longa), known for its active compound curcumin (Fuloria et al., 2022), Triphala, a classic rejuvenating antioxidant formulation (Bairwa et al., 2025) and Pippali (Piper longum), a well-known bio-enhancer (Unnikrishnan Meenakshi et al., 2024). The formulation is further processed with a Vincristine solution, integrating a known chemotherapeutic agent into the traditional matrix (Garg et al., 2024).

    The objective of this research was to prepare Swarnavari Bhasma according to classical Ayurvedic procedures, characterize its physicochemical and structural properties using modern analytical techniques, and evaluate its in vitro anti-carcinogenic activity against a panel of human cancer cell lines (Figures 1-9).

    Figure 1: Comparative IC₅₀ values of Swarnavari Bhasma and 5-FU across all tested cell lines.
    Figure 2: Dose-response curves for A549 cells. The graph shows the percentage of cell viability as a function of the concentration of Swarnavari Bhasma and 5-FU.
    Figure 3: Dose-response curves for MCF-7 cells. The graph shows the percentage of cell viability as a function of the concentration of Swarnavari Bhasma and 5-FU.
    Figure 4: Dose-response curves for Hep G2 cells. The graph shows the percentage of cell viability as a function of the concentration of Swarnavari Bhasma and 5-FU.
    Figure 5: Dose-response curves for PC-3 cells. The graph shows the percentage of cell viability as a function of the concentration of Swarnavari Bhasma and 5-FU.
    Figure 6: Morphological changes in A549 (Lung Cancer) cells. (A) Untreated control cells showing a healthy, confluent monolayer. (B) Cells treated with 5-FU showing significant cell death and detachment. (C) Cells treated with Swarnavari Bhasma (100 µL/mL) showing reduced cell density, rounding, and shrinkage.
    Figure 7: Morphological changes in MCF-7 (Breast Cancer) cells. (A) Untreated control cells. (B) Cells treated with 5-FU. (C) Cells treated with Swarnavari Bhasma (100 µL/mL), showing pronounced cytotoxicity.
    Figure 8: Morphological changes in Hep G2 (Liver Cancer) cells. (A) Untreated control cells. (B) Cells treated with Swarnavari Bhasma (100 µL/mL).
    Figure 9: Morphological changes in PC-3 (Prostate Cancer) cells. (A) Untreated control cells. (B) Cells treated with 5-FU. (C) Cells treated with Swarnavari Bhasma (100 µL/mL).

    MATERIALS AND METHODS

    Preparation of Swarnavari Bhasma

    The formulation was prepared in a multi-step process involving the preparation of intermediate components followed by their final integration. All raw drugs were authenticated prior to use.

    The Raw Swarna Makshika (SM) first underwent Shodhana (purification) via Nirvapa, where it was repeatedly heated to a red-hot state and quenched in a freshly prepared Triphala decoction (kwatha). Subsequently, the purified SM was subjected to Marana (incineration) for 12 times by triturating it with purified Gandhaka (sulphur) and Nimbu Swarasa (lemon juice), forming pellets, and heating them in an Electric Muffle Furnace at 650ºC until a fine reddish-brown Bhasma was obtained (Pathiraja et al., 2020).

    The Raw Kapardika (cowrie shells) were purified (Shodhana) by the Swedana (steaming) method in Kanji (sour gruel) using a Dolayantra for 3 hr. The purified shells were then incinerated (Marana) 3 times using a classical GajaPuta to obtain a fine, white Bhasma (Rasheed and Shivashankar, 2017).

    The prepared Swarnamakshik Bhasma (40 g), Kapardika Bhasma (40 g), and Haridra Churna (20 g) were combined. This mixture underwent three successive cycles of Bhavana (levigation), where it was triturated with Triphala kwath, Pippali Phanta (hot infusion), and a Vincristine solution, respectively, until completely dry after each cycle.

    Analytical Characterization

    The final product and its intermediates were analyzed using both classical and modern methods.

    The prepared Bhasmas were evaluated for classical quality parameters, including Varitara (floating on water), Rekhapurnatva (fineness to enter fingerprint lines), and Slakshnatva (smoothness) (Mohaptra and Jha, 2010; Pal et al., 2014).

    Standard parameters such as Loss on Drying (LOD), Total Ash, Acid-Insoluble Ash, and pH were determined as per the Ayurvedic Pharmacopoeia of India (Mangal et al., 2023).

    The final Swarnavari Bhasma was characterized using Particle Size Analysis (Dynamic Light Scattering), Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), and Field Emission Scanning Electron Microscopy (FESEM) to determine its particle size distribution, functional groups, crystalline structure, and surface morphology.

    Cell Lines and Culture

    Four human cancer cell lines were obtained from the National Center for Cell Sciences (NCCS), Pune-A549 (lung carcinoma), MCF-7 (breast adenocarcinoma), Hep G2 (liver carcinoma), and PC-3 (prostate adenocarcinoma). Each cell line was cultured in its specific growth medium supplemented with 10% Fetal Bovine Serum (FBS) and maintained in a humidified incubator at 37ºC with 5% CO₂ to ensure optimal growth conditions.

    In vitro Cytotoxicity Assay (MTT)

    The cytotoxic potential of the formulation was evaluated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and allowed to adhere for 24 hr. Subsequently, they were exposed to different concentrations of Swarnavari Bhasma (10, 40, and 100 µL/mL) and the standard reference drug 5-Fluorouracil (10, 40, and 100 µg/mL) for another 24 hr. After incubation, MTT reagent was added and plates were further incubated for 4 hr to enable viable cells to reduce MTT into insoluble purple formazan crystals. These crystals were solubilized using DMSO, and the absorbance was recorded at 570 nm to determine cell viability (Freimoser et al., 1999; Ghasemi et al., 2021; Tonder et al., 2015).

    Statistical Analysis

    Experiments were performed in triplicate. Cell viability was calculated as a percentage of the untreated control. Cell viability was expressed as a percentage relative to the untreated control group. The Half-Maximal Inhibitory Concentration (IC₅₀), representing the concentration of the sample required to inhibit 50% of cell proliferation, was derived from the corresponding dose response curves.

    RESULTS

    Pharmaceutical Preparation and Standardization

    The traditional pharmaceutical processes successfully transformed the raw minerals into fine Bhasmas. The Shodhana of Swarna Makshika resulted in a color change from brassy golden-yellow to blackish, indicating successful purification. The final Swarnavari Bhasma was a homogenous, brown powder with a characteristic bitter, astringent taste. The prepared Bhasmas passed all classical Bhasma Pariksha tests, confirming their fine particle size and proper preparation (e.g., Varitara, Rekhapurnatva).

    Physicochemical and Instrumental Characterization

    Physicochemical analysis showed low moisture content (LOD=2.1%) and high purity (Acid-Insoluble Ash=0.2%). A key finding was the high alkalinity of the intermediate Kapardika Bhasma (pH 12.1).

    FESEM images revealed a unique morphology of porous, quasi-spherical micro-aggregates formed by the sintering of smaller, irregular particles. Particle size analysis confirmed a bimodal distribution with a significant nanoparticle population (mean size 131.8 nm) and a larger microparticle population (mean size 6.67 µm).

    XRD analysis confirmed the final product was a highly crystalline, multi-phase composite. The dominant phases were identified as Calcite from Kapardika and Chalcopyrite from Swarna Makshika, validating the chemical transformation during processing.

    The FTIR spectrum provided a chemical fingerprint confirming the integration of all components. Key peaks corresponded to carbonate ions from Kapardika Bhasma (1407.38 cm⁻¹), hydroxyl groups from the herbal ingredients (3358.72 cm⁻¹), and a distinct ester carbonyl peak (1742.89 cm⁻¹) attributed to the Vincristine used in processing.

    In vitro Cytotoxic Activity

    Swarnavari Bhasma exhibited a clear dose-dependent cytotoxic effect on all four cancer cell lines, as evidenced by the detailed absorbance data from the MTT assay. As the concentration of the Bhasma increased, the mean absorbance, which corresponds to metabolic activity, decreased, indicating a reduction in cell viability. The formulation was most effective against the MCF-7 (breast cancer) cell line and least effective against the PC-3 (prostate cancer) cell line. While the standard chemotherapeutic agent 5-FU was significantly more potent in all cases, the intrinsic bioactivity of Swarnavari Bhasma was clearly established. The calculated IC₅₀ values are summarized in Table 1.

    Table 1: Summary of IC₅₀ Values against Human Cancer Cell Lines.
    Cell LineCancer TypeIC₅₀ of Swarna Vari Bhasma (µL/mL)IC₅₀ of 5-Fluorouracil (µg/mL)
    MCF-7Breast65.146
    Hep G2Liver70.9614.22
    A549Lung92.1425.27
    PC-3Prostate121.289.75

    The bar chart illustrates the differential sensitivity of the cell lines to the formulation, with MCF-7 being the most sensitive. Note the logarithmic scale on the Y-axis to accommodate the large difference in potency between the Bhasma and 5-FU.

    The detailed raw data and dose-response relationship for each cell line are presented below.

    Morphological Analysis of Cell Lines

    Morphological examination under a microscope confirmed the cytotoxic effects, showing cell rounding, shrinkage, and detachment from the culture plate surface in treated wells compared to the healthy, confluent monolayer in control wells (Tables 2-5).

    Table 2: Raw Absorbance and Viability Data for A549 (Lung Cancer) Cells.
    Treatment GroupConcentrationAbsorbance (570 nm) ReplicatesMean Absorbance% Cell Viability% Inhibition
    Control0 µL/mL2.2262.2252.2242.225
    Swarnavari Bhasma10 µL/mL1.8961.8951.8921.894
    40 µL/mL1.7531.7561.7591.756
    100 µL/mL0.9980.9970.9940.996
    5-Fluorouracil (Std.)10 µg/mL1.2631.2641.2641.263
    40 µg/mL0.9620.9630.9610.962
    100 µg/mL0.4260.4230.4210.423
    Table 3: Raw Absorbance and Viability Data for MCF-7 (Breast Cancer) Cells.
    Treatment GroupConcentrationAbsorbance (570 nm) ReplicatesMean Absorbance% Cell Viability% Inhibition
    Control0 µL/mL2.1072.1142.112.11
    Swarnavari Bhasma10 µL/mL1.4561.4521.4531.453
    40 µL/mL1.1121.1131.1161.113
    100 µL/mL0.8740.8760.8710.873
    5-Fluorouracil (Std.)10 µg/mL0.5120.5130.5150.513
    40 µg/mL0.410.4120.4160.412
    100 µg/mL0.3060.3070.3030.305
    Table 4: Raw Absorbance and Viability Data for Hep G2 (Liver Cancer) Cells.
    Treatment GroupConcentrationAbsorbance (570 nm) ReplicatesMean Absorbance% Cell Viability% Inhibition
    Control0 µL/mL2.3262.3252.3282.326
    Swarnavari Bhasma10 µL/mL1.8961.8951.8931.894
    40 µL/mL1.4561.4551.4521.454
    100 µL/mL0.8560.8520.8510.853
    5-Fluorouracil (Std.)10 µg/mL1.2531.2531.2541.253
    40 µg/mL0.8560.8510.8540.853
    100 µg/mL0.4210.4220.4230.422
    Table 5: Raw Absorbance and Viability Data for PC-3 (Prostate Cancer) Cells.
    Treatment GroupConcentrationAbsorbance (570 nm) ReplicatesMean Absorbance% Cell Viability% Inhibition
    Control0 µL/mL2.0322.0332.0342.033
    Swarnavari Bhasma10 µL/mL1.9631.9621.9651.963
    40 µL/mL1.7521.7531.7591.754
    100 µL/mL0.8540.8560.8510.853
    5-Fluorouracil (Std.)10 µg/mL1.1121.1131.1141.113
    40 µg/mL0.6890.6810.6830.684
    100 µg/mL0.5120.5130.5170.514

    DISCUSSION

    This study successfully prepared, standardized, and demonstrated the in vitro anti-carcinogenic potential of Swarnavari Bhasma. The results indicate that the formulation's bioactivity is not due to a single component but is an emergent property of the complex synergy between its herbo-mineral constituents, realized through traditional Ayurvedic processing.

    The analytical characterization provides a strong scientific basis for its potential mechanism. The presence of a significant nanoparticle population (131.8 nm) is crucial, as nano-sized particles are known to enhance cellular uptake, bioavailability, and therapeutic efficacy (Banerjee et al., 2016; Chenthamara et al., 2019; Hoshyar et al., 2016). The unique porous, micro-aggregate structure seen in FESEM images provides a large surface area, which may act as a carrier matrix for the active organic compounds, facilitating their delivery to cancer cells (Bhanja et al., 2018; Ezati et al., 2025; Shee et al., 2024).

    The cytotoxic effect is likely multifactorial. The formulation combines several agents with known anticancer mechanisms. The processing with Vincristine directly introduces a potent mitotic inhibitor that arrests cancer cells in the M-phase of the cell cycle, leading to apoptosis (Dhyani et al., 2022; Kothari et al., 2016). Curcumin from Haridra is known to induce apoptosis and suppress key pro-cancer signaling pathways like NF-κB and STAT3 (Sultana et al., 2021; Wang et al., 2019; Wilken et al., 2011). Triphala contributes potent antioxidants like gallic acid, which protect against oxidative DNA damage and can selectively induce apoptosis in cancer cells (Charucharana et al., 2025; Patra et al., 2020; Prasad and Srivastava, 2020; Sripunya et al., 2024). Piperine from Pippali increases the bioavailability of other active compounds, particularly curcumin, thereby potentiating the overall effect of the formulation.The high alkalinity of Kapardika Bhasma (pH 12.1) suggests a potential to neutralize the acidic tumor microenvironment, a condition known to promote tumor invasion and chemoresistance (Pratti et al., 2024).

    The differential sensitivity observed across the cell lines (MCF-7>Hep G2>A549>PC-3) suggests that the formulation's efficacy may be dependent on the specific molecular pathways active in different cancer types. The highest sensitivity in the ER-positive MCF-7 cell line is a particularly noteworthy finding that warrants further investigation.

    While Swarnavari Bhasma was less potent than the pure cytotoxic agent 5-FU, this is expected for a multi-component formulation designed for safety and synergy. Its significant intrinsic activity suggests its potential not as a replacement for chemotherapy, but as an adjuvant or integrative therapy. Its multi-targeted mechanism could help overcome drug resistance, enhance the efficacy of conventional drugs, or allow for reduced dosages, thereby mitigating dose-limiting toxicities.

    The primary limitation of this study is it’s in vitro nature, which cannot account for pharmacokinetics, metabolism, or interaction with the tumor microenvironment and immune system in a living organism.

    CONCLUSION

    This research provides a robust scientific validation for Swarnavari Bhasma as a potential anti-carcinogenic agent. The study successfully demonstrated that this rationally designed, standardized Ayurvedic herbo-mineral formulation exhibits significant, dose-dependent cytotoxic activity in vitro against human lung, breast, liver, and prostate cancer cells. The integration of classical pharmaceutical methods with modern analytical techniques confirmed its unique nano-micro particulate structure and complex chemical composition, providing a basis for its multi-targeted mechanism of action. These promising findings establish a strong proof-of-concept and justify further preclinical investigation, including in vivo animal studies, to evaluate its efficacy, safety, and potential as an integrative therapy in oncology.

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    Dudhat, K., & Wanjari, A. (2026). In vitro Evaluation of the Anti-Carcinogenic Activity of Swarnavari Bhasma: An Ayurvedic Herbo-Mineral Formulation. Pharmacognosy Research, 18(4), 1360–1368. https://doi.org/10.5530/pres.20260310