IN-VITRO CYTOTOXIC STUDY OF NATURAL POLYSACCHARIDE ISOLATED FROM T. CORDIFOLIA FRUIT

IN-VITRO CYTOTOXIC STUDY OF NATURAL POLYSACCHARIDE ISOLATED FROM T. CORDIFOLIA FRUIT

Noopur Verma and Ajay Kumar Gupta *

School of Pharmaceutical Science (formerly University Institute of Pharmacy), Chhatrapati Shahu Ji Maharaj University (formerly Kanpur University) campus, Kanpur, Uttar Pradesh, India.

ABSTRACT: The polysaccharide-derived polymers can play an integral and versatile role in the novel drug delivery system for a variety of therapeutic agents in the biological system. In this research, natural polysaccharide isolated to evolve natural polymer-based innovative drug delivery carriers to provide maximum therapeutic effect and minimize clinical complications, which could also be a step towards biological environment-friendly drug delivery systems since the envisaged natural polymer could be a good substitute compared to the synthetic polymers currently used for novel drug delivery systems with modified drug delivery properties. Tinospora cordifolia is a tropical herb of the Menispermaceae family, also known as 'Guduchi', well-known for its therapeutic benefits in the Indian traditional system. This plant's fruits cluster in groups of one to three, and the mucilage contains complex polysaccharides.  In-vitro cytotoxicity studies on fibroblast cells revealed about 80% cell viability, confirming its biocompatibility as a natural excipient with potential applications in green and advanced pharmaceutical drug delivery systems.

Keywords: Polymer, Polysaccharide, Tinospora cordifolia, NDDS, Cytotoxicity

INTRODUCTION: Polymer-based drug delivery systems have emerged as a major focus in contemporary pharmaceutical research due to their capacity to enhance drug stability, regulate release profiles, and improve therapeutic efficacy ¹. Polymers are extensively employed as carriers in advanced delivery platforms such as nanofibers, nanoparticles, hydrogels, and microspheres. However, before these systems can be deemed suitable for biomedical applications, their biological compatibility must be rigorously assessed ².

In this context, in-vitro cytotoxicity studies serve as a critical step in evaluating the safety of polymers and drug-loaded formulations. In-vitro cytotoxicity testing involves examining the toxic effects of materials on cultured cells under controlled laboratory conditions ^{3, 4}. These studies provide essential insights into cell viability, proliferation, morphology, and metabolic activity following exposure to polymers, drugs, or drug-polymer combinations. Since polymers in drug delivery systems directly interact with biological tissues, it is imperative to determine their potential to induce cellular damage, inflammation, or growth inhibition ^{5, 6}. Primarily, cytotoxicity evaluation must consider not only the polymer itself, but also the final drug polymer formulation. Drug polymer interactions can significantly influence toxicity, either mitigating harmful effects through controlled release or exacerbating them due to chemical incompatibility. Thus, in-vitro studies are indispensable for optimizing polymer concentration, drug loading, and formulation parameters to achieve maximal therapeutic benefit with minimal adverse cellular effects. Another key role of cytotoxicity studies is screening novel biodegradable and biocompatible polymers. Materials such as poly (lactic acid), polycaprolactone, chitosan, alginate, and polyethylene glycol are routinely tested to confirm their safety in supporting cell growth ⁷. These evaluations also align with regulatory requirements, as cytotoxicity testing is a prerequisite before advancing to in-vivo studies and eventual clinical applications. Overall, in-vitro cytotoxicity studies are a foundational step in developing polymer-based drug delivery systems. They provide crucial information on safety, compatibility, and therapeutic potential, thereby guiding researchers in the rational design of effective and biocompatible formulations for pharmaceutical and biomedical use.

MATERIAL AND METHOD: Dulbecco’s modified Eagle’s medium (DMEM), trypsin-EDTA solution, penicillin–streptomycin–Amphotericin solution, and fetal bovine serum (FBS) were procured from Himedia Laboratories Pvt. Limited, India.

Tinospora cordifolia fruits were collected from a neem tree planted in the health center of the Indian Institute of Technology (IIT), Kanpur, Uttar Pradesh (India) in the months of April-June. Authentication of plant material was carried out by Dr. K.M. Prabhukumar, Senior Scientist and Herbarium Curator, Plant Diversity, Systematics and Herbarium Division of CSIR-National Botanical Research Institute, Lucknow (PDSH/LWG/Authentication/Ang./2024-25/06) dated 08 April 2024. Authenticated plant fruits were then washed and dried in the shade for further use.

Antioxidant Studies: The Antioxidant activity of the mucilage of Tinospora cordifolia fruit was evaluated by the radical-scavenging of the DPPH (diphenyl-2-picryl-hydrazyl) method. A 1mg/ml solution of mucilage with various concentrations (60-100 ug/ml) was mixed with an equal amount of 0.1 mM DPPH in methanol and incubated in the dark at room temperature for 30 minutes. The absorbance was measured at 517 nm against methanol as a blank. A control containing methanol and DPPH was prepared, and Ascorbic acid was used as a reference standard ^{8, 9, 10}. The percentage inhibition was calculated using the following formula:

% inhibition = [(A _control − A_sample) / A_control] × 100

In-vitro Cytotoxic Study: The biocompatibility of the T. cordifolia fruit mucilage was assessed using the indirect leach- out method according to ISO 10993-5 standards (Biological evaluation of medical devices—Part 5, 2009), employing NIH3T3 fibroblast cells. For preparing the conditioned medium, mucilage samples were incubated with serum- free Dulbecco's Modified Eagle's Medium (DMEM) at a concentration of 10 mg/mL for 24 hours at 37 ° C. After incubation, the mixture was centrifuged at 1000 rpm for 10 minutes at room temperature, and the supernatant was collected and filtered through a 0. 22 µm syringe filter. Two serial dilutions were made from the 10 mg/mL stock to obtain concentrations of 1 mg/mL and 0. 1 mg/mL. Each filtrate was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin to prepare complete conditioned media.NIH3T3 fibroblasts were seeded at a density of 1. 1.5 × 10 ⁴ cells per well in 48- well plates and incubated for 24 hours to promote cell attachment. The culture medium was then replaced with 200 µL of conditioned medium corresponding to each concentration, and the cells were further incubated for 48 hours at 37 ° C in a humidified atmosphere containing 5% CO₂. All experiments were performed in triplicate ^11-15.

Cell viability was evaluated using the Alamar Blue assay. After exposure to the conditioned media, 200 µL of 0. 01 mg/mL Alamar Blue reagent was added to each well and incubated for 4 hours at 37°C. Fluorescence intensity was recorded at excitation and emission wavelengths of 530 and 590 nm, respectively, using a Biotek Gen 5 microplate reader. Results were expressed as the percentage of viability relative to untreated controls as shown in Fig. 1. For qualitative analysis, cytocompatibility was further assessed by live/dead staining with Calcein- AM and Propidium Iodide (PI).

Viable cells emitted green fluorescence (Calcein- AM positive), while non- viable cells showed red fluorescence (PI positive). Stained cells were observed using a fluorescence microscope, and images were analyzed with Image J software over 48 hours at 37°C in a humidified atmosphere with 5% CO₂. All experiments were conducted in triplicate.

FIG. 1: SCHEMATIC DIAGRAM OF THE ALAMAR STUDY

RESULT AND DISCUSSION:

Antioxidant Activity: The antioxidant properties of the isolated fruit pulp mucilage were evaluated at concentrations ranging from 60 to 100 µg/mL, demonstrating a concentration-dependent increase in percentage inhibition for both ascorbic acid (R² = 0.9918) and the Tinospora cordifolia fruit pulp mucilage (R² = 0.9958). Ascorbic acid consistently showed higher inhibition percentages across all concentrations, increasing from 77% at 60 µg/mL to 90% at 100 µg/mL Fig. 2. In contrast, acetone-precipitated mucilage exhibited moderate antioxidant activity, with inhibition levels rising from approximately 30% at 60 µg/mL to 55% at 100 µg/mL. The IC₅₀ value for ascorbic acid 1.64 µg/ml reflects its strong free radical scavenging ability, requiring a relatively low concentration to achieve 50% inhibition.

Meanwhile, the IC₅₀ value for Tinospora cordifolia fruit mucilage (10.54 µg/mL) indicates measurable antioxidant activity due to a reduction in the polyphenolic and flavonoid constituents, subsequently decreasing free radical scavenging activity.

FIG. 2: ANTIOXIDANT ACTIVITY OF ISOLATED MUCILAGE

In-vitro Cytotoxic Study: Cell viability of fibroblast (NIH3T3) cells exposed to mucilage-infused culture media was assessed using the Alamar Blue assay. It was observed that treatment with conditioned media at concentrations of 0.1, 1, and 10 mg/mL did not significantly reduce cell viability compared to the untreated control Fig. 3. In all tested concentrations, cell viability remained above 80%, indicating that the mucilage extract exhibited no cytotoxic effects, which is consistent with the ISO 10993-5 criteria for biocompatibility. Live/dead fluorescence imaging further confirmed these results, showing a predominance of green-fluorescent viable cells with minimal red-fluorescent dead cells across all treatment groups ^{15, 19, 20}. The results demonstrated that the mucilage is non-cytotoxic and biocompatible toward fibroblast cells, highlighting its potential as a polysaccharide-based carrier for drug delivery and as a regenerative component in biomedical formulations.

FIG. 3: (A). MICROSCOPIC IMAGES OF FIBROBLAST (NIH3T3) CELLS IN MUCILAGE DILUTIONS, NO DILUTION AND CONTROL (A, B, C, D) BEFORE ALAMAR BLUE ASSAY. FLUORESCENCE IMAGES OF LIVE CELLS (E, F, G, H) AND DEAD CELLS (I, J, K, L) ARE SHOWN IN GREEN AND RED, AND CONTROL. (B) RESULT OF THE INDIRECT LEACH-OUT METHOD USING ALAMAR BLUE ASSAY FOR 48 H

CONCLUSION: The present research successfully isolated a natural polymer from the fruit pulp mucilage of Tinospora cordifolia, a widely recognized medicinal herb in the Ayurvedic system. The extraction process yielded a polysaccharide-rich mucilage using alkaline extraction, followed by purification and characterization. In extraction and purification some solvents were used that can be toxic and cause cellular damage, beyond basic safety, these assays are vital for mapping complex dose-response dynamics, determining therapeutic windows, and understanding cellular mechanisms of action. The Alamar Blue assay and free radical scavenging activity demonstrated that the mucilage-based formulation maintained high cell viability, indicating its cytocompatibility and safety for biomedical applications. These findings demonstrate the potential of Tinospora cordifolia derived polymers as promising natural excipients for use in novel drug delivery systems. Their ability to provide controlled and/or targeted release of therapeutic agents makes them attractive alternatives to synthetic polymers. Moreover, this approach aligns with the growing demand for sustainable, biocompatible, and environmentally friendly materials in the pharmaceutical, cosmeceutical, and food industries. This study represents a significant step toward the development of innovative, nature-derived drug delivery platforms.

ACKNOWLEDGEMENT: The authors gratefully acknowledge the Vice-Chancellor of Chhatrapati Shahu Ji Maharaj University, Kanpur, for providing the institutional support and facilities essential for this research. The authors also sincerely thank Prof. Vivek Verma, IIT Kanpur, for his consistent support throughout the study. In addition, the authors appreciate (Dr) Shreya and Ms. Harsita, for their timely support and contributions in the completion of this work

CONFLICTS OF INTEREST: The authors declare no conflict of interest.

REFERENCES:

1. Liechty WB, Kryscio DR, Slaughter BV and Peppas NA: Polymers for drug delivery systems: Annual Review of Chemical and Biomolecular Engineering 2010; 1:149-73. DOI: 10.1146/annurev-chembioeng-073009-100847
2. Lu H, Zhang S, Wang J and Chen Q: A review on polymer and lipid-based nanocarriers and its application to nano-pharmaceutical and food-based systems. Frontiers in Nutrition 2021; 8: 783831.doi: 10.3389/fnut.2021.783831
3. Mary DL, Lawrance A, Felicita AE and Arockianathan PM: Characterization and antioxidant effect of mucilage in leaves from Cocculus hirsutus. Bioinformation 2024; 20:439.doi: 10.6026/973206300200439
4. Wichayapreechar P, Charoenjittichai R, Prasansuklab A, Vinardell MP and Rungseevijitprapa W: Exploring the In Vitro Antioxidant, Anti-Aging, and Cytotoxic Properties of Kaempferia galanga Rhizome Extracts for Cosmeceutical Formulations. Cosmetics 2024; 11: 97.https://doi.org/10.3390/cosmetics11030097
5. Kurutas EB: The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state. Nutrition Journal 2015; 15: 71. DOI: 10.1186/s12937-016-0186-5
6. Sazonova EV, Chesnokov MS, Zhivotovsky B and Kopeina GS: Drug toxicity assessment: cell proliferation versus cell death. Cell Death Discovery 2022; 8: 417.doi: 10.1038/s41420-022-01207-x
7. Wong KH, Lu A, Chen X and Yang Z: Natural ingredient-based polymeric nanoparticles for cancer treatment. Molecules 2020; 25(16): 3620. doi: 10.3390/ma18214960
8. Vulić J, Čanadanović-Brunet J, Ćetković G, Tumbas V, Djilas S, Četojević-Simin D and Čanadanović V: Antioxidant and cell growth activities of beet root pomace extracts. Journal of Functional Foods 2012; 4(3): 670-8. DOI:10.1016/j.jff.2012.04.008
9. Oh S and Kim DY: Characterization, antioxidant activities, and functional properties of mucilage extracted from Corchorus olitorius polymers 2022; 14(12): 2488.doi: 10.3390/polym14122488
10. Ahmad Nejhad A, Alizadeh Behbahani B, Hojjati M, Vasiee A and Mehrnia MA: Identification of phytochemical, antioxidant, anticancer and antimicrobial potential of Calotropis procera leaf aqueous extract. Scientific Reports 2023; 13(1): 14716. doi: 10.1038/s41598-023-42086-1
11. Põhako-Palu K, Lorenz K, Randmäe K, Putrinš M, Kingo K, Tenson T and Kogermann K: In-vitro experimental conditions and tools can influence the safety and biocompatibility results of antimicrobial electrospun biomaterials for wound healing. Plos one 2024; 19(7): 0305137. doi: 10.1371/journal.pone.0305137.
12. Negroiu G, Piticescu RM, Chitanu GC, Mihailescu IN, Zdrentu L and Miroiu M: Biocompatibility evaluation of a novel hydroxyapatite-polymer coating for medical implants (in-vitro tests). Journal of Materials Science: Materials in Medicine 2008; 19(4): 1537-44. doi: 10.1007/s10856-007-3300-6. Epub 2007 Nov 8.
13. Longhin EM, El Yamani N, Rundén-Pran E and Dusinska M: The alamar blue assay in the context of safety testing of nanomaterials. Frontiers in Toxicology 2022; 4: 981701.doi: 10.3389/ftox.2022.981701.
14. Sandjo LP, Zingue S, dos Santos Nascimento MV, de Moraes MH, Vicente G, Amoah SK, Dalmarco EM, Frode TS, Creczynski‐Pasa TB and Steindel M: Cytotoxicity, antiprotozoal, and anti‐inflammatory activities of eight curry powders and comparison of their UPLC‐ESI‐QTOF‐MS chemical profiles. Journal of the Science of Food and Agriculture 2019; 99(6): 2987-97. https://doi.org/10.1002/jsfa.9512
15. Ahmad N: In-vitro and in-vivo characterization methods for evaluation of modern wound dressings. Pharmaceutics. 2022; 15(1): 42. https://doi.org/10.3390/pharmaceutics15010042
16. Rawat V, Dewangan S and Tiwle R: Characterization, Antimicrobial Activity and Antioxidant Properties of Grewia tiliifolia Root Mucilage as a Binder Excipient. Research Journal of Pharmacy and Technology 2025; 18(7): 3037-44. 10.52711/0974-360X.2025.00435
17. Yang J, Kim JS, Kwon YS, Seong ES and Kim MJ: Antioxidant and antiproliferative activities of Eclipta prostrata (L.) L. extract and isolated compounds. Molecules 2023; 28(21): 7354.https://doi.org/10.3390/molecules28217354
18. Chandimali N, Bak SG, Park EH, Lim HJ, Won YS, Kim EK, Park SI and Lee SJ: Free radicals and their impact on health and antioxidant defenses: A review. Cell Death Discovery 2025; 11(1): 19. https://doi.org/10.1038/s41420-024-02278-8
19. Qiao Z, Kim JY, Zhang K, Park CO and Shin Y: Chia Seed Mucilage-Based Bilayer Sponges Containing Zinc Oxide Nanoparticles for Wound Dressing. ACS Applied Bio Materials. 2026 Feb 11. https://doi.org/10.1021/acsabm.5c02565
20. Amhan SA, Khalaf YH and Mohammed AM: Eco-friendly zinc oxide nanoparticles from Opuntia ficus-indica: a dual antibacterial and anticancer approach. Results in Chemistry 2025; 102648. https://doi.org/10.1016/j.rechem.2025.102648
    

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    How to cite this article:

    Verma N and Gupta AK: In-vitro cytotoxic study of natural polysaccharide isolated from T. cordifolia fruit. Int J Pharm Sci & Res 2026; 17(4): 1340-45. doi: 10.13040/IJPSR.0975-8232.17(4).1340-45.

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