INTRODUCTION:

Nanotechnology deals with the manipulation of materials at the nanoscale (1–100 nm), which possess unique physicochemical properties entirely distinct from those of bulk materials^1,2. The field of nanotechnology has revolutionized medicine through applications in cancer therapy, biosensing, targeted drug delivery, and diagnostics^3,4. Silver nanoparticles (AgNPs) have gained importance due to their broad spectrum of biological activities, including antibacterial⁵, antiviral⁶, antifungal⁷, anticancer⁸, anti-inflammatory⁹, antidiabetic¹⁰, and wound-healing effects¹¹.Their functional properties can be modified by controlling parameters such as size, shape, and surface chemistry.

Conventional chemical methods for nanoparticle synthesis are often associated with hazardous byproducts. Green synthesis provides an eco-friendly alternative by employing biological resources such as plants¹², bacteria¹³, fungi¹⁴, and algae¹⁵. Among these, plants are especially attractive for synthesis as they contain phytochemicals (e.g., flavonoids, terpenoids, phenolics) that act simultaneously as reducing and stabilizing agents¹⁶. Importantly, AgNPs have demonstrated the ability to generate reactive oxygen species (ROS), thereby inducing oxidative stress and apoptosis in cancer cells, making them promising candidates for cancer therapy and as alternatives to conventional antibiotics amid rising resistance^17,18.

Curcuma angustifolia, a traditional medicinal herb, has been used in treating skin disorders, respiratory ailments, and digestive problems¹⁹. To the best of our knowledge, this is the first report on the green synthesis of AgNPs using Curcuma angustifolia leaf extract. The synthesized nanoparticles were characterized and evaluated for their antioxidant potential, cytotoxicity, and anticancer efficacy against L929 (normal) and Hep-G2 (liver cancer) cell lines.

The objective of this study was to develop an eco-friendly approach for the synthesis of AgNPs using Curcuma angustifolia and to assess their biomedical potential, particularly their antioxidant and anticancer activities.

MATERIALS AND METHODS:

Materials:

All chemicals employed were of analytical grade. Millipore water was used for the preparation of all aqueous solutions. Distilled ethanol was used for Soxhlet extraction. Silver nitrate purchased from Merck Chemicals, India was used for the synthesis of silver nanoparticles. Ascorbic acid, Phosphate buffer, Potassium ferricyanide, Trichloro acetic acid, Ferric chloride purchased from Fischer scientific were used for antioxidant (FRAP) analysis. Dulbecco's Modified Eagle's Medium (Sigma), Foetal Bovine Serum(Thermo Fisher), Phosphate Buffered Saline (HiMedia),Trypsin EDTA (Thermo Fisher),12 well plates, Cell culture flask(T25cm²) (Tarsons), Penicillin-Streptomycin antibiotic solution (Sigma, India), MTT (HiMedia), Dimethyl sulphoxide (HiMedia), Hep-G2 cells (NCCS, Pune) were used for MTT assay.

Substrate Used:

Curcuma angustifolia, commonly known as East Indian arrowroot or narrow-leaved turmeric^20,21, is a member of the Zingiberaceae family and is native to Kerala, India. This perennial flowering plant features a rhizome and a pseudostem formed by the leaf sheath. Its leaves are arranged oppositely with parallel venation and have a scent similar to turmeric. The flowers are bisexual and zygomorphic²². This study focuses on utilizing Curcuma angustifolia leaves for the preparation of silver nanoparticles (AgNPs).

Preparation of Leaf extract:

The leaves were washed with double distilled water; small pieces of leaves were dried in shade²³ for 20days.10 gm of dried sample is subjected to Soxhlet extraction using 250ml of ethanol. After 48 hours, the extract is collected, cooled and filtered through Whatman number 1 filter paper and stored in refrigerator at 4⁰C.

Green Synthesis of AgNPs:

The bio synthesis of silver nanoparticles was carried out using1mM solution of AgNO₃and Curcuma angustifolia leaf extract. The two solutions were mixed in a 1:10 ratio at room temperature. A colour change from yellow to brown was observed, indicating the bio reduction of silver. The resulting colloidal solution was centrifuged, and the obtained AgNPs were dried. The phytoconstituents in the leaf act as capping agents, stabilizing the nanoparticles thus preventing aggregation.

Characterization of AgNPs:

The reduction of Ag+ ions was monitored by recording the UV-Visible spectra of the solution over a 24-hour period using a Shimadzu UV-1780 UV-Visible spectrophotometer. To examine the crystalline nature of the AgNPs, X-ray diffraction (XRD) studies were performed using a Shimadzu XRD-6000/6100, operating at 30 kV, 30 mA with Cu-Kα radiation at various 2θ angles. Transmission Electron Microscopy (TEM) analysis was conducted to determine the particle size distribution and average size of the nanoparticles using a JEOL JSM 2100 microscope with a LaB6 electron source, operating at 200 kV.

Antioxidant Assay -FRAP Assay:

Antioxidant activity of AgNPs was evaluated using Ferric Reducing Antioxidant Power (FRAP) assay. Stock solutions of AgNPs at concentrations of 0, 0.078, 0.156, 0.3125, 0.625, 1.25, 2.5, and 5 µL/mL, as well as standard ascorbic acid at concentrations of 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, and 50 µg/mL, were prepared, and 10 µL of each was used for the assay. To each sample and control 0.04ml of phosphate buffer (pH 6.6) and 0.05ml of 1% potassium ferricyanide [K₃Fe (CN)₆] solution were added. The reaction mixture was vortexed well and then incubated at 50°C for 20 min. At the end of the incubation, 0.5 ml of 10% trichloroacetic acid was added to the mixture. Then 50μl of deionised water and 50μl of 0.1% ferric chloride were added. The coloured solution was read at 700 nm against the blank using micro plate reader (iMark, BioRad). IC50 was calculated by using software Graph Pad prism 6²³.Radical scavenging activity (RSA) was calculated by the following formula,

A_control – A _sample

RSA = -------------------------------- x 100………….... (1)

A_control

RSA =Radical Scavenging Activity, A Control =Absorbance of Control and ASample=Absorbance of sample²⁴

In Vitro Anticancer Efficiency of AgNPs:

The anticancer activity and cytotoxicity of AgNPs were assessed using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl tetrazolium Bromide) assay^25-28 with human liver cancer cell line (Hep-G2) obtained from the National Centre for Cell Science (NCCS), Pune. Hep-G2 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS and 1% Penicillin-Streptomycin under conditions of 37°C, 5% CO₂, and 90% humidity. When the cells reached confluence, they were harvested using Trypsin-EDTA (0.25%) and seeded into multi-well plates for experimentation. All cell culture work was performed under sterile conditions.

Test samples were prepared by dissolving in 5% DMSO to achieve a final concentration of 1 µl/ml. The solution was filtered through a 0.22 µm Millipore syringe filter to ensure sterility. The test samples were then added to the culture medium to achieve final concentrations of 6.25µl/ml, 12.5µl/ml, 25µl/ml, 50µl/ml, and 100µl/ml. The culture medium without any test sample served as the cell control for the cytotoxicity assay. Confluent cells (2 days old) were trypsinized and suspended in 10% growth medium. A 100 µl suspension of 5 x 10³cells/well was seeded into a 96-well tissue culture plate and incubated at 37°C in a humidified 5% CO₂ incubator.

After 24 hours, the growth medium was removed; freshly prepared test samples in complete growth medium were added to achieve final concentrations of 6.25µl/ml, 12.5µl/ml, 25µl/ml, 50µl/ml, and100 µl/ml in 100µl of the medium in each well. The samples were added in triplicates to the respective wells and incubated at 37°C in a humidified 5% CO₂incubator. Control cells were maintained parallel to the test system.

For the MTT assay, 15 mg of MTT was reconstituted in 3 ml PBS and sterilized by filter sterilization. After 24 hours of incubation, the contents of the wells were removed and 30 µl of the reconstituted MTT solution was added to all test and cell control wells. The plate was gently shaken and incubated at 37°C in a humidified 5% CO₂ incubator for 4 hours. After incubation, the supernatant was discarded and 100 µl of MTT solubilisation solution (DMSO) was added to each well. The wells were gently mixed by pipetting up and down to dissolve the Formazan crystals. Absorbance values were measured using a micro plate reader at a wavelength of 540 nm

The percentage of growth inhibition was calculated using the formula³⁰:

Mean OD of Sample

% Of Cell viability = ------------------------- x 100…..(2)

Mean OD of Control

Mean OD of Sample =Absorbance of treated cells, Mean OD of Control =Absorbance of untreated cells

RESULTS AND DISCUSSION:

UV –Visible Spectroscopy:

The AgNPs exhibit a distinct surface plasmon resonance (SPR) peak in the range of 380 nm to 450 nm³¹. The position of this peak is influenced by the size and shape of the nanoparticles. When silver nitrate and leaf extract are mixed in a 1:10 ratio, the colour changes from yellow to brown. This colour change indicates the reduction of silver ions to nanosilver, a process facilitated by the phytochemicals present in the leaf. The UV-Visible absorption spectrum, as shown in Figure 1, displays a peak at 430 nm, which corresponds to the surface plasmon resonance band of AgNPs.

Figure 1: UV-Visible Spectra of Curcuma angustifolia leaf Extract and AgNPs

Powder -XRD Analysis:

Powder -XRD Analysis helps to analyze the crystalline nature of silver nanoparticles. The XRD pattern of Ag NPs (Fig 2) shows 5 distinct peaks .The 2θ values of the peaks are 38.10, 44.010, 64.380, 77.440 and 82.010.These five peaks correspond to (111), (200), (220), (311), (222)³² planes of face centred cubic structure of AgNPs with respect to Joint Committee on Powder Diffraction Standards (JCPDS), File No 04-0783.Peak corresponding to (111) plane is the more intense one. From this it is clear that the AgNPs are oriented in (111) plane.

Figure 2 XRD Spectrum of AgNPs

TEM:

Transmission electron microscopy (TEM) was employed to analyze the morphology, size, and shape of the synthesized AgNPs. The TEM images (Figure 3) revealed that the nanoparticles were predominantly flower-shaped and well-dispersed. A thin organic layer surrounding the particles was also observed which can be attributed to phytoconstituents from the C. angustifolia leaf extract acting as stabilizing agents. The particle size was found to range between 10 and 50 nm. The crystallinity of the sample was confirmed by the lattice fringes visible in the images. The Selected Area Electron Diffraction pattern (SAED) exhibited circular rings, which can be attributed to face centred cubic (fcc) lattice of AgNPs. The intense circular ring close to the centre is due to (111) reflections, the second ring is due to (200), third due to (220), fourth is due to (311) planes and the fifth one is due to (222) reflections. The clear circular ring in the image also suggests a high crystallinity of the synthesized nanoparticles (Fig 3d) ³³.

Figure 3 TEM images of AgNPs (a) 10 nm, (b & c) 50nm, (d) SAED

Antioxidant Assay-FRAP Analysis:

The human body is continuously exposed to an imbalance between antioxidants and pro-oxidants, a condition usually refereed to as oxidative stress. Excessive free radical generation has been implicated in the pathogenesis of numerous disorders, including gastric ulcers, Parkinson’s disease, Alzheimer’s disease, autism, diabetes, obesity, autoimmune disorders, renal and hepatic dysfunctions, cardiovascular diseases, and cancer. Antioxidants play a crucial role in counteracting oxidative stress by neutralizing free radicals and inhibiting their formation, thereby delaying or preventing cellular damage³⁴.

In recent years, considerable attention has been directed toward the green synthesis of metal nanoparticles using plant-derived materials rich in natural antioxidants. Such nanoparticles often exhibit enhanced antioxidant potential due to the synergistic effects of phytoconstituents involved in their synthesis. Antioxidant activity is commonly evaluated using in vitro assays such as 2,2-diphenyl-1-picrylhydrazyl (DPPH)³⁵ and ferric reducing antioxidant power (FRAP)^36,37.

In this study, the FRAP assay was employed to assess the antioxidant potential of AgNPs. This method is highly reproducible and widely accepted for evaluating the antioxidant capabilities of various compounds. In the FRAP assay, a colourless Fe³⁺ complex reacts with an antioxidant, reducing it to a blue-coloured complex. The results from various concentrations of the formulations (0–50) μg/ml) showed inhibition percentages ranging from 0 to 316.81, as depicted in Figure 4.

Figure 4: Antioxidant Activities of a) Standard Ascorbic acid b) AgNPs

Table 1: Antioxidant activity of AgNPs by using FRAP Method

Sample	Concentration	% of radical scavenging activity	IC50
Ascorbic acid	0	0	2.381 ± 0.022 μg/ml
	0.78	3.48
	1.56	28.70
	3.125	63.48
	6.25	94.78
	12.5	243.91
	25	790.87
	50	1200
AgNPs	0	0	0.6769 ± 0.14 μl/ml
	0.08	7.96
	0.16	11.95
	0.31	20.35
	0.63	40.27
	1.25	75.22
	2.50	150.00
	5.0	316.81

The antioxidant properties of the biosynthesized AgNPs can be attributed to the phytochemicals present in Curcuma angustifolia leaf extract, particularly flavonoids and phenolic compounds that act as capping and stabilizing agents. These phytoconstituents provide functional groups capable of donating electrons or hydrogen atoms, thereby neutralizing free radicals and reducing oxidative stress. The results of the FRAP assay demonstrated that the AgNPs exhibited strong, dose-dependent antioxidant activity (Table 1). The half-maximal inhibitory concentration (IC50), defined as the concentration required to scavenge 50% of free radicals, was calculated to be 0.6769 ± 0.14 µL/mL for AgNPs. In comparison, the IC50 for the standard antioxidant, ascorbic acid, was 2.381 ± 0.022 µg/mL. These findings clearly indicate that the AgNPs synthesized from Curcuma angustifolia possess a superior radical scavenging potential compared to the reference standard. The enhanced antioxidant activity may be explained by the presence of phenolic hydroxyl groups in the extract. These free –OH groups, located on aromatic rings, can readily donate protons or electrons to neutralize reactive free radicals, thus preventing the initiation and propagation of oxidative chain reactions^38,39. Additionally, the nanoscale dimensions and high surface-to-volume ratio of AgNPs may further amplify their interaction with free radicals, enhancing their overall scavenging capacity.

Cytotoxicity Studies and Anticancer Studies:

Anticancer Studies -MTT Assay:

In the current study, silver nanoparticles synthesized using Curcuma angustifolia leaf extract were evaluated for their anticancer activity against Hep-G2 cell lines (hepatocellular carcinoma), and cytotoxicity was assessed using the normal fibroblast-like L929 cell line, which was obtained from the subcutaneous connective tissue of a mouse. The cell viability was determined using the MTT assay, which measures the activity of mitochondrial dehydrogenase enzymes in viable cells. A decrease in cell viability indicates the anticancer efficacy of the AgNPs. Various concentrations of the AgNPs (6.25, 12.5, 25, 50, and 100 µg/ml) were tested. The results demonstrated a dose-dependent cytotoxic effect of AgNPs against Hep-G2 cells. As shown in Figures 5 and 6, cell death was minimal (12.71%) at the lowest dose of 6.25µg/ml, while it significantly increased at higher concentrations, reaching a substantial level at 100 µg/ml (Figure 5). Even at lower concentrations, cytotoxicity in Hep-G2 cells could be attributed to intracellular oxidative stress.The IC50 value for L929 cell line was found to be 87.66μg/ml whereas Hep-G2 shows IC50 of 50.43 μg/ml (Figure 6) . Since the lower IC50 value corresponds to greater toxicity, these results shows that silver nanoparticles are more toxic to Hep-G2 cancer cell lines than normal L929 cell lines. Figure 7 illustrates the morphological changes observed in the Hep-G2 cell lines.

Figure 5: Percentage of Cell viability and Cell death with concentration of AgNPs

Several studies have reported IC50 values for AgNPs synthesized from plant sources⁴¹, showing cytotoxicity against Hep-G2 cell lines in the range of 11-69 μg/mL. The variation in IC50 values may be attributed to differences in the bioactive compounds present in the plant sources, as well as the size, shape, and surface area of the AgNPs⁴². The exact mechanism underlying the cytotoxicity effect on Hep-G2 cell lines is not fully understood, but it may involve physicochemical interactions between the silver atoms and intracellular protons, as well as with the phosphate and nitrogen bases in DNA, ultimately leading to DNA damage⁴³.

Figure 6: Dose Dependant Cytotoxicity effect of Biosynthesized AgNPs on Hep-G2 Cell line by MTT Assay

Figure 7: Morphological Changes Induced By AgNPs on Hepato Cellular Carcinoma (Hep-G2) Cell line; (a) Before Treatment (b) After Treatment

CONCLUSION:

In conclusion, the present study successfully demonstrated the synthesis of unique flower-shaped silver nanoparticles using Curcuma angustifolia leaf extract. The results indicate that the AgNPs have a flower-like morphology. The antioxidant activity of the AgNPs was evaluated, revealing strong radical scavenging properties. Notably, AgNPs also displayed significant cytotoxicity against Hep-G2 cell lines.

CONFLICT OF INTEREST:

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

ACKNOWLEDGMENTS:

The authors would like to thank Amrita Vishwa Vidyapeetham, Amritapuri campus and Amrita Centre for Advanced Research in Ayurveda lab for providing research facilities.

REFERENCES:

1. Nasrollahzadeh M, Sajadi SM, Sajjadi M, Issaabadi Z. An introduction to nanotechnology. In: Interface Sci Technol. 2019; 1–27. https://doi.org/10.1016/b978-0-12-813586-0.00001-8

2. Choudhury A, Laskar RE, Deka D, Sonowal K, Saha S, Dey BK. A review on nanoparticle: Types, preparation and its characterization. Res J Pharm Technol. 2021; 14(3): 1815-22. https://doi.org/10.5958/0974-360x.2021.00322.x

3. Fritea L, Banica F, Costea T, Moldovan L, Dobjanschi L, Muresan M, Cavalu S. Metal nanoparticles and carbon-based nanomaterials for improved performances of electrochemical (bio)sensors with biomedical applications. Materials. 2021; 14(21): 6319. https://doi.org/10.3390/ma14216319

4. Alphandéry E. Nanomaterials as ultrasound theragnostic tools for heart disease treatment/diagnosis. Int J Mol Sci. 2022; 23(3): 1683. https://doi.org/10.3390/ijms23031683

5. Xue X, Wang Y, Yang H. Preparation and characterization of boron-doped titanianano-materials with antibacterial activity. Appl Surf Sci. 2012; 264: 94–9. https://doi.org/10.1016/ j.apsusc.2012.09.128

6. Kubo A, Rausalu K, Savest N, Žusinaite E, Vasiliev G, Viirsalu M, et al. Antibacterial and antiviral effects of Ag, Cu and Zn metals, respective nanoparticles and filter materials thereof against coronavirus SARS-CoV-2 and influenza A virus. Pharmaceutics. 2022; 14(12): 2549. https://doi.org/10.3390/ pharmaceutics14122549

7. Sidorowicz A, Margarita V, Fais G, Pantaleo A, Manca A, Concas A, et al. Characterization of nanomaterials synthesized from Spirulinaplatensis extract and their potential antifungal activity. PLoS One. 2022; 17(9): e0274753. https://doi.org/10.1371/ journal.pone.0274753

8. Ullah A, Saadullah M, Alvi F, Sherin L, Ali A, Shad NA, et al. Synergistic effect of silver doped ZnOnanomaterials enhances the anticancer potential against A459 lung cancer cells. J King Saud Univ Sci. 2021; 34(1): 101724. https://doi.org/10.1016/ j.jksus.2021.101724

9. Hwang SJ, Jun SH, Park Y, Cha S, Yoon M, Cho S, et al. Green synthesis of gold nanoparticles using chlorogenic acid and their enhanced performance for inflammation. Nanomedicine. 2015; 11(7): 1677–88. https://doi.org/10.1016/j.nano.2015.05.002

10. Sher N, Ahmed M, Mushtaq N. Plant-based synthesis of AuNPs using Hippeastrum hybridum (L.): Their ex vivo anti-acetylcholinesterase property. BioNanoScience. 2023; 13(4): 1766–78. https://doi.org/10.1007/s12668-023-01227-6

11. Solanki AK, Autefage H, Rodriguez AR, Agarwal S, Penide J, Mahat M, et al. Cobalt containing glass fibres and their synergistic effect on the HIF-1 pathway for wound healing applications. Front BioengBiotechnol. 2023; 11: 1125060. https://doi.org/10.3389/ fbioe.2023.1125060

12. Bao Y, He J, Song K, Guo J, Zhou X, Liu S. Plant-extract-mediated synthesis of metal nanoparticles. J Chem. 2021; 2021: 1–14. https://doi.org/10.1155/2021/6562687

13. Abbas R, Luo J, Qi X, Naz A, Khan IA, Liu H, et al. Silver nanoparticles: synthesis, structure, properties and applications. Nanomaterials. 2024; 14(17): 1425. https://doi.org/10.3390/ nano14171425

14. Rai M, Bonde S, Golinska P, Trzcińska-Wencel J, Gade A, Abd-Elsalam KA, et al. Fusarium as a novel fungus for the synthesis of nanoparticles: mechanism and applications. J Fungi. 2021; 7(2): 139. https://doi.org/10.3390/jof7020139

15. Alprol AE, Mansour AT, El-Beltagi HS, Ashour M. Algal extracts for green synthesis of zinc oxide nanoparticles: promising approach for algae bioremediation. Materials. 2023; 16(7): 2819. https://doi.org/10.3390/ma16072819

16. Zulfiqar Z, Khan RRM, Summer M, Saeed Z, Pervaiz M, Rasheed S, et al. Plant-mediated green synthesis of silver nanoparticles: synthesis, characterization, biological applications, and toxicological considerations: a review. BiocatalAgricBiotechnol. 2024; 57: 103121. https://doi.org/10.1016/j.bcab.2024.103121

17. Shreyash N, Bajpai S, Khan MA, Vijay Y, Tiwary SK, Sonker M. Green synthesis of nanoparticles and their biomedical applications: a review. ACS Appl Nano Mater. 2021; 4(11): 11428–11457. https://doi.org/10.1021/acsanm.1c02946

18. Prasad S, Gupta SC, Tyagi AK. Reactive oxygen species (ROS) and cancer: role of antioxidativenutraceuticals. Cancer Lett. 2016; 387: 95–105. https://doi.org/10.1016/j.canlet.2016.03.042

19. Sharma S, Ghataury SK, Sarathe A, Dubey G, Parkhe G. Curcuma angustifoliaRoxb. (Zingiberaceae): ethnobotany, phytochemistry and pharmacology: a review. J Pharmacogn Phytochem. 2019; 8(2): 1535–1540.

20. Jyotirmayee B, Mahalik G. Traditional uses and variation in curcumin content in varieties of Curcuma – the saffron of India. Ambient Sci. 2021; 9(1): 6–12. https://doi.org/10.21276/ ambi.2022.09.1.rv01

21. Elhawary EA, Moussa AY, Singab ANB. Genus Curcuma: chemical and ethnopharmacological role in aging process. BMC Complement Med Ther. 2024; 24(1): 43. https://doi.org/10.1186/ s12906-023-04317-w

22. Sandhyarani ND, Imotomba R, Thokchom R. Surface sterilization protocol for Curcuma angustifolia Roxb. micropropagation. J ExpBiolAgric Sci. 2018; 6(5): 890–894. https://doi.org/10.18006/ 2018.6(5).890.894

23. Yadav AR, Mohite SK. Antioxidant activity of Malvastrumcoromandelianum leaf extracts. Res J Top Cosmet Sci. 2020; 11(2): 59–61. https://doi.org/10.5958/2321-5844.2020.00010.2

24. Qubtia M, Ghumman SA, Noreen S, Hameed H, Noureen S, Kausar R, et al. Evaluation of plant-based silver nanoparticles for antioxidant activity and promising wound-healing applications. ACS Omega. 2024; 9(10): 12146–12157. https://doi.org/10.1021/ acsomega.3c10489

25. Lydia E, John S, Mohammed R, Sivapriya T. Investigation on the phytochemicals present in the fruit peel of Carica papaya and evaluation of its antioxidant and antimicrobial property. Res J PharmacognPhytochem. 2016; 8(4): 217–222. https://doi.org/ 10.5958/0975-4385.2016.00032.7

26. Joseph R, Azeez S, Bhageerathy C. Anticancer effect of sequential extracts from Curcuma caesia rhizomes on human cancer cell lines and characterization of selected polyphenols in active extracts by LC-MS/MS. Asian J Pharm Res. 2023; 13(4): 219–226. https://doi.org/10.52711/2231-5691.2023.00041

27. Singh MK, Prathapan A, Nagori K, Ishwarya S, Raghu KG. Cytotoxic and antimicrobial activity of methanolic extract of Boerhaaviadiffusa L. Res J Pharm Technol. 2010; 3(4): 1061–1063.

28. Menon S, Agarwal H, Rajeshkumar S, Kumar SV. Anticancer assessment of biosynthesized silver nanoparticles using Mucunapruriens seed extract on lung cancer treatment. Res J Pharm Technol. 2018; 11(9): 3887–3891. https://doi.org/10.5958/ 0974-360X.2018.00712.6

29. Nanditha R, Saravanan J, Praveen TK, Deepa S, Rymbai E. Evaluation of anti-cancer, antioxidant and antimicrobial activities of Alstoniascholaris L. Res J Pharm Technol. 2020; 13(9): 4153–4157. https://doi.org/10.5958/0974-360X.2020.00733.7

30. Swarnalatha Y. Isolation of flavonoids and their anticancer activity from Sphaeranthusamaranthoides in A549 cell line. Res J Pharm Technol. 2015; 8(4): 462–467. https://doi.org/10.5958/ 0974-360X.2015.00077.3

31. Sanganna B, Kulkarni AR. Antioxidant and anti-colon cancer activity of fruit peel of Citrus reticulata essential oil on HT-29 cell line. Res J Pharm Technol. 2013; 6(2): 216–219.

32. Barabadi H, Mobaraki K, Jounaki K, Sadeghian-Abadi S, Vahidi H, Jahani R, et al. Exploring the biological application of Penicilliumfimorum-derived silver nanoparticles: in vitro physicochemical, antifungal, biofilm inhibitory, antioxidant, anticoagulant, and thrombolytic performance. Heliyon. 2023; 9(6): e16853. https://doi.org/10.1016/j.heliyon.2023.e16853

33. Daphne J, Francis A, Mohanty R, Ojha N, Das N. Green synthesis of antibacterial silver nanoparticles using yeast isolates and its characterization. Res J Pharm Technol. 2018; 11(1): 83–92. https://doi.org/10.5958/0974-360X.2018.00016.1

34. Raj S, Mali SC, Trivedi R. Green synthesis and characterization of silver nanoparticles using Enicostemmaaxillare (Lam.) leaf extract. BiochemBiophys Res Commun. 2018; 503(4): 2814–2819. https://doi.org/10.1016/j.bbrc.2018.08.045

35. Anandalakshmi K, Venugobal J, Ramasamy V. Characterization of silver nanoparticles by green synthesis method using Pedalium murex leaf extract and their antibacterial activity. Appl Nanosci. 2015; 6(3): 399–408. https://doi.org/10.1007/s13204-015-0449-z

36. Kharat SN, Mendhulkar VD. Synthesis, characterization and studies on antioxidant activity of silver nanoparticles using Elephantopusscaber leaf extract. Mater SciEng C. 2016; 62: 719–724. https://doi.org/10.1016/j.msec.2016.02.024

37. Datir SB, Patel AM, Patel AK, Patil PP, Rohit FI, Thorat VD, et al. Evaluation of antioxidant activity of the aerial parts of Abutilon indicum (Linn) Sweet (Malvaceae). Res J Pharmacol Pharmacodyn. 2010; 2(5): 324–327.

38. Al Asaad N, Al Diab D. Determination of total antioxidant activity of fruit juices widely consumed in Syria. Res J Pharm Technol. 2017; 10(4): 957–962. https://doi.org/10.5958/0974-360X.2017.00174.3

39. Priya RS, Geetha D, Ramesh P. Antioxidant activity of chemically synthesized AgNPs and biosynthesized Pongamiapinnata leaf extract mediated AgNPs a comparative study. Ecotoxicol Environ Saf. 2015; 134: 308–318. https://doi.org/10.1016/j.ecoenv.2015.07.037

40. Madhanraj R, Eyini M, Balaji P. Antioxidant assay of gold and silver nanoparticles from edible Basidiomycetes mushroom fungi. Free RadicAntioxid. 2017; 7(2): 137–142. https://doi.org/10.5530/ fra.2017.2.20

41. Karpagam T, Firdous J, Revathy N, Priya S, Varalakshmi B, Gomathi S, et al. Anti-cancer activity of Aloe veraethanolic leaves extract against in vitro cancer cells. Res J Pharm Technol. 2019; 12(5): 2167. https://doi.org/10.5958/0974-360x.2019.00360.3

42. Shyamalagowri S, Charles P, Manjunathan J, Kamaraj M, Anitha R, Pugazhendhi A. In vitro anticancer activity of silver nanoparticles phyto-fabricated by Hylocereusundatus peel extracts on human liver carcinoma (HepG2) cell lines. Process Biochem. 2022; 116: 17–25. https://doi.org/10.1016/j.procbio.2022.02.022

43. Gunatilaka A, Kingston DG. DNA-damaging natural products with potential anticancer activity. Stud Nat Prod Chem. 1997; 21: 457–505. https://doi.org/10.1016/s1572-5995(97)80036-1

Received on 06.05.2025 Revised on 09.09.2025

Accepted on 03.12.2025 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2303-2309.

DOI: 10.52711/0974-360X.2026.00331

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.