INTRODUCTION:

Using plants for medicinal purposes stretches back to the earliest civilizations. Ancient Hindu texts like the Rigveda and Bhagawad Gita showing reference for plant-based healing practices. Ayurveda, the traditional Indian medical system, dedicates eight branches to the study of plants and their therapeutic applications, highlighting their deep connection to life science and well-being. Today, medicinal plants remain a vital part of healthcare systems across the globe, from developing nations to the most advanced economies.

Their continued use as medicine and dietary supplements underscores the enduring value of this natural bounty¹. Northeastern India boasts a rich tradition of using various Curcuma species for medicinal purposes. A thorough examination of existing literature has revealed six particularly significant Curcuma species used in ethnomedicine: Curcuma longa: This is the widely known turmeric, a popular spice with a long history of medicinal use. Curcuma caesia: Commonly called black turmeric, this species has unique properties valued in traditional medicine. Curcuma zedoaria: Known as zedoary, this Curcuma species has its own set of medicinal applications. Curcuma amada: Also known as mango ginger, this species offers distinct medicinal benefits. Curcuma angustifolia: This is the east Indian arrowroot, another Curcuma species with recognized medicinal uses. Curcuma aromatica: Widely known as wild turmeric, this species plays a significant role in traditional northeastern Indian medicine². Black turmeric is traditionally used for piles, leprosy, bronchitis, asthma, cancer, epilepsy, fever, wounds, impotency, toothache, and vomiting. This herb is applied externally to heal leprosy wounds, white spots on the skin, and wounds in general. ³. It has a wide range of medicinal qualities, such as anti-inflammatory, anti- tubercular, anti-asthmatic, anti-hyper triglyceridemic, antioxidant, antimicrobial, analgesic, anti-cancer, anti- ulcer, antibiotics, antiviral, antitumor, and anti-diabetic properties ⁴. These days, biological nanomaterials are a handy tool for drug carriers in the pharmaceutical and medical fields. Therefore, there is a pressing need for research to create ecological, biological, or eco-friendly processes for the synthesis of NPs. Scientists now prefer the green synthesis of nanoparticles over different synthesis techniques ⁵. Copper was the first metal used by humans on their journey of civilization. It has been a tradition to use copper in our daily lives since ancient times. Copper-based items are more commonly used in the modern world due to their affordability and versatility in various applications ⁶. Copper oxide nanoparticles (CuO NPs) have emerged as a captivating area of investigation in cancer research due to their potential anticancer properties and biocompatibility. While research in this field is still developing, initial studies have shown promising results. One exciting application of CuO NPs involves their incorporation into nanocomposites. These composite materials offer several advantages: controlled metal concentration, cost-effective production, reduced agglomeration, and incompatible polymers, such as alginate, are gaining particular attention as key components in these nanocomposites. Alginate's biodegradability and biocompatibility further contribute to the safety profile of these novel materials. ^{7, 8}. Cancer, a formidable foe, can invade any part of the body. It's characterized by the uncontrolled growth of abnormal cells with the ability to spread to other tissues. This devastating disease remains a leading cause of death globally, second only to heart disease. While various treatment options exist – radiation therapy, surgery, immunotherapy, and chemotherapy – chemotherapy often takes center stage due to its accessibility. However, this approach has limitations. Despite reaching affected areas with ease, chemotherapy comes with built-in drawbacks. The significant side effects of chemotherapy pose a major challenge ⁹. Breast cancer is classified into four distinct types based on molecular and histological evidence, including estrogen receptor-expressing (ER+), progesterone receptor-expressing (PR+), human epidermal receptor 2 expressing (HER2+), and triple-negative (ER-, PR-, HER2-). Standard treatments for breast cancer include lumpectomy, hormone therapy, radiation therapy, and chemotherapy. Hormone therapy is the most common treatment for hormone receptor expression type, and hormone receptor inhibitors are sometimes used. For HER2 type, monoclonal antibodies combined with chemotherapy are used, whereas triple-negative type presents significant challenges for treatment. The use of hormone receptor inhibitors and monoclonal antibodies in treating breast cancer has been observed to yield positive results. However, triple-negative breast cancer remains a particularly difficult type to treat, and new approaches are needed to improve outcomes for patients ¹⁰. Plants have played a significant role in the search for cancer-fighting drugs, but extracting and purifying active compounds can be challenging. However, with ongoing research and technological improvements, harnessing the power of plants has the potential to revolutionize cancer treatment in the future ¹¹. In this present study, copper nanocomposite is checked for anticancer effect against MCF-7 cell lines.

Materials and Methods Extract Preparation:

Curcuma caesia, or black turmeric, is purchased from local markert, authenicated and processed for its rhizomes. Five grams of black turmeric rhizomes (Curcuma caesia) were washed, finely crushed into a thin paste, then diluted with 100 mL of distilled water and stirred using a magnetic stirrer at a temperature of 60- 70℃ for 20 minutes. The mixture was then filtered. It was observed that the colour of the extract, which was initially a darker shade of yellow. The prepared extract was stored at 4°C for further use.

Synthesis of copper nanoparticles:

1M copper sulphate is prepared. For synthesising copper nanoparticle, copper sulphate and C. caesia extract were taken in different ratio (1:1, 1:2, and1:3) and heated in a mantle at 70°C for 30 minutes. The mixture was observed for any colour changes and turned to a dark green shade, indicating the successful synthesis of copper nanoparticles (CuNPs). The synthesized CuNPs were collected and washed thrice by centrifuging at 6,000 rpm for 10 minutes at 25°C. After washing, the copper nanoparticles were dried at room temperature for 12 hours. Also different parameters like pH (5.5, 7 and 8), and temperature (30, 50 and 70°C) were maintained to determine the effective synthesis of nanoparticle ^12,13.

Characterization of Copper nanoparticles:

For the characterization of the synthesized copper nanoparticles UV spectrometry, X-ray diffraction (XRD), and SEM analysis were done.

Copper alginate nanocomposite preparation:

Copper nanoparticles were immobilized by entrapment in calcium alginate beads. To prepare a 1% (w/v) solution of sodium alginate, sodium alginate was dissolved in deionized water and kept in a magnetic stirrer at ^{60°C to 75°C. A 200 ml solution of 0.1 M CaCl}2 ^{was
prepared by dissolving calcium chloride in deionized water.}Two grams of Cu nanoparticles were added to the solution to synthesize beads containing Cu. Using a micropipette, the sodium alginate solution was added dropwise to the Cu-calcium chloride solution and each drop ^{formed a bead on which Cu was
entrapped. The beads were maintained in CaCl}2^{solution at 4°C
overnight. The}beads were then rinsed with 250 ml of deionized water in a 500 ml conical flask. The excess water in the beads was removed by blotting with filter paper. The beads were washed with distilled water and dried. Finally, the beads were ground into powder using a mortar and pestle for characterization of the synthesized copper nanoparticles encapsulated in alginate beads. SEM analysis was performed ¹⁴.

Cytotoxicity Test:

The cytotoxicity effect of copper nanoparticles, Curcuma caesia extract, and copper alginate nanocomposite is checked by MTT assay using an MCF-7 cell line. MCF-7 cell lines were procured from National Center of Cell Sciences (NCCS), Pune, India and cultured in Dulbecco’s modified Eagle’s minimal medium. Cell survival is measured at 620nm by checking the reduction of MTT.

MTT Assay:

The fibroblast cells were cultured into 96-well plates, and drugs were applied to the cell monolayer. The cells were then observed for 24 hours to examine the morphology of the cells. Following this, the cells were twice washed with 1XPBS and incubated for three hours with 0.2 mg/ml of MTT (3 -(4,5-dimethylthaizole-2-yl)-2,5- diphenyl tetrazolium bromide). The purple-coloured product formed was then dissolved with isopropyl alcohol and the optical density was measured at 620nm using ELISA Reader ^{15, 16}.

Calculation for cell viability

OD sample

Percentage of cell viability (%) = --------- X 100

OD Control

Tryphan blue dye exclusion assay:

The cells were trypsinized using 0.25% trypsin-EDTA solution, then resuspended in phosphate buffered saline (PBS) and stained with 0.4% Trypan blue dye solution (v/v in PBS). After two minutes, the cells were placed in a Neubauer chamber, and the number of live and non-viable cells per 1 x 1 mm square was counted using a phase contrast microscope. Dead cells retained the blue dye due to membrane semipermeability and are thus colored, but viable or live cells remained unstained ¹⁷.

RESULTS AND DISCUSSION:

Nanomaterials have gained popularity in fields like biomedicine, catalysis, energy storage, and sensing due to their distinct physicochemical features compared to bulk materials. Silver nanoparticles, have generated a lot of interest, especially in biomedicine. AgNPs are widely recognized for their potent, broad-spectrum antibacterial and anticancer effects. AgNPs have also been studied for their potential biological effects on bone mending, wound healing, vaccination immunogenicity, and anti-diabetic effects. Greater effectiveness in medical applications will result from better understanding of the biological mechanisms and potential cytotoxicity of AgNPs ¹⁸.

Green nanoparticles are a more ecologically friendly alternative for treating cancer and antibiotic- resistant bacteria, as well as developing sensors to detect chemicals in biological samples. In an indirect biological application, nanoparticles generated with plants, bacteria, or fungi can be utilized as catalysts in the production of novel compounds having anticancer and antibacterial characteristics. Using these catalysts, we can lessen environmental pollution, which is a challenge in our time.

Nanoparticles have the potential to revolutionize medicine. Metallic nanoparticles produce oxidative stress due to their small size and reactive surface area, triggering biological reactions. Their primary method of action is to induce apoptosis by producing ROS and causing cell death due to oxidative stress. Nanoparticles biosynthesized from natural extracts is the scope for the development of nanoparticles in medicine. They have reduced toxicity for normal cells and stronger anti-tumour action for cancer cells. They also eliminate the toxicity caused by harmful compounds used to stabilize chemically produced nanoparticles. Gold and silver nanoparticles have cytostatic and bactericidal capabilities, and the degree of their activity is determined by the natural extract used—that is, the chemicals adsorbed on their surface. Biosynthesized palladium, copper, and platinum nanoparticles may serve as renewable catalysts for the synthesis of novel pharmaceuticals ¹⁹.

Copper nanoparticle (Figure:1) was synthesised using C. caesia extract, copper sulphate, and the formed nanoparticle was observed as green colour. In this synthesis process, various parameters like concentration, pH, temperature, and time period were standardized to check for the maximum nanoparticle synthesis. Three different concentration ratios of copper sulphate and C. caesia extract were added (Table: 1). It was found that the highest amount of nanoparticle synthesis was seen with 1: 1 ratio. In the remaining 2 concentration ratios, nanoparticle synthesis was less even though with the increased amount of copper sulphate. It may be due to the less availability of phytochemicals for reduction.

Table 1: Copper nanoparticle synthesis with variable concentrations

Concentration ratio	pH of copper sulphate	Temperature (℃)	Time period	Production of nanoparticles (ml)
1: 3	5.5	70	2days	0.5
1: 2	5.5	70	1 hour	1
1: 1	5.5	70	30 minutes	2

Similarly, the effect of pH in copper nanoparticles was checked by maintaining three different pH 5.5, 7and 8. It was found that the nanoparticle synthesis was high with acidic pH (5.5). Among the 3 different temperature (30, 50 and 70 ℃), the synthesis was high at 70 ℃.

Figure: 1 Copper nanoparticle synthesis

C. caesia Rhizome:

Recently, the development of carbon nanostructures coated with various types of nanoparticles has shown promise for a variety of applications. Copper materials, in particular, have been shown to alter cancer cell metabolism and cause cell death. Furthermore, research involving these nanostructures revealed the generation of reactive oxygen species (ROS) and DNA damage. As a result, delivering copper ions to the desired tissues with great efficiency and minimal loss is critical ²⁰. Globally, cancer burden is expected to reach 24 million individuals by 2035. The strong technological developments in using nanoparticles for medicinal purposes are the new hope for avoiding the impending health crisis. However, the single-modal drug delivery approach is limited by low bioavailability (about 5-10%), burst release, and low target efficiency. Recently, there has been a lot of interest in multifunctional theranostic nanoparticles that can release drugs in response to an external magnetic field and help with tissue regeneration, bioimaging (magnetic resonance imaging), and thermal ablation ²¹.

CuO-NPs have a greater potential for use because they are among the materials that are biocompatible and rarely toxic. Since CuO-NPs are less expensive than silver and gold-NPs and have a wider range of biological activities, they are now being reported for use in a variety of medicinal applications. Nevertheless, one major issue that needs to be addressed is the stability of the particles. The CuO-NPs free particles congregate into a bigger cluster due to the high surface energy of the NP, which results in NPs precipitation ^22,23.

Synthesised copper nanoparticle was characterised by UV-Spectrometry, XRD, and SEM analysis. UV- spectrometry result showed that the nanoparticle was having maximum absorption at 350nm. XRD analysis showed that the board peak was observed between 10-30, which indicates that the synthesised nanoparticle is amorphous in nature (Figure: 2, 3, 5). SEM analysis showed plate shaped nanoparticle formation. Sodium alginate beads observed by SEM showed spherical appearance (Figure :6). Copper alginate nanocomposite (Figure: 4) was analysed by SEM, which confirmed that the nanocomposite prepared was with the combination of copper nanoparticle and calcium alginate (Figure:7)

Figure 2: UV absorption spectrum of a copper nanoparticle.

Figure 3: XRD analysis of synthesised copper nanoparticle

Figure 4: Copper alginate nanocomposite preparation

Figure: 5 SEM analysis of Copper nanoparticle

Figure 6: SEM analysis of Sodium alginate beads.

Figure 7: SEM analysis of Copper alginate nanocomposite

Figure 8: MTT assay of extract, copper nanoparticle and copper alginate nanocomposit

p<0.05 was considered statistically significant

Mukuthan et al., 2017 ²⁴, reported the anticancer activity of hexane extract of dried rhizome of C. caesia. In their study, 3 cancer cell lines such as Hep 2, HepG2, and HT 29 were used to check the anticancer potential of C. caesia. It was found that the C. caesia hexane extract was effective against all the cancer cell lines in dose dose-dependent manner. Reenu et al., studied the anticancer effect of fresh and dried rhizome of black turmeric using six different solvent extracts against skin melanoma, colon carcinoma and lung adenocarcinoma cell line. They also found the dose-dependent effect of the extracts against cell line, dry rhizome extracts were more effective than fresh rhizome extract and among the extracts chloroform and ethylacetate extracts were shown an effective percentage of inhibition than the other extracts ²⁵.

The cytotoxicity effect of C. caesia extract, synthesized copper nanoparticle, and copper alginate nanocomposite was checked with MCF-7 cell line by MTT assay. Copper alginate polymer nanocomposite was prepared by following the protocol of Gangarapu and Arava, 2017. It was found that the prepared nanocomposite, synthesized copper nanoparticle, and plant extract were not toxic to normal cell lines. At the same time, all three (extract, nanoparticle, and nanocomposite) when checked against the MCF-7 cell line showed dose-dependent cytotoxicity (Figure: 8). Also, trypan blue dye exclusion study confirmed the same. It was found that the copper alginate nanocomposite was highly effective against the MCF-7 cell line, which was followed by synthesised copper nanoparticle and plant extract (Figure:9).

MCF-7 cell line treated with C. caesia extract

MCF-7 cell line treated with synthesized copper nanoparticle

MCF-7 cell line treated with copper alginate nanocomposite

Figure 9: Tryphan blue dye exclusion assay MCF-7 cell line treated with C. caesia extract

CONCLUSION:

Nanotechnology presents a promising avenue for advancements in anticancer therapies, particularly with the development of copper alginate nanocomposites. This study highlights a green and effective method of producing copper nanoparticles from Curcuma caesia extract by utilizing its abundant phytochemical content. Green synthesized copper nanoparticle incoorpated into sodium alginate polymer, could be the best choice of drug delivery strategy. Additionally, comprehensive characterization and cytotoxicity testing using the MTT assay are crucial steps in developing effective and targeted breast cancer therapies. The findings of the present study, demonstrate the potential of copper nanoparticles derived from Curcuma caesia, especially when combined with alginate, as effective agents in cancer therapy, providing a fresh, biocompatible method for the targeted treatment of cancer.

CONFLICT OF INTEREST:

There is no conflict of interest.

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Received on 24.06.2024 Revised on 20.11.2024

Accepted on 04.02.2025 Published on 01.10.2025

Available online from October 04, 2025

Research J. Pharmacy and Technology. 2025;18(10):4667-4672.

DOI: 10.52711/0974-360X.2025.00671

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

Vijaya Bharathi. S*, Dhanush Kumar S