Introduction
The term “Nanotechnology” was first coined by the great scientist Norio Taniguchi in 1974. Studying and manipulating data under incredibly micro sizes is called nanotechnology. Nanotechnology is part of innovation in science and engineering that will transform many sectors such as medicine, biomedical, and biomaterial. Particles that are ranging between 1 and 100 nanometers in size are called nanoparticles. Nanoparticles due to their specific advantages such as biocompatibility, reduced toxicity, stability, permeability, and retention effect are widely used in cancer treatment and diagnosis. The biosynthesis of metal nanoparticles, such as gold and silver, has recently hooked the attention of various biomed and biotech fields.
The development of abnormal cells and their ability to divide uncontrollably often leads to the condition known as cancer. Cancer is a leading cause of death worldwide and accounts for nearly one in six deaths across the globe. Gold nanoparticles have attracted wide applications due to their electrical conductivity and high chemical and thermal stability. AuNPs are set to possess minimum cytotoxicity and also have the potential ability to serve as an antioxidant. It is also emerging as an important drug delivery vector due to minimal cytotoxicity when placed in an in vivo condition.
Effects of AuNP’s
The monolayer culture is made with 10% medium, dimethyl ethanol amine (DMEA), and fetal bovine serum (FBS) along with the antimicrobes. Then, the culture medium is incubated in a CO2 incubator at 37°C for growth. The dead cells are removed the next day by phosphate-buffered saline (PBS), and the live cells are detached using trypsin, which is a proteolytic enzyme. Now the cells are seeded in a 96-well plate along with the culture medium and allowed for growth. Now different concentrations of AuNPs are added to individual wells, and a calorimetric assay for accessing cell metabolic activity (MTT assay) is performed. MTT assay is a method used to detect cell toxicity against cancer and non-cancer cells.
● Cell viability refers to the proportion of healthy cells in a sample population.
Sample collection
Spices have high antioxidant content and are highly beneficial for human health. Spices such as clove, cinnamon, turmeric, and cardamom are rich sources of powerful antioxidants. Turmeric has antibacterial, antiviral, antifungal, antioxidant, and anticancer activities and can reduce the risk of various malignant diseases. Turmeric extract is highly cytotoxic to mammalian cells, and it is effective in reducing animal tumors. Curcumin from turmeric has been tried to treat cancer lesions in human participants.
Synthesis of nanoparticles
Biosynthesized nanoparticles from curcumin have higher efficacy in cancer cells and less toxicity toward normal cells. The biosynthesis of gold nanoparticles is considered to be more eco-friendly than the physical and chemical methods which are expensive. The nanoparticle size should range from 70 nm to 200 nm for efficient drug delivery. Gold nanoparticles synthesized from plant extracts have a particle size ranging from 15 to 80 nm and also show that the activity becomes quicker as they get smaller in size.
Objectives
● To synthesize the metal nanoparticle (gold) using cinnamon, clove, turmeric, and cardamom extracts.
● Screening of gold nanoparticles synthesized.
● To check the stability of AuNPs.
● Studying antioxidant activity.
● To characterize the synthesized nanoparticles (UV, TEM, EDX, XRD, and FTIR).
● In vitro cytotoxicity of AuNPs using normal cell line.
● In vitro anti-cancer activity of AuNPs against breast cancer.
Apoptosis analysis of AuNPs using a fluorescence microscope.
Experiment
Aim: To synthesize the gold nanoparticles from turmeric and to study their effects against the colon cancer cell lines (Figure 1).
Figure 1. The solution of cardamom, turmeric, clove, and cinnamon.
(I) Sample Collection
Materials required:
○ Fresh samples of cinnamon, clove, turmeric, and cardamom are collected and ground up to 70% fine powder.
Procedure: (Extraction)
2° g of powder is dissolved in 30 ml of distilled water and well mixed. The mixture is then boiled for 20 min at 60°C. The solution is filtered using filter paper.
1. Cardamom
2. Turmeric
3. Clove
4. Cinnamon
(II) Synthesis of Gold Nanoparticles
Materials required:
10° ml of different sample extracts.
Gold chloride (Aucl4) (Figures 2, 3).
Figure 2. Solution for synthesis of gold nanoparticles.
Figure 3. Synthesis of gold nanoparticle confirmed via UV-visible spectrometer.
Procedure:
(1) 8 ml of double distilled water is mixed with 2 ml of extract.
(2) To this 40 microliter of gold chloride solution is added.
(3) Test tubes are incubated for 24 h.
Stability analysis of AuNP’s using turmeric extract
● Antiviral. A recently published study in the Journal of General Virology describes how curcumin helps prevent transmissible gastroenteritis virus (TGEV).
● Antioxidant. When the body is exposed to free radicals or unstable molecules reacting to the environment and other triggers, cells become damaged.
● Anti-inflammatory. Help to reduce inflammation in the body.
● The antioxidant activity of synthesized stable gold nanoparticles is studied by DPPH scavenging activity and phosphomolybdenum assay.
● The DPPH scavenging activity of the sample was measured using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method.
Procedure
● 0.4 mM solution of DPPH in methanol was prepared, and 2 ml of this solution was added to different concentrations of the sample and was allowed to stand at room temperature for 20 min (Figures 4, 5).
Figure 4. Stability check of AuNPs synthesized from turmeric at different time antioxidants by DPPH scavenging activity.
Figure 5. DPPH scavenging activity of synthesized stable AuNPs.
● Absorbance was read at 517 nm against blank samples.
● The lower absorbance of the reaction mixture indicated higher free radical scavenging activity.
● The percentage of the DPPH radical scavenging is calculated.
● Radical scavenging activity (%) = (Abs control - Abs sample/Abs control) × 100 (Table 1).
Table 1. Scavenging activity of gold nanoparticles.
(I) Phytochemical analysis confirmed the presence of constituents such as alkaloids, carbohydrates, glycosides, saponins, proteins, phenol, flavonoids, and terpenoids.
● Carbohydrates:
○ Fehling’s test: One ml of filtrate was boiled in the water bath with 1 ml each of Fehling solutions I and II. A red precipitate indicates the presence of sugar.
○ Fehling’s solution I: Copper sulfate (34.66 g) was dissolved in distilled water and made up to 500 ml with distilled water.
○ Fehling’s solution II: Potassium sodium tartrate (173 g) and sodium hydroxide (50 g) were dissolved in water and made up to 500 ml.
● Proteins:
○ Biuret test–1 ml of the filtrate was treated with 2% of copper sulfate solution. To this 1 ml of ethanol (95%) was added followed by excess potassium hydroxide pellets. The pink color in the ethanolic layer indicates the presence of proteins.
● Terpenoids:
○ Salkowski test–1 ml of extract and 2 ml of chloroform were added and mixed well. A little concentrated H2SO4 was carefully added to form a reddish-brown layer.
● Steroids:
○ To 1 ml of extract, 2–3 drops of sulfuric acid are added. The blue color indicates its presence.
● Flavonoids:
○ 3 ml of dilute ammonia was added to 1 ml of filtrate followed by the addition of 1 ml of concentration of sulfuric acid. The yellow color shows the presence of flavonoids.
● Glycosides:
○ Borntrager’s test–To 1 ml of filtrate, 3 ml of chloroform was added and shaken. The chloroform layer was separated, and 10% ammonia solution was added to it. The pink color indicates the presence of glycosides (Figure 6).
Figure 6. Phytochemical analysis.
Carbohydrates −−−
Proteins −−−
Terpenoids + + +
Steroids −−−
Flavonoids + + +
Glycosides −−−
Cell toxicity
Toxicants that can cause cell death and serious organ dysfunction.
● Cells are seeded in a 96-well plate with an appropriate medium for growth.
● Different concentrations of gold nanoparticles are added to each well.
● Plate is incubated at 37°C for 24–36 h.
● Medium is removed, and plates are viewed under a microscope.
● PBS wash is done.
● 20 ml of MTT assay solution is added to each well.
● Viability of the cells is checked.
Cell line
Permanently established cell culture that can proliferate indefinitely given appropriate fresh medium and space.
● Check cells in a T flask under a microscope for their growth.
● Remove the culture media carefully.
● Add PBS for the removal of dead cells.
● Trypsin is added for the detachment of cells in the flask.
● Flask is kept undisturbed for 5 min.
● Check cells under the microscope to confirm the detachment of cells from the surface.
● Fresh medium is added for growth (usually 10% medium is used along with DMEA, FBS, and antimicrobes) (Figure 7).
Figure 7. Detachment of cells after trypsin treatment.
Result and conclusion
Synthesized gold nanoparticles from spices exhibit higher efficacy in destroying the cancer cells and inducing apoptosis while offering less toxicity toward normal cells is observed.
Author contributions
All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.
References
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2. Kuttan R, Sudheeran PC, Josph CD. Turmeric and curcumin as topical agents in cancer therapy. Tumori. (1987) 73:29–31. doi: 10.1177/030089168707300105
3. Liu XY, Wang JQ, Ashby CR Jr., Zeng L, Fan YF, Chen ZS. Gold nanoparticles: synthesis, physiochemical properties and therapeutic applications in cancer. Drug Discov Today. (2021) 26:1284–1292. doi: 10.1016/j.drudis.2021.01.030