Hydrothermally green synthesized ginger derived carbon nanodots showing antioxidant, catalytic reducing and anticancer properties*
Carbogenic carbon nanodots containing Curcuminoids and 6-gingerol layers with bulk of resonating non bonded electrons were synthesized using simple and green hydrothermal method from natural herb Ginger. As synthesized C nanodots were characterized using UV-Vis spectroscopy, IR, DLS, and TEM analysis. The antioxidant, catalytic reducing and anticancer properties of C dots were studied using ex vivo KMnO4 reduction assay, catalytic 4-nitrophenol reduction test, and in vitro MTT assay on MCF-7 cell line respectively. These carbogenic carbon nanoparticles shown quantum particle size of 4 nm. The green synthesized C dots shown excellent in vitro biological anti oxidant and anticancer properties along with reducing nature. This study exhibited the novelty of these green synthesized bioactive carbon nanodots for tagging and coating of bioactive materials for drug vectorization, biodetection, biocompatible cell targeting and biological applications.
Keywords: Ginger Carbon Dots, Antioxidant, Reducing, Anticancer, MCF-7 cell line.
Carbon nanodots (CNDs), as a new member of carbon nanomaterial family, have aroused great interest because of their outstanding water solubility, high sensitivity and selectivity to target analytes, low toxicity, favorable biocompatibility, and excellent photo stability1-3. Lots of methods for the production of CNDs have been reported such as hydrothermal and solvothermal technology and needs simple equipments. Due to their excellent fluorescence, CNDs have made impressive strides in sensitivity and selectivity to a diverse array of salt ions, organic/biological molecules and target gases4-8. The development of CDs as nano probes is still in its infancy, but continued progress may lead to their integration into environmental and biological applications. CNDs mainly have two major categories as carbogenic and graphitic carbon nanodots. These carbon nanomaterials can be derived from natural resources, biomolecules as carbohydrates, proteins using hydrothermal, solvothermal and microwave synthetic methods. As synthesized polymeric layer structured CNDs contain N, S, O hetero atoms with Carbon as main elemental composition with SP2 hybridization and along with conjugation and plenty of mobile electrons. Carbogenic carbon quantum dots or CNDs can be derived from natural herbs and wastes and contain mainly SP2 hybridization and conjugation of Carbon atoms and or with S, N, O atoms. CNDs are conjugated systems which have sp2 and sp3 hybridized carbons atoms with plenty of oxygen containing groups. CNDs obtained by the hydrothermal treatment reaction contains ionization, condensation, polymerization, and carbonization by bottom-up method. Such carbon dots may contain plenty of mobile electrons in polymeric layers limiting in size of 2 to 8 nm. and can show excellent non blinking photoluminescence and UV-VIS absorption of radiations9-17. So these CNDs can be used for coating biomaterials, nano probes, nano vectors for bio applications18-22. Ginger is one of the most widely used herb condiments in the world and is used as a traditional medicinal herb in eastern countries like India, China, due to its antioxidative, anti-inflammatory, and anti-carcinogenic properties. Curcumin, a hydrophobic polyphenol [(1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl) hepta-1,6-dienne-3,5-dione], is a yellow ingredient of ginger, which exhibits many biological activities such as antibacterial, anticancer, and hepatoprotective activities. Curcumin can inhibit the growth of the human cancer cells and change the cell-surface morphology, and trigger pro-apoptotic factor (e.g., mitochondrial damage and caspase activation) to promote cell apoptosis, with low toxicity to other cells. In addition to curcumin, 6-gingerol (a natural analog of curcumin; [5-hydroxy-1-(40-hydroxy-30-methoxyphenyl)3-decanone]) is another abundant constituent of ginger, which exhibits antimetastatic and anti-invasive pharmacological activities on cancer cells. Such active ingredients of ginger can be accommodated in quantum dot polymeric level by carbonization of ginger to CNDs for use of biological activities and applications. Here in this paper we have synthesized carbogenic carbon nanodots by use of natural herb ginger. After physicochemical characterization of these CNDs, their reducing nature, catalytic activities, biocompatibility, anti oxidant nature and anticancer potential have been checked by various biological screening tests.
2. Materials and Methods
All the chemicals used for synthesis of CNDs and their biological screening such as NaOH, KMnO4, 2,4-DNP, NaBH4, 4-Nitrophenol, Vitamin-C, 5-Fluorouracil, MTT reagent were of A. R. grade from S. D. fine chem. and Merck ltd. Cell culture medium DMEM, 10% fetal bovine serum, Human breast cancer cell line (MCF-7) were procured from NCCS center, Pune, India. The double distilled water from Millipore system was used throughout the synthesis and testing.
2.2.1. Hydrothermal synthesis of Carbogenic CNDs from ginger
The carbogenic carbon nanoparticles (CNDs) were synthesized using hydrothermal green method with some modification from natural herb ginger. In brief fresh tenders of rhizomes of ginger were purchased from local market and washed with boiled water. Then the surface cover of tenders peeled and cut into small pieces. These pieces were crushed by mortar and pestle and aqueous extract was taken in appropriate volume(25 ml.) in a beaker. 0.01 M NaOH (10 Ml.) and 15 ml. double distilled water was added in to beaker and basic extract was carbonized at 200oC for 3 Hours. over hot plate under air atmosphere. As formed carbon residue was diluted with double distilled water to 100 ml. and filtered through filter paper no.1. then finally the filtrate was dialyzed through dialysis membrane with porosity 2 nm. for 8 hrs. with stirring. The transparent brown liquid containing carbon nano dots stored in refrigerator for further use.
2.2.2. Structural and morphological characterization of CNDs
The structure, hybridization, morphology, particle size and types of atoms of CNDs were confirmed on the basis of physicochemical characterization on the basis of UV-VIS and IR spectral analysis, TEM, DLS analysis. Systronic double beam spectrometer was used for UV-VIS spectral analysis of CNDs with solution conc. of 10 µg./ml. in water prepared after drying CNDs suspension at 100oC with water as blank. TEM image and DLS scattering for particle size of CNDs determined with original CNDs aqueous suspension. IR spectra of CNDs determined using KBr pallet method on Perkin Elmer series spectrometer.
2.2.3. Reducing catalytic activity of CNDs
The reducing nature of CNDs along with catalytic activity was determined by reduction of 4-nitrophenol to 4-aminophenol in presence of CNDs with sodium borohydride (NaBH4). The role of CNDs on reduction rate was studied with UV-VIS spectrophotometry. The time required for reduction in presence of CNDs studied by wavelength scan spectra of 4-nitrophenol reduction to 4-aminophenol. Briefly, 2 ml. of 4-nitrophenol(0.01M) and 1 ml. NaBH4 (0.01M) with 1ml. water taken in cuvette and 1 ml. of 10 µg./ml. of CNDs added to this mixture. Suddenly UV-VIS spectra was recorded from 2 minutes after reaction up to 12 minutes. The online real time UV-VIS scan was performed until completion of reaction of 4-NP to 4-AP.
2.2.4. Antioxidant property of CNDs by ex vivo KMnO4 assay
The antioxidant activity of CNDs were tested by ex vivo KMnO4 reduction assay with Vitamin-C as standard control antioxidant drug by UV-VIS spectrometer optometric absorbance measurement. Briefly 5 ml. 0.01M KMnO4 reacted with 5 ml. 1mg./ml. CNDs in a hard glass test tube sealed at open end with cotton and incubated in dark for 30 min. at 37oC in CO2 environment of incubator. The absorbance of bare potassium permanganate solution was determined before and after incubation with antioxidant material as OD1 and OD2 respectively. Same test was performed for CNDs and Std. Vitamin-C as control for various concentrations of 0.12, 0.25, 0.5 and 1 mg./ ml. finally the percent antioxidant activity of material determined by using ex vivo assay formula as,
Percent antioxidant activity = OD1-OD2 / OD1 X 100 %
2.2.5. Anticancer potential of CNDs by in vitro MTT assay on MCF-7
Cancer cell cultures – MCF-7 (human breast cancer) cell lines were purchased from NCCS, Pune, India. All cell lines were grown and maintained in suitable (DMEM -media and were grown and subcultured in medium supplemented with 10% fetal bovine serum,1% L-Glutamine.1% penicillin streptomycin antibiotic solution. All cells were trypsinated using trypsin-EDTA solution and seeded in 96- well plates.
The newly synthesized CNDs were evaluated for their in vitro cytotoxic effects against MCF-7 (Breast cancer cell line), by the standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay using 5-FU (5-Fluorouracil) drug as a positive control in aqueous form.
The MCF cell line was maintained in DMEM medium supplemented with 10 % fetal bovine serum. The cells were plated at a density of 1 ? 105 cells per well in a 96-well plates, and cultured for 24 h at 37°C. The cells were subsequently exposed to 10 µM CNDs.The plates were incubated for 48 h, and cell proliferation was measured by adding 10µL of MTT (thiazolyl blue tetrazolium bromide) dye (5 mg ml-1 in phosphate-buffered saline) per well. The plates were incubated for a further 4 h at 37 °C in a humidified chamber containing 5% CO2. Formazan crystals formed due to reduction of dye by viable cells in each well were dissolved in 200 µl DMSO, and absorbance was read at 490 nm. The results were compared with the standard drug inhibitors 5 fluorouracil. (10µg/Ml.) Lastly percent cytotoxicity of CNDs was calculated by using following formula.
Percent Cytotoxicity = Reading of control – Reading of treated cells / Reading of control X 100
3. Results and Discussion
3.1. Morphological and structural characterization of carbon nanodots
3.1.1. IR analysis
The IR spectra of CNDs shown peaks in both the regions of functional and fingerprint signals. The functional group signal region of spectra exhibited the presence of aromatic and conjugated –OH groups, diketone, aromatic conjugated system, while fingerprint region of spectral signals shown presence of aromatic –OCH3 aromatic-H, -CH2 stretch, and presence of sp2 hybridized carbon groups. The IR signal frequency at 3486 cm-1 is due to the aromatic –OH groups. Peak at 2930 cm-1 show presence of conjugated diketonic group. Peak at 1644 cm-1 may be attributed to aromatic -OCH3. All other peaks of spectra in fingerprint area may be due to presence of conjugated –CH groups, aromatic-H, -C=O etc. (Fig.1). hence all these evidences prove the presence of curcuminoids and 6-gingerol layer of CNDs. Hence these CNDs are carbogenic carbon nanoparticles containing sp2 hybridized conjugated carbon atoms containing plenty of Pi and non bonded electrons with chain sizes in nanometer range.
3.1.2. UV-VIS absorption spectra of CNDs
The UV-VIS spectra of CNDs shows two absorption peaks at 210 nm. and 315 nm. and with long tailing in visible spectra. These observations clearly indicates the presence of Pi and non bonding electrons in carbogenic CNDs. The absorption peak at 210 nm. shows n to ?* transition and peak at 315 is due to ? to ?* transition and electron radiation relaxation (Fig. 2). Hence these spectral analysis indicates the presence of conjugated carbon system with n and ? electrons probably due to curcuminoids and 6-gingerol in CNDs with SP2 hybridized carbon in conjugation. So CNDs could contain aromatic conjugated natural carotenoid like diketonic molecular systems of these active ingredients of ginger.
The DLS scattering spectra of CNDs reveal that, the average particle size of the carbon nanoparticles is 4 nm. which matched with the TEM image of CNDs and with size of these carbon quantum nanoparticles. The sizes of CNDs varies from 2 to 12 nm. (Fig. 3), but maximum CNDs shows size between 2 to 6 nm. hence these are quantum dot carbon nanoparticles with abundance of mobile electrons responsible for light scattering in DLS and electron scattering in TEM. The TEM image of CNDs proved that there is some aggregation showing amorphous nature and circular morphology of CNDs material.
3.2.1. Reducing catalytic activity of CNDs by reduction of 4-NP to 4-AP
The reducing and catalytic nature of CNDs tested by reduction of 4-nitrophenol(4-NP) to 4-aminophenol(4-AP) in presence of NaBH4 by absorbance measurement with time lag of reaction. The initial absorption spectra of 4-NP and peak at 330 nm. vanished after reduction by CNDs in presence of NaBH4 as hydrogen source and CNDs as catalyst. After 12 min. 4-NP is totally converted to 4-AP showing new absorption spectra with peak at 460 nm. the catalytic reduction cycle of reaction completes after 12 min. UV-VIS real time online reaction wavelength scan performed from 2 min. to 12 min. until completion of reaction in cuvette. The formation of 4-AP take place with shifting and dampening of peak of 4-NP and formation of new peak of 4-AP in UV-VIS wavelength scan spectra (Fig.4).
Very good antioxidant activity is shown by CNDs derived from natural herb ginger compared with Vitamin-C by ex- vivo KMnO4 assay. The antioxidant activity determined for 0.12, 0.25, 0.5 and 1 mg./ml. concentrations of CNDs and Vit.-C as standard control is represented in Fig.5. The CNDs shows higher antioxidant activity than Vit.-C which increases with increase in concentration of drug. The color of KMnO4 fade after treatment of material and incubation in biological environment conditions, which elaborates the reducing as well as antioxidant nature of control Vit.-C and material CNDs. EC50 minimal inhibition concentration value or half reducing antioxidant activity of Vit.-C and CNDs determined by triplicate absorbance measurement are 0.62 and 0.48 mg./ml. respectively. So CNDs are better option for antioxidant material than Vit.-C for bio applications with minimum concentration for high antioxidant activity.
3.2.3. Anticancer potential of CNDs by in vitro MTT assay on MCF-7 cell line
MTT assay performed on MCF-7 human breast cancer cell line for anticancer potential of CNDs with 5-Fluorouracil as control drug shows moderate to good activity against these cells. As CNDs are reducing, antioxidant and contain free mobile electrons they can inhibit growth of MCF-7 by generation of ROS (Reactive Oxygen Species) at acidic pH inside cells. The cell viability of MCF-7 decreased by CNDs up to 64 % at concentration of 5 µg./ml. in sterile phosphate buffer saline with pH=7.4 and up to 78 % at 10 µg./ml. compared with 5-FU to 15 % at 5 µg./ml. and 19 % at 10 µg./ml. respectively. So the EC50 = 5 µg,/ml. shown by CNDs prove that a good candidate drug for anticancer application on selected cell lines without toxicity on normal cells.
The green, hydrothermal and cost effective synthesis of carbon nano dots from natural herb ginger has been reported in this paper. The synthesized CNDs had shown presence of layers of curcuminoids and 6-gingerol as drug ingredients from ginger with conjugation and plenty of mobile electrons. These CNDs had explored reducing catalytic, antioxidant, anticancer activities. Hence these carbogenic CNDs with 4 nm. mean quantum size exhibited important biological activities. So these water soluble CNDs derived from natural herb and in basic medium by carbonization process can be used as tagging and coating material on bioactive nanomaterials for cell vectorization or probing and for biocatalytic, antioxidant and anticancer applications. Overall the CNDs derived from natural herb ginger exhibit characteristics of:
better reducing, catalytic, antioxidant and anticancer activities
good water and phosphate buffer solubility hence these are bioavailable drug candidate
good stability and low toxicity on normal cells and moderate cytotoxicity on cancer cells
quantum dot size and presence of curcuminoids and or 6-gingerol natural multiactive drugs
So the CNDs can be better options for toxic quantum dot materials as CdS, and CdSe in biological applications.