Abstract
This study was aimed to determine the effectiveness of synthesized gold nanoparticles of an ethnobotanically and medicinally important plant species Cassia tora against colon cancer cells and to find its antibacterial and antioxidant activities. In order to improve the bioavailability of C. tora, we synthesized gold nanoparticles through green synthesis, by simple mixing and stirring of C. tora leaf powder and tetrachloroauric acid (HAuCl4) solution which gave a dispersion of gold nanoparticles conjugate with C. tora secondary metabolites (SMs) with characteristic surface plasmon resonance. It was characterized by Fourier transform infrared spectroscopy, zeta sizer, zeta potential and transmission electron microscopy. Antibacterial activity was carried out for gold nanoparticles conjugated with C. tora SMs, using well-diffusion method. The MTT assay for cell viability and markers such as catalase, nitric oxide and lipid peroxidation was predictable to confirm the cytotoxicity and antioxidant properties. The treatment of gold nanoparticles conjugated with C. tora SMs on Col320 cells showed reduction in the cell viability through MTT assay, and it also significantly suppressed the release of H2O2, LPO and NO production in a dose-dependent manner. C. tora SMs conjugate gold nanoparticles showed enhanced bioavailability, antioxidant and anticancer effect against colon cancer cell line (Col320).
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Introduction
The nature has provided the storehouse of remedies to cure many ailments of mankind. The traditional herbal medicines are still practiced in large part of our country mostly in tribal and rural areas. In many developing countries, large section of population relies on traditional medicinal plants since they contain active constituents that are used in the treatment of many human diseases (Stary and Hans 1998). Cassia tora Linn. Family: Caesalpiniaceae is an annual herb, 30–39 cm high, growing in India as wasteland rainy season weed. These herbs have been reported for their usefulness in the form of decoctions, infusions and tinctures in traditional system of medicines for treating skin diseases like psoriasis, leprosy and many more (Horvath 1992; Zahra et al. 2000; Cordova et al. 2002; Harrison 2003). C. tora was also reported for the presence of phytochemical such as anthraquinones, carbohydrates, glycosides, cardiac glycosides, amino acid, phytosterols, fixed oils and fats, phenolic compounds, tannins, flavonoids, steroids and saponins (Sathya and Ambikapathy 2012). And with high anthraquinone content, including chrysophanol, physcion and obtusin may aid in cancer prevention (Qi 2011). At present scenario, the pharmaceutical research has focused on the potent activities of C. tora, including its anti-aging, anticancer and antioxidant effects (Chen et al. 2014). Hence, the current study was intended to use C. tora as a bioreductant and capping agent for the synthesis of gold nanoparticles as a promising carrier and therapeutic drug for cancer.
The biologically inspired experimental processes for the synthesis of nanoparticles are evolving into an important branch of nanotechnology. Gold nanoparticles have advantages over other metal nanoparticles due to their biocompatibility and non-cytotoxicity. Nanoparticles are nanometers in size. Gold is used internally in human for the last five decades due to their chemical inertness. These are also used in chemotherapy and diagnosis of cancer cell (Cai and Chen 2007). Biological synthesis of nanoparticles appears to be simple, cost-effective, non-toxic and easy to use for controlling size, shape and stability, which is unlike the chemically synthesized nanoparticles (Gurunathan et al. 2014). Gold nanoparticles occur in various size ranges from 2 to 100 nm, but 20–50 nm particles size range shows the most efficient cellular uptake. Specific cell toxicity is shown by 40–50 nm sized particles. These 40–50 nm particles diffuse into tumors and can be easily recovered. But the larger particles 80–100 nm do not diffuse into the tumor and stay near the blood vessels (El-Sayed et al. 2006). These have a great extinction coefficient (Alvarez et al. 1997 ). Thus, gold nanoparticles have a great contribution in cancer therapy, diagnosis of cancerous cell and importance in the therapy of HIV (Bowman et al. 2008). Colon cancer is the third most common cancer and the third leading cause of cancer death in men and women. In this study, we took colon cancer cell line and treated it with AuNPs–C. tora SMs conjugate to find its anticancer activity.
Materials and methods
Collection of plant material
Leaves of C. tora were collected from Cuddalore and Poonamallee, Chennai, Tamil Nadu, South India. It was authenticated by a plant taxonomist Preetam Raj JP from the Department of Plant Biology and Biotechnology, Loyola College, Chennai.
Bio-reduction of gold nanoparticles by C. tora leaves
Glasswares were rinsed with deionized water before starting the synthesis. HAuCl4 was obtained from Sigma-Aldrich Chemicals. One gram of leaf powder was boiled in 10 ml of deionized water and subsequently filtered through Whatman No. 1 filter paper. An aliquot of 500 µl was taken and mixed with 99 ml of deionized water to that 1 ml of 1 mM HAuCl4 was added. The mixture was kept on magnetic stirrer for 24 h.
Characterization of synthesized AuNPs
UV–Visible spectroscopy studies
UV–Vis spectroscopy measurements of the HAuCl4 solution and C. tora SMs capped gold colloidal solution were carried out on a UV–Vis spectrophotometer (Model UV2450-Elico). Scanning was done from 200 to 800 nm.
Particle size analysis and zeta potential determination
The particle size ranges of the nanoparticles were determined using particle size analyzer (ZetasizerNano ZS-Malvern), and potential was measured using zeta potential. Particle sizes were calculated based on measuring the time-dependent fluctuation of scattering of laser light by the nanoparticles.
FT-IR spectroscopy measurements
After complete reduction of HAuCl4 ions by C. tora SMs, the colloidal gold nanoparticles were centrifuged at 12,000 rpm for 10 min at room temperature. The gold nanoparticle pellet obtained after centrifugation was resuspended in deionized water and centrifuged again to remove the traces of unbound leaf extract present in the solution prior to FT-IR analysis. FT-IR spectrum was taken to assess the involvement of possible capping by the plant extract (both aqueous and gold nanoparticles). Measurements were carried out on a Perkin–Elmer FT-IR Spectrum One spectrophotometer.
Dark field and HR-TEM measurements
Dark-field microscopy enables detailed studies of the plasmonic features of noble metal nanoparticles. AuNPs–C. tora SMs nominally sized 40–60 were used due to their stability. These particle qualities were diluted in essentially particle-free double-distilled H2O and mixed to obtain suitable bimodal particle suspensions (Wagner et al. 2014). Olympus BX51 microscope was used which was illuminated with dark-field condenser. TEM samples of the gold nanoparticles were prepared by placing a drop over carbon-coated copper grid and allowing the solvent to evaporate. TEM measurements were performed on a TECHAN Philips model 2400EX instrument operated at an accelerating voltage between 80 and 200 kV.
Antibacterial activity
Test microorganisms
Bacillus subtilis (MTCC 441), Enterococcus faecalis (ATCC 29212), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (MTCC 3615), Escherichia coli (ATCC 25922) and Proteus vulgaris (MTCC 1771) were used for the experiment.
Well-diffusion assay
Antimicrobial activity was carried out using well-diffusion method. Petri plates were prepared with 20 ml of sterile Mueller-Hinton agar (MHA) (Hi-Media, Mumbai) for bacteria. The test cultures were swabbed on the top of the solidified media and allowed to dry for 10 min. The aqueous extract of C. tora leaves and synthesized C. tora–AuNPs (100 and 150 µl) were loaded and left for 30 min at room temperature for compound diffusion. HAuCl4 3H2O was used as control. The plates were incubated for 24 h at 37 °C for bacteria to grow, and the zone of inhibition was measured in millimeters (mm). The experiment was repeated twice.
Anticancer activity
Col320 cell lines were obtained from National Centre for Cell Sciences, Pune, India, and grown on Dulbecco’s modified essential medium supplemented with 10 % (v/v) fetal calf serum, penicillin (100 μ/ml) and streptomycin (100 μg/ml). Cells were seeded in 24 well plates at a concentration of 3 × 104 cells/ml of DMEM/well and incubated for 48 h at 37° C under 5 % CO2 to attain confluence. The cells were then treated with various concentrations (25, 50 and 75 μg/ml). Then, the cells were incubated for 24 h, and cell supernatants (100 μl) were analyzed for leakage of catalase levels, using commercial spectrophotometric kits. The cells were used to analyze viability, NO production and lipid peroxidation. The experiments were carried out in triplicates in each group.
Estimation of cell viability—MTT assay
The MTT assay is a colorimetric assay for measuring the activity of cellular enzymes that reduce the tetrazolium dye, MTT, to its insoluble formazan, giving a purple color. MTT (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole) is reduced to purple formazan in living cells (Gerlier and Thomasset 1986; Mossman 1983). A solubilization solution (usually dimethyl sulfoxide, an acidified ethanol solution or a solution of the detergent sodium dodecyl sulfate in diluted hydrochloric acid) is added to dissolve the insoluble purple formazan product into a colored solution. The absorbance of this colored solution can be quantified by measuring at a certain wavelength (usually between 500 and 600 nm) by a spectrophotometer. Cell proliferation was measured using MTT assay. After 24 h of treatment, 20 μl of MTT solution (5 mg MTT in 1 ml PBS) was added per well and incubated at 37 °C for 4 h in 5 % CO2 atmosphere. Then, the medium was removed and washed with PBS; 200 μl of DMSO was added to each well. The intensity of the colored product was measured using an ELISA microplate reader at 570/620 nm. The results were expressed as the percent optical density of treated cells to that of the control cell.
Estimation of catalase
To 0.1 ml of sample, 1 ml of phosphate buffer and 1 ml of hydrogen peroxide was added, and the timer was started. The reaction was arrested by the addition of 2 ml dichromate–acetic acid reagent. Standard hydrogen peroxide (20 µM) was taken and treated similarly. The tubes were heated in the boiling water bath for 10 min. The green color developed was read at 570 nm using spectrophotometer.
Assay for NO production
Nitric oxide (NO) plays an important role in neurotransmission, vascular regulation, immune response and apoptosis. NO is rapidly oxidized to nitrite and nitrate which are used to quantitate NO production. NO is a reactive radical that plays an important role in many key physiological functions. NO, an oxidation product of arginine by nitric oxide synthase (NOS), is involved in host defense and development, activation of regulatory proteins and direct covalent interaction with functional biomolecules. Simple, direct and non-radioactive procedures for measuring NOS are becoming popular in research and drug discovery. Nitric oxide synthase assay involves two steps: a NOS reaction step during which NO is produced followed by an NO detection step. Since the NO generated by NOS is rapidly oxidized to nitrite and nitrate, the NO production is measured following reduction of nitrate to nitrite using an improved Griess method. The procedure is reduced to as short as 40 min. This assay determines nitric oxide based on the enzymatic conversion of nitrate to nitrite by nitrate reductase. The reaction is followed by a colorimetric detection of nitrite as an azo dye product of the Griess reaction. The Griess reaction is based on the two-step diazotization reaction in which acidified NO2 produces a nitro-sating agent which reacts with sulfanilic acid to produce the diazonium ion. This ion is then coupled to N-(1-naphthyl) ethylenediamine to form the chromophoric azo derivative, which absorbs light at 540 nm. After 24 h of incubation, the level of NO production was monitored by measuring the nitrite concentration in the supernatant of cultured medium using the Griess reagent.
Estimation of lipoperoxides
Quantification of lipid peroxidation is essential to assess oxidative stress in pathophysiological processes. Lipid peroxidation forms malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), as natural by-products. Measuring the end products of lipid peroxidation is one of the most widely accepted assays for oxidative damage. Lipid peroxidation assay provides a convenient tool for sensitive detection of the MDA in a variety of samples. The MDA in the sample is reacted with thiobarbituric acid (TBA) to generate the MDA-TBA adduct. The MDA-TBA adduct can be easily quantified colorimetrically (λ = 532 nm) or fluorometrically (Ex/Em = 532/553 nm). This assay detects MDA levels as low as 1 nmol/well colorimetrically and 0.1 nmol/well fluorometrically. After treatment, the cells were trypsinized, suspended in 0.5 ml of PBS and sonicated for 10 s. To this, 0.5 ml of TCA–TBA reagent was added and heated to 100 °C for an hour and centrifuged. The extent of lipid peroxidation was quantified by estimating the levels of malondialdehyde. The absorbance was measured at 535 nm, and the results were expressed as nmol/mg protein (Wu and Cederbaum 2008).
Results and discussion
Characterization of synthesized AuNPs
Plasmon resonance
It is a useful technique to study the optical and electrical properties of the materials based on Beer–Lambert law which governs the absorption of light by molecules. Upon mixing the HAuCl4 solution and C. tora leaf powder in deionized water, there was no significant change in the UV–Vis spectra (at 0 h), only the characteristic signature of C. tora leaf around 250 nm was seen, which we speculate may be due to complexation of Au3+ with C. tora leaf powder. Spectrophotometric absorption measurements in the wavelength ranges of 400–450 nm (Huang et al. 2007 ) and 500–550 nm (Shankar et al. 2004) are used in characterizing the silver and gold nanoparticles, respectively. As a function of time, we observed an emerging peak around 538 ± 2.82 nm (Fig. 1a), which was in the expected range. At this point of time, the color of the reaction mixture changed from gold to light purple color which is an indication of the formation of AuNPs. The formation of gold nanoparticles was visually confirmed by the color change (Fig. 1b). Such a color transition is often indicative of changes in the metal oxidation state (Fujiwara et al. 2007). The increase in the background may be due to the aggregation happened in the synthesized non-purified AuNPs–C. tora leaf conjugates (Choudhury et al. 2013). It is well known that the particles aggregate the background scattering. As one can observe the NPs formation after 2 h, however, clear light purple color was observed by 24 h, indicating the completion of the reaction. More studies are required to prove the intermediates involved in the reaction. Following the UV–Vis spectroscopic study, we carried out FT-IR study to elucidate the possible structural modification in the C. tora leaf molecule, followed by TEM imaging, and zeta potential measurements were done to find the size and charge present on the slip plane of AuNPs.
Zeta sizer and potential
Zeta potential is the key parameter that controls electrostatic interactions in particle dispersions (Kaszuba et al. 2010). The zeta sizer and potential of AuNPs–C. tora conjugate were 41 nm and −12.5 mV (Fig. 2a, b). The negative potential is considered as an incipient stability for colloids (Ostolska and Wisniewska 2014). The AuNPs–C. tora conjugate colloidal solution was found to be stable for 3 month under refrigeration.
FT-IR
The qualitative aspects of infrared spectroscopy are one of the most powerful attributes of this diverse versatile analytical technique (Coates 2000). It is useful for characterizing the surface chemistry (Chithrani et al. 2006). We carried out FT-IR measurements in order to decipher the structural modifications occurred in C. tora leaf upon the formation of AuNPs conjugate. Conversely, one may be able to identify indirectly which moiety of C. tora leaf is bound to the AuNPs. IR spectroscopy, a vibrational spectroscopic technique, is known for elucidating the finger print of chemical compounds. We observed conspicuous peaks around 3464.15, 2926.01, 2395.59, 2011.76, 1622.13, 993.34, 844.82 and a peak around 561.29 cm−1. The peaks around 3400 cm−1 are likely due to the alcohol OH stretch are usually a broad and strong absorption near 3400 and presence of residual water molecules in synthesized AuNPs. The peaks around 2750–3000 cm−1 are likely to originate from carbonyl C=O group which is present in the synthesized AuNPs. The peaks around 2300–2100 cm−1 indicate C≡X stretch region, the peaks around 1600–1750 cm−1 are likely due to the amide present in the synthesized AuNPs, and the peaks around 1500–400 cm−1 fingerprint region arise from complex deformations of the molecule. They may be characteristic of molecular symmetry, or combination bands arising from multiple bonds deforming simultaneously or the presence of peaklets at the lower wave numbers following it also could likely suggest the presence of aromatic C=C, which is present in synthesized AuNPs, also the disappearance of the peaklets at the lower wave number region after the formation of AuNPs suggests the possible structural change. Hence, peak around 1620 cm−1 is likely to be aliphatic (alkene) C=C also, whose absorption is converged with aromatic and amide absorption bands (Fig. 3a, b). Another potential structure is sharp, medium peak around 1400, and it signifies aromatic C=C group, but it shifted in synthesized AuNPs.
Dark field and HR-TEM
Dark-field microscopy is a widely unknown method to measure the particle size distribution of diffusing nanoparticles by particle tracking (Fig. 4a). TEM is used for morphological characterization at the manometer to micrometer scale. TEM measurements divulged that the resultant product after 24 h was comprised of nearly spherical AuNPs with 57 nm (Fig. 4b).
Antibacterial activity of synthesized AuNPs and aqueous C. tora leaf extract
The C. tora leaf may serve as a source of natural bactericidal agents to be used in medicinal systems (Koffi-Nevry et al. 2012). This study was designed to assess the antibacterial activity of synthesized AuNPs and aqueous C. tora leaf extract against gram-positive and gram-negative bacteria. The green synthesized AuNPs and aqueous C. tora leaf extract showed no inhibition against the gram-positive bacteria and gram-negative bacteria.
Anticancer activity
We intended to use AuNPs capped with C. tora leaf extract on cancer cells to observe the enhanced bioavailability and anticancer activity of C. tora focusing on colon cancer cell line. Col320 is widely used due to its relatively high steady state functioning of the free radical production and antioxidant defenses; therefore, variations of responses at different conditions are more easily detected.
Estimation of cell viability
The anticancer activity of the extract was confirmed by MTT assay. The measurement of cell viability plays a fundamental role in all forms of cell culture (Stoddart 2011). Promoting appropriate cell life and death is a key part of cell culture. When cells are put into contact with a SM, their viability may be affected. Some materials are cytotoxic, i.e., deadly to cells. In the present study, the treatment of Col320 cell line with the AuNPs capped with C. tora leaf extract suppressed the cell viability of cancerous cell up to (71.2 %) at (75 µg) concentrations when compared to the untreated cells (Fig. 5a). Cell morphological changes such as cell shrinkage, loss of surface contact and blebbing were observed in treated cancer cells Col320 cell lines after 24 h of incubation [Fig. 6a(i, ii)]. Cells were also observed in dark-field microscope [Fig. 6a(iii, iv)]. And we also checked the toxicity of the AuNPs capped C. tora leaf extract on normal cell line (Vero), and it is found that our sample does not affect the viability of normal cell (Fig. 5b). Hence, our results confirm that the C. tora leaf extract conjugated with AuNPs shows higher activity and can be used to enhance activity of colon cancer.
Catalase assay
To confirm the anticancer activity further, catalase was carried out; catalase is a ubiquitous antioxidant enzyme that degrades hydrogen peroxide into water and oxygen (Loewen et al. 1985), and H2O2 is very dangerous to the cells and tissues. In our study, when the concentration of the AuNPs capped C. tora leaf extract increased, it showed gradual increase in catalase enzyme, and when enzyme increases, it breaks H2O2 present in the cancerous cells, and hence, H2O2 is reduced into non-toxic molecules like water and oxygen (Fig. 6b).
Assay for NO production
Production of NO molecules is an important cytotoxic function that macrophages use to resolve infection by several obligate intracellular protozoan and bacterial parasites (De Groote and Fang 1995). The inducible form of the NO synthase enzyme (iNOS) produces large amounts of reactive nitrogen molecules by oxidizing the terminal guanidino nitrogen of l-arginine (Dong et al. 1995; Park et al. 1996). Since reactive nitrogen molecules can damage host tissues as well as the invading microbe (Park et al. 1996), nitric oxide at appropriate levels is involved in normal function of organs, whereas uncontrolled release can cause destruction of target tissue during inflammation and septic shock (Saravanan et al. 2012). The treatment with AuNPs–C. tora leaf conjugates (75 µg) significantly reduced the release of NO (69.12 µM) (Fig. 6c). Low level of NO is important in protecting organs; hence, our sample is capable for reducing NO in the cells and helps in protecting the cells.
Estimation of lipoperoxide
The major reactive aldehyde resulting from the peroxidation of biological membranes is malondialdehyde (MDA; Vaca et al. 1988). It is used as an indicator of tissue damage by a series of chain reactions (Ohkawa et al. 1979). Malondialdehyde, being a major breakdown product of lipid peroxides, is a useful index of lipid peroxidation (Saravanan et al. 2013). The treatment with AuNPs–C. tora leaf conjugates reduced the release of LPO (3.23). Thus, the study confirmed the anticancer property of AuNPs–C. tora through modulating the free radicals release and also established anti-inflammatory potential (Fig. 6d).
Conclusion
Aqueous extract of C. tora lacks in bioavailability and permeability, mainly due to its poor solubility of extract in water. To improve the bioavailability of C. tora, it is conjugated to the surface of metal nanoparticles. In the present study, we report the binding of C. tora to the surface of AuNPs. The nanoparticles were characterized by zeta sizer, TEM and FT-IR. We found that C. tora acts both as a reducing and capping agent, stabilizing the gold solution. The synthesized AuNPs–C. tora SMs conjugate showed enhanced bioavailability, antioxidant and anticancer effect against colon cancer cell line (Col320). To the best of our knowledge, this is the first report to describe the synthesis of biocompatible and soluble AuNPs with an average size of 41 nm using C. tora. Therefore, this study could provide valuable insight in using AuNPs capped C. tora leaf extract as a potential anticancer agent. Further, various parts of this medicinal plant could be explored using nanotechnology for assessing its therapeutic potential.
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Abel, E.E., John Poonga, P.R. & Panicker, S.G. Characterization and in vitro studies on anticancer, antioxidant activity against colon cancer cell line of gold nanoparticles capped with Cassia tora SM leaf extract. Appl Nanosci 6, 121–129 (2016). https://doi.org/10.1007/s13204-015-0422-x
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DOI: https://doi.org/10.1007/s13204-015-0422-x