DOI: http://dx.doi.org/10.26510/2394-0859.pbe.2017.35

Research Article

Green synthesis of silver nanoparticles from Morinda tinctoria Roxb and scrutiny of its multi facet on biomedical applications

Geetha Paramasivam, Revathy Kannan, Sugapriya Menaga Paulraj, Jeyaraj Pandiarajan*

Department of Biotechnology, Ayya Nadar Janaki Ammal College, Sivakasi, Tamil Nadu, India

*For correspondence

Dr. Jeyaraj Pandiarajan,

Department of Biotechnology, Ayya Nadar Janaki Ammal College, Sivakasi, Tamil Nadu, India.

Email: pandiarajan@ anjaconline.org

 

 

 

 

 

 

 

Received: 21 August 2017

Accepted: 26 September 2017

ABSTRACT

Objective: This research work focus on synthesis of silver nanoparticles from Morinda tinctoria Roxb leaves. The synthesis of nanoparticles from biological processes is evolving a new era of research interests in nanotechnology. Silver nanoparticles are usually synthesized by chemicals. M. tinctoria Roxb leaves are reputed plant in traditional system of medicine and it is used for the treatment of illness such as arthritis, cancer, gastric ulcer and other heart disease etc.

Methods: The present study the leaves were collected, air dried and subjected to synthesis of silver nanoparticles (MtNps). The obtained nanoparticles from M. tinctoria Roxb leaves was characterized and antimicrobial activity, antidiabetic and anticancer activity was analyzed by standard procedures.

Results: M. tinctoria silver Nanoparticles study showed the evaluation of antioxidant and anticancer activity. Antioxidant activities were done using DPPH antioxidant assay and hydrogen peroxide assay. The anticancer study was conducted to evaluate the in-vitro anticancer activity of green synthesized nanoparticles using human hepatic carcinoma cell lines (HepG2).

Conclusions: M. tinctoria silver Nanoparticles evaluation showed higher antioxidant activity and anticancer activity in M. tinctoria Roxb.

Keywords: Silver nanoparticles, M. tinctoria leaves, Antimicrobial, Antioxidant, Anticancer

Introduction

Recently, metal nanoparticles have gained a lot of attention due to their unique chemical, optical, magnetic, mechanical, and electric magnetic properties. Thus metallic nanoparticles are used in different applications such as electronics, catalysis and photonic.1 The silver metal has a great toxicity against a wide range of microorganisms, particularly; silver nanoparticle which has promising antimicrobial properties. Silver nanoparticles are found to be effective as anti-inflammatory, anti-angiogenesis, antiviral, anti-platelet activity and against cancer cells which makes them vital.2-7 Accordingly, an environmental process for the synthesis of silver nanoparticles is important. Plant extract solutions and bio-organisms have been in spot light for their extreme ability to synthesis nanoparticles, including silver and gold nanoparticles.8 Biosynthesis of silver nanoparticles has already been reported as clean, cost effective and non-toxic to environmental routes. Green synthesis offers improvement over synthetic, chemical or micro-organisms methods as it is cost effective, environmentally friendly and can easily be scaled up for large scale synthesis.9 The methods used for the synthesis of silver nanoparticles and toxic chemicals are used for the reduction process of substances such as citrates, NaBH4, or ascorbates.

Morinda tinctoria (MTR) belongs to the family of Rubiaceae that grows wild and is distributed throughout Southeast Asia. It is commercially known as Nunaa and is indigenous to tropical countries. MTR is considered as an important folklore medicine. The tribes of Australia have used the ripe fruits of MTR for the treatment of respiratory infections. Further, it has been reported to have a broad range of therapeutic and nutritional values. There is a greater demand for its fruit juice in treatment for arthritis, cancer, gastric ulcer and heart diseases.10 The major components identified in the Nunaa plant include octoanic acid, potassium, vitamin C, terpenoids, scopoletin, flavones glycosides, lineoleic acid, anthraquinones, morindone, rubiadin, and alizarin.11 M. tinctoria are used as astringent, deobstrent, and to relive pain in the gout.12

There is a greater demand for fruit extract of Morinda species in treatment for different kinds of illness such as arthritis, cancer, gastric ulcer and other heart disease. Anti convulsant, analgesic, anti-inflammatory, anti-oxidant activity and cytoprotective effect of M. tinctoria leaves has been reported.13,14

Figure 1: Photography of M. tinctoria leaves.

Materials and Methods

Collection of plants     

M. tinctoria Roxb plant was collected from Vemboor of Tuticorin District, Tamil Nadu.

Preparation of leaf extract

Fresh and healthy leaves were collected locally and rinsed thoroughly first with tap water followed by distilled water to remove all the dust and unwanted visible particles, cut into small pieces and dried at room temperature. About 10 g of these finely incised leaves of each plant type were weighed separately and transferred into 250 mL beakers containing 100 mL distilled water and boiled for about 20 min. The extracts were then filtered thrice through Whatman No. 1 filter paper to remove particulate matter and to get clear solutions which were then refrigerated (4°C) in 250 ml Erlenmeyer flasks for further experiments.

Synthesis of silver nanoparticles

10 ml of leaf extract was added into 90 ml of aqueous solution of 1 Mm silver nitrate (AgNo3) for the reduction of silver nitrate into Ag+ ions and kept in dark room at 37o C for 24 hours. After 24 hours, color of the solution changed from green to dark brown indicating the formation of silver nanoparticles. The bioreduced silver nanoparticles solution was measured using UV- Visible absorbance.

Purification of silver nanoparticles

The silver nanoparticles solution thus obtained was purified by repeated centrifugation at 7000 rpm for 20 minutes. It is followed by redispersion of the pellet in deionized water to get rid of any uncoordinated biological molecules.

Characterization of silver nanoparticles

UV- Visible Spectrophotometry

Initial characterization of silver nanoparticles was carried out using UV-Visible spectroscopy. Change in color was visually observed in the silver nitrate solution incubated with leaf extract of M. tinctoria Roxb. The bio-reduction of precursor silver ions was monitored by sampling of aliquots (silver nanoparticles diluted with distilled water) at different time intervals. Absorption measurements were carried out on UV-Visible Spectrophotometer at a resolution 1nm between 200 and 800 nm. Distilled water was used as a blank. The spectrum recorded was then plotted.

Scanning electron microscopy (SEM)

Scanning electron microscopy (SEM) analysis was done using Hitachi S-4500 SEM machine. Thin films of synthesized and stabilized silver nanoparticles were prepared on a carbon coated copper grid by just dropping a very small amount of the sample on the grid, extra solution was removed using a blotting paper and then the film on the SEM grid were allowed to dry by putting it under a mercury lamp for 5 min and the sample was analyzed for morphology and size of the silver nanoparticles.

Energy dispersive x-ray spectroscopy (EDAX)

M. tinctoria leaf extract reduced silver solution was dried; drop coated on to carbon film, and tested using Hitachi S-4500 SEM instrument equipped with a thermo EDAX attachments. Energy dispersive x-ray spectroscopy (EDAX) analysis for the conformation of elemental silver was carried out for the detection of elemental silver. EDAX was sample composition of the analyzed for the sample composition of synthesized nanoparticles.

Fourier transfrom infrared spectroscopy (FTIR)

For FTIR measurements, the Ag nanoparticle solution was centrifuged at 10000 rpm for 30 min. The pellet was washed three times with 20 ml de-ionized water to get rid of the free proteins/ enzymes that are not capping the silver nanoparticles. The samples were dried and grinded with KBr pellets and analyzed.

XRD analysis

A thin film of the silver nanoparticle was made by dipping a glass plate in a solution and carried out for X-ray diffraction studies. The crystalline silver nanoparticle was calculated from the width of the XRD peaks, using the Debye-Scherrer formula,

D=0.94λ/ β cosθ

Where, D is the average crystallite domain perpendicular to the reflecting planes, λ is the X-ray wave length and β is the full width at half maximum and θ is the diffraction angle.

Antimicrobial activity assay

Bacterial and fungicidal activity was using standard agar well diffusion method against human pathogenic bacteria (Corynebacterium glutamicum, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa) and fungi (Aspergillus japonicas, Penicillium citrinum, Aspergillus niger, Aspergillus flavus, Aspergillus ochraceus).

Procedure

Nutrient agar (NA) and Potato Dextrose Agar (PDA) were prepared for cultivation of the bacteria and fungi respectively. Approximately 20 ml of molten and cooled media was poured in sterilized petri dishes. The plates were left overnight at room temperature to check for any contamination to appear. Then under an aseptic condition, placed a sterile swab into the broth culture of a fresh overnight grown cultures of the bacteria and fungi then gently removed the excess liquid by gently pressing or rotating the swab against the inside of the tube and spread it on nutrient agar and potato dextrose agar containing petriplates respectively. With a help of sterile borer, 5mm wells punched in the solid agar medium and then different concentration of the solution (50 µl, 100 µl, 150 µl) containing nanoparticles, 1 mM silver nitrate was inoculated in these wells and the plates were incubated at 370C for 12-24 hours and 2 or 3 days for bacteria and fungi respectively.15 Further, the plates were examined for evidence of zone of inhibition, which appear as a clear area around the wells surrounding bacteria and fungi growth. The diameter of such zones of inhibition was measured using a meter ruler and the mean value for each organism was recorded and expressed in millimeter.

Antibacterial activity of pus ample against silver nanoparticles

Collection of pus samples

A pus sample was collected from diabetic wound of patients at government hospital, Sivakasi. Wound samples were collected using sterile cotton swabs. The pus specimen was inoculated on blood and MacConkey agar plates. The streaked plates were incubated at 37o C for 24 hours.

Preparation of Bacterial Strains

The bacterial strains of pus samples were inoculated into the 50 ml of nutrient broth and the inoculums were incubated at 37°C for 24 hours.

Procedure

Antibacterial assay was carried out by disc diffusion technique. The plant extracts and synthesized nanoparticles were tested against pus samples using the Mueller Hinton agar. Medium was prepared, pH was adjusted and poured 20 ml per plate. After solidification, the pus sample culture was swabbed on the plates. Then sterile discs were loaded with the plant extract and nanoparticles (100 µl). The plates were incubated for 24 hrs. After incubation period the zone of inhibition was measured.

DPPH radical scavenging assay

The antioxidant activity of synthesized nanoparticles was determined on the basis of their scavenging activity of the stable 1, 1- diphenyl-2-picryl hydrazyl (DPPH) free radical.16 The sample and ascorbic acid were mixed with 95% ethanol to prepare the stock solution (5 mg/ml). Here ascorbic acid was taken as standard.17

Procedure

At first, 5 tubes were taken to make aliquots of 5 concentrations (20-100 µl) with the samples. DPHH was weighed and dissolved in ethanol to make 0.004% (w/v) solution and to dissolve homogeneously magnetic stirrer was used. After making the desired concentrations 3 ml 0.004% DPPH solution was applied on each test tube by pipette. The room temperature was recorded and kept the test tubes for 30 minutes to complete the reactions. DPPH was also applied on the blank test tubes the same where only ethanol was taken as blank. After 30 minutes, the absorbance of each test tube was taken by a UV spectrometer at 517 nm.

DPPH radical scavenging activity (%) = [(control absorbance)-(sample absorbance)]/ [control absorbance]*100

Hydrogen peroxide assay

The antioxidant of the synthesized nanoparticles was determined on the basis of their scavenging activity of the stable hydrogen peroxide free radicals. The samples and hydrogen peroxide were mixed with phosphate buffer (pH-7.4) to prepare stock solution. Here ascorbic acid was taken as standard. At first, 5 test tubes were taken with aliquots of 5 concentrations (20-100 µl) of the synthesized silver nanoparticles. To that, 0.6 ml of H2O2 in phosphate buffer was added. The reaction mixture was incubated at room temperature for 10minutes. Absorbance was read at 230 nm against the blank. Then the percentage of inhibition was calculated by the following equation.

H2O2 radical scavenging activity (%) =[(control absorbance)-(sampleabsorbance)]/[control absorbance]*100.

Anticancer activity

Cell line

The human hepatic carcinoma cell lines (HepG2) were obtained from National Centre for Cell Science (NCCS), Pune and grown in Eagles Minimum Essential Medium containing 10% fetal bovine serum (FBS). The cells were maintained at 37oC, 5% CO2, 95% air and 100% relative humidity. Maintenance cultures were passaged weekly, and the culture medium was changed twice a week.

Cell treatment procedure

The monolayer cells were detached with trypsin-ethylenediamine tetra acetic acid (EDTA) to make single cell suspensions and viable cells were counted using a hemocytometer and diluted with medium containing 5% FBS to give final density of 1x105 cells/ml. One hundred microlitres per well of cell suspension were seeded into 96-well plates at plating density of 10,000 cells/well and incubated to allow for cell attachment at 370C, 5% CO2, 95% air and 100% relative humidity. After 24 hrs the cells were treated with serial concentrations of the test samples. The nanoparticles M. tinctoria was dispersed in phosphate buffered saline. An aliquot of the sample solution was diluted to twice the desired final maximum test concentration with serum free medium. Additional four serial dilutions were made to provide a total of five sample concentrations. Aliquots of 100 µl of these different sample dilutions were added to the appropriate wells already containing 100 µl of medium, resulting in the required final sample concentrations. Following sample addition, the plates were incubated for an additional 48 hrs at 370C, 5% CO2, 95% air and 100% relative humidity. The medium containing without samples were served as control and triplicate was maintained for all concentrations.

MTT assay

3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetra-zolium bromide (MTT) is a yellow water soluble tetrazolium salt. A mitochondrial enzyme in living cells, succinate-dehydrogenase, cleaves the tetrazolium ring, converting the MTT to an insoluble purple formazan. Therefore, the amount of formazan produced is directly proportional to the number of viable cells.

After 48 h of incubation, 15 µl of MTT (5 mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 370C for 4 h. The medium with MTT was then flicked off and the formed formazan crystals were solubilized in 100 µl of DMSO and then measured the absorbance at 570 nm using micro plate reader. The % cell inhibition was determined using the following formula.

% Cell Inhibition=100- Abs (sample)/ Abs (control) x100.

Nonlinear regression graph was plotted between % cell inhibition and log concentration and IC50 was determined using GraphPad Prism software.

Results and Discussion

Visible observation of silver nanoparticles synthesis

The silver nanoparticles were synthesized using leaf extract of M. tinctoria plant. The reduction of silver ions into silver particles during exposure to the plant extract is followed by colour change. As the leaf extracts was mixed in the aqueous solution of silver ion complex, it started to change the colour from yellowish brown to dark brown (Figure 2). Almost all the herbal mediated silver nanosolutions after incubation time were showed the colour change from light to dark colour. In this present study, the leaf extracts of M. tinctoria has the potential to reduce silver nitrate to silver nanoparticles. The color of the reaction medium changed into dark brown colour after 24 hours. In the present study the colour changes was observed in both plants within 30 mintues. The quick conversion of solution color showed the formation of silver nanoparticles by observing color change from colorless to yellowish-brown color in bamboo leaves extract.10

Figure 2: Visible observation of silver nanoparticlefrom M. tinctoria leaf extract. A) Silver nitrate, B) plant extract C) Initial reaction mixture D) Reaction mixture after 24 hours Silver nanoparticles of M. tinctoria.

UV-Visible spectroscopy analysis

The synthesis of silver nanoparticles in the mixture of solution was further analyzed and confirmed by UV-visible spectroscopy. The UV spectra peaks of M. tinctoria showed in Figure 3. The broad Plasmon resonance was observed after 24 hrs and 48 hrs at 420 nm for M. tinctoria.

Figure 3: UV- Visible absorption spectrum of silver nanoparticles synthesized from.

M.tinctoria leaf extract

AgNps synthesized by using M. tinctoria were formed at 409 nm with polydispersed.18 Generally, UV-VIS spectroscopy can be used to examine size and shape of the controlled nanoparticles in aqueous suspense. The results of the UV-VIS absorption showed increasing colour intensity with increased time intervals and this might be due to the production of the silver nanoparticles and the formation of the brownish yellow colour might be due to the excitation of the surface plasmon vibration of the synthesized AgNps.19,20

SEM analysis of silver nanoparticles

SEM image was showed that the information about the morphology and size of the synthesized silver nanoparticles of M. tinctoria in Figure 4 (A-D) respectively.

From the SEM images the nanoparticles are more or less crystal in shape in the maginification of 20.00KX, 50.00KX, 100.00KX. The M.tinctoria exhibit the uniform sized cubic grains, the size of the cubes range from 10-20 nm. Similarly, silver nanoparticles synthesized from leaves of Allium cepa exhibited crystal shape.22

Figure 4: SEM images of silver nanoparticles synthesized from M. tinctoria. A) 20.00KX B) 50.00KX C) 10.00KX D) 100.00KX.

EDAX analysis of silver nanoparticles

Energy dispersive absorption spectroscopy (EDAX) of synthesized nanoparticles of M. tinctoria was showed in the Figure 5. The presence of expected element in the final products Ag was confirmed. The present analysis revealed that the nano-structures were formed as peak at 3 keV solely of silver nanoparticles for M. tinctoria.The other spectral signals such as Ca, K, Cl, O, Mg and Si were also noticed in the EDAX spectrum.

Figure 5: EDAX spectrum of silver nanoparticles from M. tinctoria.

FTIR spectrum

FTIR spectrum of produced silver nanoparticles were showed many absorption bands and the absorption bands for The absorption bands for M. tinctoria was 3362.66 cm-1, 2921.96 cm-1, 2850.59 cm-1, 2360.71 cm-1, 1739.67 cm-1, 1455.19 cm-1, 1374.19 cm-1, 1228.57 cm-1, 1163.00 cm-1, 1034.74 cm-1, 993.27 cm-1, 676.00 cm-1, 658.65 cm-1 were assigned to the N–H stretch 1°, 2° amines, amides, C–H stretch alkanes, C–H stretch alkanes, H–C=O: C–H stretch aldehydes, C=O stretch aldehydes, N–O asymmetric stretch nitro compounds, C–H rock alkanes, C–N stretch aliphatic amines, C–N stretch aliphatic amines, C–N stretch aliphatic amines, C–N stretch aliphatic amines, =C–H bend alkenes, C–H stretch aromatics, –C≡C–H: C–H bend alkynes (Figure 6). FTIR spectrum indicates the leaf extract of M. tinctoria assisted production of silver nanoparticles by showing functional groups like amide, alkanes, carbonyls, alkyl halides, alkynes, aliphatic amines, nitro groups, aldehydes present at different position. From this observation, the identified biomolecules present in the plant extract of M. tinctoria was responsible for reduction and stabilization of silver nanoparticles. Hence, the terpenoids are proved to have good potential activity to convert the aldehyde groups to carboxylic acids in the metal ions. Further, amide groups are also responsible for the presence of the enzymes and these enzymes are responsible for the reduction synthesis and stabilization of the metal ions, further, polyphenols are also proved to have potential reducing agent in the synthesis of the AgNps.23,24

Figure 6: FTIR spectrum of silver nanoparticles synthesized by using M. tinctoria leaf extract.

XRD analysis

The XRD pattern showed the intense peaks in the whole spectrum of 2Ө values ranging from 10-70 for the silver nanoparticles. The synthesized silver nanoparticles were in the form of nanocrystals of M. tinctoria was 28.274, 32.703 and 46.642 corresponding to the diffraction exhibited from 10 to 70 range of 2Ө (Figure 7). XRD pattern obtained for the silver nanoparticles showed number of Bragg's reflections that may be indexed on the basis of the face centered cubic structure of silver.25

Figure 7: XRD patterns of silver nanoparticles synthesized from M. tinctoria.

Antimicrobial activity

Silver is said to be a universal antimicrobial substance for centuries. Though, silver ions or salts have limited usefulness as an antimicrobial agent. Such as, the interfering effects of salts and antimicrobial mechanism of continuous release of enough concentration of Ag ions from the metal form. This kind of limitation can be overcome by using silver nanoparticles. However, to use silver against microorganisms, it is essential to prepare it with environmentally friendly and cost- effective methods. Besides, it is also important to enhance the antimicrobial effects of silver ions.21,26

Antibacterial activity of silver nanoparticles from leaf extract of M. tinctoria was performed by Agar-well diffusion method. In this study, five bacterial cultures were used namely E. Coli, K. pneumonia, P. vulgaris, S. aureus, and C. glutamicum in 50 µl, 100 µl, 150 µl, 200 µl. In that plates the silver nanoparticles were added and incubated for 24 hrs. After incubation, the zone of inhibition well was measured (Figure 8 and 9).

Figure 8: Zone of inhibition in diameter of M. tinctoria silver nanoparticles against antibacterial activity. 1) E. coli 2) K. pneumoniae 3) P. vulgaris 4) P. aeruginosa 5) C. glutamicum.

Figure 9: Antibacterial activity of silver nanoparticles of M. tinctoria with different concentrations at 50 µl (1), 100 µl (2), 150 µl (3), 200 µl (4).

Antifungal activity of M.tinctoria derived silver nanoparticles were performed by Agar-well diffusion method. In this study, five fungal cultures were used namely A.japanicus, P.citrinum, A.niger, A.flavus, A.oxrasicus. The silver nanoparticles were taken in different concentration such as 50µl, 100µl, 150µl, 200µl (Figure 11). After the incubation of 48 hrs, the zone of inhibition was found in Figure 10.

Figure 10: Zone of inhibition in diameter of M. tinctoria silver nanoparticles against antifugal activity. 1) A. japonicus 2) P. citrinum 3) A. niger 4) A. flavus 5) A. ochraceus.

Figure 11: Antifungal activity of silver nanoparticles of M. tinctoria with different concentrations at 50 µl (1), 100 µl (2), 150 µl (3), 200 µl (4).

Antibacterial activity of pus sample

The antidiabetic activity of the silver nanoparticle was performed by disc diffusion method. In this study, the pus sample was brought from Government hospital of Sivakasi from the diabetic patients. The patients sample was inoculated separately in blood agar and MacConkey agar plates. Then the pus sample was plated with the silver nanoparticles were added and incubated for 24 hours in incubator. After incubation, the zone of inhibition was around the disc was measured and it was showed Figure 12. The zone of inhibition against the pus sample of diabetic patient was 11 mm in diameter. Wound infections are a major complication in diabetic patients and also a major healthcare burden.25

Patients with chronic diabetic conditions are prone to showed process of wound healing and seem to be at particularly high risk for soft tissue infections, urinary tract infections and surgical site infections caused by S. aureus, S. haemolyticus, P. aeruginosa.26-28

Figure 12: Antibacterial activity of pus sample against M. tinctoria derived silver nanoparticles.

Radical scavenging activity of DPPH Assay

The free radical scavenging activity of silver nanoparticles of M.tinctoria was determined by DPPH (1,1-diphenyl-2-picrylhydrazyl) method. The DPPH radical scavenging effects of nanoparticles of M .tinctoria was showed in Figure 13.

Figure 13: Percentage of antioxidant activity of ascorbic acid and silver nanoparticles by DPPH Assay.

The silver nanoparticles showed the significant free radical scavenging activities when compared to with standard ascorbic acid. In various concentration of synthesized silver nanoparticles (20 µl to 100 µl) about 35% to 42% 0f free radicals was observed in M. tinctoria's silver nanoparticles.

Scavenging activity of hydrogen peroxide

The H2O2 scavenging activity of the silver nanoparticles of M. tinctoria. H2O2 scavenging activity of silver nanoparticles compared with standard ascorbic acid. The percentage of inhibition of free radicals increased with increase in concentrations of substrates. The concentration from 20 µl to 100 µl of silver nanoparticles showed 30% to 68% for M. tinctoria's silver nanoparticles (Figure 14).

Figure 14: Percentage of antioxidant activity by hydrogen peroxide assay.

Hydrogen peroxide is a weak oxidizing agent and can inactivate a few enzymes directly, usually by oxidation of essential thiol (-SH) groups. Hydrogen peroxide can cross cell membranes rapidly, once inside the cell, H2O2 can probably react with Fe2+, and possibly Cu2+ ions to form hydroxyl radical and this may be the origin of many of its toxic effects.29

Anticancer activity

The present study was conducted to screen cytotoxic potential against human hepatic carcinoma cell line (HepG2) with silver nanoparticles of leaf extracts of M. tinctoria plant (Figure 15) The microscopic examination of the human hepatic carcinoma cell line (HepG2) revealed characteristic growth pattern after 24 hrs were treated with serial concentrations of test sample (0.25, 2.5, 25, 50, 100 μg) of M. tinctoria. Separate control was maintained to check the viability of the procedure. The present study clearly led to the confirmation that, the % of inhibition was increased in the reaction mixture. In the concentration of 100 µg of M. tinctoria's silver nanoparticles were completely inhibited. Simultaneously, % of cell inhibition was also evaluated.

Figure 15: Anticancer activity of silver nanoparticles of M. tinctoria and different concentration A) 0.25 µg, B) 2.5 µg, C) 25 µg, D) 50 µg, E) 100 µg, F) Control.

In the cytotoxic potential of M. tinctoria's silver nanoparticles were evaluated. 99.7189% of the cells were inhibited when the concentration of silver nanoparticles has reached around 50µg. Least inhibition was observed in the concentration like 0.25 µg, it gives 0.913563% of cell inhibition (Table 1 and Figure 16).

Table 1: Different concentration of silver nanoparticles of M. tinctoria and% of cell inhibition.

Conc (µg/ml)

 

% Cell inhibition

 

0.25

0.913563

IC50

24.89 µg/ml

2.5

2.810963

25

50.9487

50

99.7189

R2

0.9991

100

100

Table 2: Different concentration of M. tinctoria silver nanoparticles and absorbance against the cell line (HepG2).

Conc

µg

2.5 µg

25 µg

50 µg

µg

Cont

ABS

0.48

0.46

0.238

0.003

0

0.483

 

0.459

0.459

0.225

0.001

0

0.469

 

0.471

0.464

0.235

0

0

0.471

Avg

0.47

0.461

0.232667

0.001333

0

0.474333

On the other hand, IC50 and R value of the silver nanoparticles suspension were also estimated. The experimental data has clearly revealed that IC50=24.89 µg/ml for M. tinctoria's silver nanoparticles. This value is quite a least concentration that could control the proliferation of cancer cell lines in the in vitro condition (Table 2).

Figure 16: Effect of M. tinctoria AgNPs on cell inhibition of human hepatic carcinoma cell line (HepG2).

Conclusions

In this study, a simple approach was attempted to obtain a green eco-friendly way for the synthesis of silver nanoparticles using aqueous M. tinctoria Roxb leaves extracts. The silver ions in an aqueous solution was exposed to the M. tinctoria leaves extracts, the biosynthesis of AgNPs were confirmed by the rapid colour change of plant extracts. The natural benign AgNPs were confirmed further by using UV-Vis spectroscopy, SEM, EDAX, FTIR and XRD. Phenols and favonoids were present in the leaves, and they serve as an effective reducing agent. AgNPs biosynthesized from M. tinctoria leaves also exhibits great antimicrobial activities against pathogenic bacterial and fungal cultures. These biosynthesis silver nanoparticles can potentially be used for different medical applications. The synthesized nanoparticles showed dose dependent free radical scavenging activity and anticancer activity against human hepatic carcinoma cell line (HepG2).

Funding: No funding sources

Conflict of interest: None declared

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