One‐pot green synthesis and structural characterisation of silver nanoparticles using aqueous leaves extract of Carissa carandas : antioxidant, anticancer and antibacterial activities

Abstract

Facile green synthesis of silver nanoparticles (AgNPs) using an aqueous extract of Carissa carandas (C. carandas) leaves was studied. Fabrication of AgNPs was confirmed by the UV–visible spectroscopy which gives absorption maxima at 420 nm. C. carandas leaves are the rich source of the bioactive molecules, acts as a reducing and stabilising agent in AgNPs, confirmed by Fourier transforms infrared spectroscopy. The field emission scanning electron microscope revealed the spherical shape of biosynthesised AgNPs. A distinctive peak of silver at 3 keV was determined by energy dispersive X‐ray spectroscopy. X‐ray diffraction showed the facecentred cubic structure of biosynthesised AgNPs and thermal stability was confirmed by the thermogravimetric analysis. Total flavonoid and total phenolic contents were evaluated in biosynthesised AgNPs. Biosynthesised AgNPs showed free radical scavenging activities against 2, 2‐diphenyl‐1‐picrylhydrazyl test and ferric reducing antioxidant power assay. In vitro cytotoxicity against hepatic cell lines (HUH‐7) and renal cell lines (HEK‐293) were also assessed. Finally, biosynthesised AgNPs were scrutinised for their antibacterial activity against methicillin‐resistant Staphylococcus aureus, Shigella sonnei, Shigella boydii and Salmonella typhimurium. This study demonstrated the biofabrication of AgNPs by using C. carandas leaves extract and a potential in vitro biological application as antioxidant, anticancer and antibacterial agents.

1 Introduction

Production of a novel drug delivery system in recent years, using the fabrication of metal nanoparticles with specific size and surface morphology has been pulling the researchers in the field of nanotechnology [1]. Since 18th centuries, metal nanoparticles are used for medicinal purpose and have fascinated for consideration in the field of cancer. Among metal nanoparticles, silver nanoparticles (AgNPs) are one of most popularised nanomaterial because of its special physico‐chemical properties, biocompatible, low toxicity and biomedical application such as radiotherapy sensitisation agent, molecular imaging agents, biological markers, antibacterial, antifungal, antidiabetic, anti‐inflammatory and anticancer property [2]. They are broadly utilised in food and pharmaceutical industries, e.g. preparation of creams, ointments and gels to treat the cut, burn and wound [3]. There are diverse physical and synthetic techniques are available for the preparation of AgNPs which includes a sol–gel–sol method, chemical reduction, photochemical reduction and other biological methods. Biofabrication or phytofabrication of nanoparticles by using biological method is an alternate and environmentally friendly approach for the preparation of AgNPs [4]. The use of plant extracts is possibly beneficial over the microorganisms due to apparent progress, cell culture preservation and protection. This method is preferred over other technique, as it toxic free and acts as natural reducing and capping agent during the biogenesis of AgNPs [5]. There are few works of the literature are available on the biofabrication of AgNPs using plant extracts of Ziziphus jujuba, Hibiscus cannabinus, Phlogacanthus thyrsiformis and Pelargonium graveolens.

AgNPs are used as antioxidant, antibacterial agent and effective drug targeted system for the cancer treatment which has currently gained extensive attention. It was testified that AgNPs using leaves extract of Artemisia vulgaris had anticancer property against human epithelial (HeLa) cancer cell line and human breast cancer (MCF‐7) cell line [6]. By Trojan effect, AgNPs could be transported into the cancerous cell and stop the activity of RNA topoisomerase enzyme and transcription of the gene through a mutual contact. The cancerous cell is more delicate to AgNPs destruction as compared to normal cells [7]. AgNPs size and surface characteristics are important tools for biomedical contemplations. AgNPs which are small in size show an effective penetration power and target cancer cells [8]. The possible mechanisms of AgNPs have not been completely known yet but the researchers are trying to find it.

Recently, exploration of natural and chemical antioxidants headed the selection and authentication of novel antioxidants from the traditional plants. Synthetic antioxidants produce various side effects such as allergic, mutagenic and carcinogenic effects. The antioxidant property in plant extracts is present due to redox impending of phytoconstituents. Antioxidants play a pivotal role in scavenging for oxygen, break down into peroxides and deactivating the free radicals. It is presumed that nanoparticles possess a higher content of antioxidant because of adsorption of phytocompounds from extract directly to the surface of AgNPs [9]. Researchers focusing on the metallic nanoparticles owing to bacterial resistance. Silver gained a much consideration among other metals because of its dynamic property as an antibacterial agent and generally used in marketed products.

Carissa carandas (C. carandas) Linn. (Karonda) is used as a therapeutic plant by tribals all over the world and prominent in a different traditional system of medicine. All parts of the C. carandas are utilised as a part of folklore medicine [10]. It is an evergreen deciduous shrub, belong to Apocynaceae family. It is also knowns as karmada in Hindi and supposes to grow near to the Himalayas region. Geographically the natural origin of this plant is Nepal to Afghanistan and also covers some regions of India. It is distributed all over the hot regions (high temperature) of India and Srilanka. It is a water scarcity tolerant plant, flourishes well in tropical and subtropical regions. It has grown in the sandy region and rocky soils, either grow wild or cultivated in the area of Punjab. Stem contains a white latex, leaves are conical, long and green in colour. Fruits are globose and contain seeds, show red colour after ripening. Flowering occurs in the month of January and matures in the months of May and June [11]. Traditionally, the plant has been used in the treatment of scabies, intestinal worms, pruritus, antiscorbutic, anthelmintic, pain relieving, cancer and hepatoprotective. Karonda tree is generally grown in the gardens and agricultural fields [12]. The leaves have triterpenoid compound as well as tannins, and another isomer of ursolic acid. It also contains carissic acid, which is responsible for bioreduction of Ag+ ions to AgNPs [13].

C. carandas aqueous extract contains different bioactive constituents including alkaloids, flavonoids, phenols, tannins, glycosides, saponins, carbohydrate, proteins and enzymes which act as a reductant and used as scaffolds during the biofabrication of AgNPs in the medium [14]. The bioactive molecules act as a strong antioxidant agent and protect the cell from oxidative stress. Owing to this property of Carissa carandas AgNPs (CCAgNPs), they are used in the therapy of cancer, bioimaging, antimicrobial, diabetes, wound healing, antioxidant and so on.

The aim of this study is to biofabricate and evaluates the AgNPs utilising aqueous leaves extract of the therapeutic plant, C. carandas. The cytotoxic effect of bioengineered AgNPs was evaluated on human hepatic cancer cell lines (HUH‐7) and human renal cancer cell lines (HEK‐293) by the MTT assay.

2 Material and methods

Silver nitrate was procured from Qualigens fine chemicals, Mumbai. 2,2‐Diphenyl‐1‐picrylhydrazyl (DPPH) {Sigma‐Aldrich (USA)} and purchased from local suppliers. All other chemicals were used in the experiment are of analytical grade and used as such.

2.1 Collection of plant materials

The fresh leaves of C. carandas were collected from the local region of Mirzapur district as shown in Fig. 1 a and kept in a polythene bag. The leaves were washed with double distilled water in order to remove extraneous matter and dried under shade for one month in the absence of sunlight. Then the leaves were grounded with the help of the blender and kept in the bottle for the further use.

Fig. 1.

Biosynthesise silver nanoparticle from plant leaves

(a) Leaves of Carissa carandas, (b) Biofabricated silver nanoparticles

2.2 Preparation of plant extract

The dried leaves about 10 g were taken into a 500 ml Erlenmeyer flask which contained 250 ml of distilled water. This solution was boiling at 80°C with continuous stirring at 200 rpm for 60 min. The mixture was allowed to cool and filtered through Whatman filter paper no.1. The filtrate termed as extract and placed in a refrigerator for further use [15].

2.3 Biosynthesis of AgNPs

To synthesised AgNPs, 80 ml of 1 mM AgNO₃ was added to the 20 ml of plant extract in vigorously shaking Erlenmeyer flask at room temperature. After 60 min, the colour of the solution was changed from orange to dark brown as shown in Fig. 1 b and indicated the formation of AgNPs. The biofabricated nanoparticles mixture was subjected to centrifugation in the centrifuge machine at 12,000 rpm for 10 min. The upper layer of the mixture was decanted, the precipitate was washed with distilled water two to three times to remove the particulate matter and dry in an oven. The AgNPs yield obtained was 63.21% and further used for the characterisation [16].

2.4 Instrumental characterisation of the AgNPs

2.4.1 UV–visible study

The UV–visible study was achieved utilising Shimadzu UV‐1700 spectrophotometer, working at 1 nm resolution. The samples were suitably diluted with double distilled water and placed in a quartz cuvette in the range of 200–800 nm.

2.4.2 Fourier transform infrared (FTIR) analysis

FTIR examination was done in order to determine the biomolecule presents inside the leaves and nanoparticles. A dried AgNP was mixed with the potassium bromate pellet to interpret the presence of functional group on Perkin Elmer FTIR spectrophotometer which was operated between 4000 and 400 cm⁻¹.

2.4.3 X‐ray diffraction (XRD) study

The level of crystallinity of the bioengineered AgNPs was inspected by XRD study. The information relating to the X‐ray pattern (AgNPs) was achieved by using PANalytical X pert PRO running (outfitted with a nickel monochromator) with Cu radiation in the 2θ window of 3°–80°.

2.4.4 Field emission scanning electron microscope (FESEM) coupled with energy dispersive X‐ray spectroscopy (EDX)

FESEM pictures of bioengineered AgNPs were taken to determine its size and surface morphology. The suspension was put on the coverslip, dried under vacuum and placed on a FESEM Carl Zeiss SMT AG (Germany) worked at an accelerated voltage of 20 kV and filtering in between 2° and 30° for its examination. The FESEM instrument was attached to the EDX device to determine the elemental composition of AgNPs.

2.4.5 Transmission electron microscope (TEM)

TEM images revealed the size and shape of biosynthesised AgNPs. A few drops of a suspension of AgNPs was put on a carbon coated copper grids worked at the voltage of 200 kV, dried at RT and analysed by Hitachi TEM instrument.

2.4.6 Thermogravimetric (TG) analysis

Effects of high temperature on biosynthesised AgNPs were assessed utilising a TG machine (EXSTAR TG/DTA 6300). For TG examination, powdered AgNPs (3.0 mg) was set in an alumina container and heated from 35 to 700°C for 10°C/min under nitrogen climate in a heating chamber.

2.5 Quantification of bioactive compounds

2.5.1 Total phenolic contents assay

Total phenolic contents of C. carandas extract (CCE) and AgNPs were measured by utilising the Folin–Ciocalteu method. In a reaction mixture, 0.1 ml of CCE and CCAgNPs were blended with 2.8 ml deionised water. This mixture was blended with 2 ml of 2% sodium carbonate and 0.1 ml of 0.1 N Folin–Ciocalteu reagents. The reaction mixture was subjected to incubate for 30 min at RT and the absorbance of the sample was recorded at 750 min UV/visible spectrophotometer [17].

2.5.2 Total flavonoid contents assay

Total flavonoid contents of CCE and AgNPs were measured by utilising the aluminium chloride colorimetric technique. In this method, 0.5 ml of CCE and CCAgNPs were blended with 1.5 ml of ethanol, 0.1 ml of 10% aluminium chloride and 2.8 ml of double distilled water. The blend was left for 30 min at room temperature and the absorbance was measured at 415 nm [18].

2.6 In vitro antioxidant assay

2.6.1 DPPH free radical scavenging assay

This activity utilised to determine the in vitro antioxidant property of silver nitrate, plant extract and AgNPs. Different dilutions of samples were prepared and 1 ml (0.1 mM) solution of DPPH which was previously dissolved in methanol was added to the samples. The samples were mixed vigorously and subjected to incubate for 30 min. 100 μl of methanol (without extracts) mixed with DPPH solution to prepare a control. The absorbance was noted at 517 nm by using a UV spectrophotometer and compared with a positive reference (ascorbic acid). The percentage of inhibition was calculated by using the following formula [19]:

$$\text{\%}\mathrm{inhibition}=\frac{\left(\mathrm{ODc}-\mathrm{ODs}\right)}{\mathrm{ODc}}\times 100,$$

where ODc is the absorbance of control and ODs is the absorbance of sample.

2.6.2 Ferric reducing antioxidant power (FRAP) assay

This assay was done to determine the in vitro antioxidant activity of plant extract, silver nitrate and AgNPs of the various concentrations. This activity was assayed by the reduction of a ferric 2, 4, 6‐tripyridyl‐triazine complex (Fe³⁺ ‐TPTZ) to the ferrous form (Fe²⁺ ‐TPTZ). In the reaction mixture, 2.5 ml of TPTZ solution (10 mM TPTZ in 40 mM HCl) and FeCl₃ (20 mM) in 25 ml of acetate buffer (0.3 M and pH 3.6), was mixed with 0.5 ml of the different test samples. In the same way, control was prepared without added an extract. All samples were subjected to incubation at 37°C for 30 min, the optical density was noted at 593 nm and compared with a positive reference [19].

2.7 In vitro cytotoxic activity

2.7.1 Preparation of cell culture

Human hepatocellular carcinoma (HUH‐7) and human kidney (HEK‐293) cell lines were procured from National centre for cell science (NCCS), Pune, India. Cells were kept in DMEM (Dulbecco's Altered Bird Medium) and 10% foetal bovine serum, 100 units/ml penicillin, 100 mg/ml streptomycin, 0.14% sodium bicarbonate and 0.1 mM sodium pyruvate. Cells were developed in an incubator in a CO₂ atmosphere at room temperature and 95% humidity.

2.7.2 Study of anticancer property

MTT reagents were utilised to measure the in vitro anticancer effect of bioactive AgNPs of the leaves of C. carandas on HUH‐7 and HEK‐293 cell lines. MTT reduces to form blue formazan by mitochondrial dehydrogenase, which demonstrates mitochondrial activities and subsequently cell growth. 3 × 10³ cells per well were seeded in 96 well plates in 100 ml cell culture medium and allowed to incubate for 24 h. After 24 h cells were treated with different dilution of the biofabricated AgNPs. After this, 5 mg/ml of MTT reagent was mixed with a fresh medium into test, and control wells and again subjected to an incubator for 4 h. MTT was dissolved in DMSO and formed formazon products. The absorbance of a mixture was recorded at 540 nm against blank and calculated as [20]

$$\text{\%}\mathrm{of}\mathrm{cell}\mathrm{inhibition}=\left(Y-\frac{X}{Y}\right)\times 100,$$

where Y is the absorbance of the control (untreated cells) and X is the mean absorbance of treated cells with AgNPs.

2.8 Antibacterial activity of AgNPs

Biofabricated AgNPs were examined by the agar well‐diffusion technique against various pathogenic bacteria (methicillin‐resistant Staphylococcus aureus (MRSA), Shigella boydii (S. boydii), Shigella sonnei (S. sonnei) and Salmonella typhimurium (S. typhimurium). The experimental bacteria were kept on the nutrient broth medium (3.0 g beef extract, 5.0 g/l peptone and a final pH of 6.8 ± 0.2; Sigma‐Aldrich, Germany) at room temperature for 48 h. The microscopic organisms were homogeneously swapped onto singular plates by utilising the sterile cotton swabs. Wells of 3 mm breadth were drawn on Mueller–Hinton agar (3.0 g beef infusion, 1.5 g starch, 17.5 g peptone, 17.0 g/l agar; pH 7.4 ± 0.2) by the utilising stopper borer. AgNP (80 μl) was transferred to each well, and three antibiotics, which serve as a control (gentamicin, ampicillin and neomycin) were utilised as controls. After a 24 h period of incubation, the inhibition zones (mm) were noted [21].

3 Result and discussion

3.1 Structural characterisation by absorbance spectroscopy

The UV–visible spectrum of the synthesised AgNPs was delineated in Fig. 2. The absorption spectrum depicts the formation of AgNPs, formed by silver nitrate and the chemical constituents found in the leaves of C. carandas [22].

Fig. 2.

UV–visible spectrum of AgNPs

It was concluded from Fig. 2 that AgNPs possess free electrons, which show mutual vibration in reverberation with the light due to surface plasmon resonance. The sample has been obtained from a plant source contains a different type of phytocompounds. It is an essential instrument which determines synthesised nanoparticle because the valence of silver from Ag¹⁺ in AgNO₃ modifying to Ag⁰ in the prepared nanoparticles [23]. The formation of metal nanoparticles involved the features of the nucleated particles. The other organic compounds which are present in the aqueous extract interact with silver ions and peak move to the shorter wavelength. With the time, the absorption peak shifted to higher wavelength and showed absorbance maxima at 420 nm. A comparable absorption spectrum at 433 nm was envisaged during biofabrication of AgNPs utilising Zizyphus xylopyrus bark extract leaf extract [24].

3.2 FTIR spectroscopy

FTIR analysis of CCAgNPs shows the possible association between silver and the bioactive moieties involved in the formation and the stability of AgNPs. The spectra reveals the intense peak at 3426, 2905, 1610, 1444, 1381, 776, 343 cm⁻¹ in AgNPs and 3370, 2960, 1620, 1444, 1317, 777, 409, 342 cm⁻¹ in plant extract. Figs. 3 a and b show the FTIR spectrum of CCE and CCAgNPs. The vibration band found at 3426 cm⁻¹ in CCAgNPs spectra assigned the OH stretching of the phenolic group, a similar result was also obtained during biosynthesis of AgNPs of Helicteres isora root extract [25]. In the same case, the vibration bands at 2905 cm⁻¹ assigned to the stretching vibration of aliphatic C–H group, 1610 cm⁻¹ attributed to the bending vibrations modes of aromatic ring C=C [26], 1444 cm⁻¹ due to aliphatic C–H group, 1381 cm⁻¹ due to aromatic stretching of C–N (the peptide bonds of proteins) [27], 776 cm⁻¹ denoted to bending of the aromatic carbon–hydrogen bond, respectively [28], observed in the past reports.

Fig. 3.

FTIR spectra

(a) C. carandas leaves, (b) AgNPs

From the FTIR study, it can be demonstrated that the functional groups show the unique features of phenols, aromatic amine and carbonyl groups of CCE. As reported by reference, carbonyl groups and hydroxyl groups in phenols, have a noteworthy capability to chelate silver ions and permits them to mount and prevent the AgNPs against accumulating in solution [29]. The shifting of peaks up and down reveals the synthesis of the AgNP. The characteristic features of the biomolecule in CCE are responsible for the formation and stabilisation of AgNPs [30].

3.3 XRD analysis

XRD analysis was performed to determine the geometric structure and gapping between atoms of AgNPs. Fig. 4 delineates the XRD pattern of the incorporated AgNPs by the leaves of CC. It was suggested from the results that crystalline planes indicate the peaks of AgNPs. It shows main characteristic peaks for silver, which are found at 38.12°, 44.18°, 64.53°, 77.48°, 81.07° and might be corresponding with (111), (200), (220) and (311) alluded from the JCPDS card no. 040783. Subsequently, AgNPs suggests the face‐centred cubic (FCC) structure which is same as silver structure. The comparable crystallographic result was developed during biosynthesis of AgNPs using the seed extract of Cydonia oblong [31].

Fig. 4.

XRD diffraction pattern of AgNPs

3.4 TG analysis

A TG graph of CCAgNPs was represented in Fig. 5. The stepwise decomposition process of AgNPs is shown in the graph. The TG was used to study the thermal stability of AgNPs which start to degrade at about 225°C. There was a steady weight loss was found at 700°C. The weight loss up to 250°C due to the evaporation of the solvent molecule (water moisture) present on the surface of nanoparticles [32]. The weight loss from 250 to 700°C represents the loss of carbohydrate and protein decomposition present in the nanoparticles. The total loss of weight of AgNPs up to 700°C was 97.41% [33]. It suggests that biomolecules are present in the plant extract act as a capping agent for the reduction of silver ion and stable the AgNPs in the solution.

Fig. 5.

TG analysis of AgNPs

3.5 FESEM with EDX analysis

FESEM provide images of samples at the submicron level and provide higher resolution pictures. The images were distinct and spherical in shape. The size of AgNP ranged between 28 and 60 nm (Figs. 6 a and b). Biosynthesised AgNPs using the aqueous seed extract of night jasmine yielded obtained nanoparticles in the size of 50–80 nm [34].

Fig. 6.

FESEM with EDX

(a), (b) FESEM images of AgNPs, (c) EDX spectrum

The qualitative and quantitative elemental detection of AgNPs was confirmed by using EDX. It shows a higher amount of silver content at 3 keV which confirms the synthesis of AgNPs (Fig. 6 b). The biofabrication of AgNPs using Saraca indica leaf extract demonstrated similar result for EDX at the 3 keV [35]. The high peaks in the region showed various valence conditions of Ag in the AgNPs. The peaks represent other elements are normally distinguished in the specimens gathered on a carbon‐coated copper grid or might be the presence of bioactive in the leaves extract [36].

3.6 TEM analysis

TEM examination provides data about the size and development of AgNPs. This technique is used to decide the morphology and the exact size of synthesised AgNPs. The images verified that morphology of fabricated AgNPs irregular and polydispersed in nature (Figs. 7 a and b) [37]. Moteriya et al. [38] reported irregular and polydispersed appearance during biofabrication of AgNPs using Cassia roxburghii leaf extract.

Fig. 7.

TEM

(a), (b) TEM images of AgNPs

3.7 Total flavonoid and total phenolic contents

It was observed that high concentration of flavonoid contents and low concentration of phenolic contents in the extract of CC when compared to CCAgNPs as represented in Figs. 8 a and b. The antioxidant property of the plant is due to the presence of total flavonoid and phenolic contents [39]. The flavonoid and phenols of CC extract reduced the silver nitrate solution to nanoparticles.

Fig. 8.

Phytochemical assay and antioxidant activity

(a) Total phenolic content of AgNPs, (b) Total flavonoid content, (c) DPPH free scavenging radical activity, (d) FRAP assay

3.8 In vitro antioxidant activity

The antioxidant property of CCE and AgNPs was assessed by DPPH free radical scavenging tests. The outcomes of antioxidant property were represented in Fig. 8 c. DPPH is a steady and very strong free radical for assessment of the antioxidant property of any therapeutic agents [40]. The antioxidant activity was observed to be higher in AgNPs when contrasted with CCE. AgNPs demonstrated most elevated level than CCE against DPPH.

A comparable effect was found in the FRAP test for AgNPs and plant extract. Reduction in the levels of FRAP assay was observed in both test samples as same happen in DPPH‐scavenging activities, at the concentration of 100 and 150 µg/ml. As appeared in Fig. 8 d, the reduced level of AgNPs and CCE reversed (increased) with increasing serial dilution (>150 µg/ml). Ascorbic acid obtained the highest reducing power when compared to both test samples.

Reactive oxygen species (ROSs) are a chemically reactive substance which contains oxygen. ROSs are used for cell signalling and homeostasis which is by‐products of metabolism of oxygen. Due to oxidative stress, there is a destruction of cell structure. The antioxidants component guards the cells against the impeding properties of ROS. The DPPH radical scavenging activity of green synthesised AgNPs might be attributed to their capability to give electrons to counteract the unsteady DPPH free radicals during reaction [41]. The principle of FRAP analysis is grounded on the capability of antioxidants to reduce Fe³⁺ to Fe²⁺ by using TPTZ to give blue Fe²⁺ ‐TPTZ compound at 593 nm [42]. In agreement with our results, Mahendran and Kumari testified FRAP assay of AgNPs from Nothapodytes nimmoniana (Graham) Mabb. fruit extracts [43]. The result of the DPPH assay and the FRAP assay demonstrated the antioxidant potential of AgNPs when compared to ascorbic acid (standard). The influential DPPH free radicals activity and FRAP assay of biosynthesised AgNPs accepted previously supported our outcomes in the current investigation.

3.9 Cytotoxic impact of AgNPs

The anti‐proliferative impact of AgNPs was appraised against HUH‐7 and HEK‐293 cell lines, by utilising MTT test. The reactions are exhibited as the percent cell inhibition, at varying dilutions of CCAgNPs and silver nitrate from 10 to 50 µg/ml for 24 h, and compared with control. CCAgNPs displayed powerful anti‐proliferative activity against both cell lines. It is obvious from Figs. 9 a and b and Table 1 that levels of % cell inhibition of HUH‐7 and HEK‐293 cell lines were elevated as there was an increment in the concentration of AgNPs. The IC₅₀ cell inhibition of CCAgNPs against HUH‐7 and HEK‐293 cell lines was found at 10.29 and 22.14 µg/ml. The IC₅₀ cell inhibition of silver nitrate against HUH‐7 and HEK‐293 cell lines was found at 57.32 and 46.60 µg/ml. The results of in vitro anticancer activity for HUH‐7 and HEK‐293 were found different, as variation in concentration. From the consequences, the cancer cell lines reveal higher sensitivity to anticancer drugs. The debilitated survival cell lines demonstrate a potential cytotoxic impact of CCAgNPs.

Fig. 9.

In vitro cytotoxic activity of AgNPs

Table 1.

MTT assay of CCAgNPs on HuH‐7 and HEK‐293 cell lines

Concentration, µg/ml	% Cell inhibition
	AgNPs (HUH‐7 cell line)	Silver nitrate (HUH‐7)	AgNPs (HEK‐293 cell line)	Silver nitrate (HEK‐293)
50	82.34	46.79	79.21	40.23
30	76.47	38.28	69.89	33.78
25	67.29	24.12	52.45	20.38
20	34.98	13.24	28.78	11.90
10	12.57	5.42	9.56	3.67
control	0	0	0	0

The potential application of biosynthesised AgNPs seems to be tremendously favourable due to their pivotal role as an antiangiogenic agent. Kim and Choi [44] explored the underlying mechanism of anticancer activity of biosynthesised AgNPs on human cell lines. They specified that AgNPs were engrossed by a different process such as pinocytosis, endocytosis and phagocytosis on human cells. AgNPs interact with cellular materials of the cell, destructs DNA of the cell and leads to cell death. In other reports, it was suggested that AgNPs which implicates commotion of the mitochondrial respiratory chain and disruption of ATP synthesis and cause DNA damage [45].

The dose‐dependent enhancement was found on cell inhibition after CCAgNPs treatment. The exploration revealed that the cell inhibition of cell lines increased with increasing concentration (10–50 μg/ml) of CCAgNPs after a time duration of 24 h. This study approved the dose graded tactic to the assessed toxicity of AgNPs on the HUH‐7 and HEK‐293 cell lines. The cancer cell lines were treated with CCAgNPs at varying concentrations shows significant morphological alteration (unique characteristics of apoptotic cells), such as disruption of cell membrane integrity, shrinkage of the cell and low cell concentration. Biosynthesised AgNPs using methanolic leaves extract of Euphorbia helioscopia were found to be effective against human epithelial cells (HeLa) and L929 cells [46].

The lower IC₅₀ values of HuH‐7 and HEK‐293 represent the prospect of CCAgNPs to be a potential anti‐proliferative agent. It was concluded from MTT assay that, data of mortality acquired from the present research displays the incidences of dead cells are notably more than in cancer cell lines and synthesised AgNPs kills both types of cells, even though, the degree of destroying cells was higher in cancer cell lines. The detected outcomes unquestionably favoured the implication that CCAgNPs provide new expectation as anticancer agents.

3.10 Antimicrobial activity of AgNPs

From long times, silver metals and silver derived compounds have been used as antibacterial agents and used to store water to provide silver elements in the body. We investigated biosynthesised AgNPs as potential antimicrobial agents. It was used to test the antimicrobial activities towards gram‐positive as well as gram‐negative bacteria displaying inhibition zones. On the basis of the inhibition zone results, AgNPs displayed significant antimicrobial activity against MRSA (22 ± 0.9 mm), S. sonnei (20 ± 0.16 mm), S. boydii (25 ± 0.34 mm) and S. typhimurium (28 ± 0.71 mm) as compared to standard drugs. The consequences of antibacterial activities of biofabricated AgNPs appraised by the agar well‐diffusion technique are portrayed in Table 2.

Table 2.

Antibacterial activity of AgNPs against various types of bacteria

Bacteria	Tetracycline	Ampicillin	Neomycin	AgNPs
		Inhibition zone, mm
MRSA	18 ± 1.62	0.0 ± 0.0	7 ± 0.1	22 ± 0.9
S. sonnei	15 ± 1.01	6 ± 0.2	12 ± 0.62	20 ± 0.16
S. boydii	21 ± 0.9	22 ± 0.61	19 ± 0.21	25 ± 0.34
S. typhimurium	18 ± 0.6	25 ± 0.28	26 ± 0.31	28 ± 0.71

This excellent capability of the biofabricated AgNPs using aqueous leaves extract of C. carandas will be key apprehension for the purpose of clinical and environmental protection. The antimicrobial activity of AgNPs on gram‐negative as well as gram‐positive bacteria may be substantial importance. The incubation period of the pathogenic microorganisms with biosynthesised AgNPs was observed to lower the colony forming bacteria. The inhibition rate was also raised after treating with AgNPs and reduction was observed in MRSA, S. sonnei, S. boydii and S. typhimurium, respectively. The improved inhibition of AgNPs because of its capability to bind the peptidoglycan content presents in the cell wall of bacteria and disrupt the membrane of bacteria. It forms pits in the membrane which cause leakage of cellular matter in the culture media [47]. The destruction produced by AgNPs caused inhibition of respiratory chain dehydrogenases and leads to cell death. When AgNPs comes in the contact of the cell, it affects the protein and phospholipid content that leads to shrinkage, putrefaction and cell death [48].

Several approaches have been suggested to enlighten the inhibitory effects of AgNPs on bacteria. It is also presumed that silver shows a high affinity towards sulphur and phosphorus, which is the main component for the antibacterial effects. The bacterial cell membrane contains a sulphur contain proteins in large quantity, AgNPs reacts with sulphur‐containing proteins present in the cell and affects the viability of cells [49]. It was also proposed that AgNPs released silver ions in the bacterial cell and interact with the phosphors molecule of DNA and inactivate the replication of DNA. It also interacts with sulphur‐containing amino acids and inhibits the functions of enzymes and resulting in the death of bacterial cell [50].

4 Conclusion

Cancer kills near about seven million people every year worldwide. In present times, reliable and potent drugs are available from a plant source which are non‐toxic, economic, fast and eco‐friendly. Facile green synthesis of AgNPs shows a significant effect against bacteria and cancerous cells and could be used as a pioneer drug in future. This investigation may offer an innovative opportunity for the survey of traditional plants for the fabrication of AgNPs to discover a therapeutic anticancer, antioxidant and antimicrobial agent. This study demonstrates the biosynthesis of AgNPs utilising the aqueous extract of leaves of C. carandas. It can be concluded from the study that it can be explored for it potential effect in the field of pharmaceutical and biomedical applications.