Biofilm reduction, cell proliferation, anthelmintic and cytotoxicity effect of green synthesised silver nanoparticle using Artemisia vulgaris extract

Abstract

Infectious diseases are caused by etiological agents. Nanotechnology has been used to minimise the effect of clinical pathogens which have resistance to antibiotics. In current research synthesis, characterisation and biological activities of green synthesised nanoparticles using Artemisia vulgaris extract have been done. The characterisation of AgNPs was carried out using Fourier transform infrared spectroscopy, UV‐Vis spectrophotometry, and scanning electron microscopy. Anti‐biofilm, cell viability, antibacterial, brine shrimp lethality, and deoxyribonucleic acid protection effects have been screened. UV‐Vis spectra showed the absorption peak of synthesised nanoparticles at 400 nm. FT‐IR indicated the involvement of the functional group in the preparation of AgNPs. SEM showed the spherical shape of AgNPs with 30 nm diameter. Biological screening results revealed the antibacterial effect against clinical bacterial pathogens. Biofilm reduction and cell viability assay also supported the antibacterial effect. Cytotoxicity effect was recorded as 100% at 200 μg/ml through brine shrimp lethality assay. Protein kinase inhibition zones recorded for AgNPs (16 mm bald) compared with A. vulgaris extract (11 mm bald). It has been concluded that green synthesised AgNPs are more effective against infectious pathogens and could be used as a potential source for therapeutic drugs.

1 Introduction

There is a severe concern about evolving contagious infections and the rising drug resistance in bacterial pathogens. Pathogenic bacteria are causative agents of various infections such as stomach pain, chill fever, diarrhoea, headache, nausea, fever and pain in muscle followed by vomiting, abdominal pain, cramps, and diarrhoea containing blood and mucus [1]. Regardless of greater awareness about microbial pathogenesis in addition to modern therapeutics applications, the rate of morbidity and mortality is still increasing day by day due to the microbial infections [2]. Thus, there is a pressing demand for the discovery of innovative approaches and identification of new antimicrobials from mineral and natural materials for the development of novel drugs and antimicrobial agents for controlling microbial infections for the next generation. Differently sized nanomaterials including organic and inorganic nanoparticles have found their applications as modifications in therapeutics, synthetic textiles, food packaging products, industrial and medicine due to the developments in the field of nanotechnology and nanoscience [3].

Silver nanoparticles are considered as most effective nanoparticles due to the good antimicrobial efficacy [4]. Silver nanoparticles are certainly the most extensively used nanomaterials amongst all, thus they are used as antimicrobial agents for water treatment, in sunscreen lotions, textile industries etc. [5]. Green synthesis is a new era of research all over the world [6]. For the production of silver nanoparticles (AgNPs) enormous section of flora had been exploited through different plant parts, i.e. leaves of neem [7], tannic acid [8], cinnamon bark [9], fruit extracts of Emblica officinalis [10] and dried leaves of Cinnamomum camphora were used for AgNPs synthesis [11, 12].

Artemisia vulgaris L. (mugwort) a medicinal plant is the member of family Asteraceae, this family comprises of over 1100 genera and possibly as many as 20,000 species [13], and has been extensively employed to cure diabetes in folk medicine. Its excerpts are employed for the treatment of depression, epilepsy, irritability, stress, psychoneurosis, uterine cancer, hepato‐protective activity and anxiety [14, 15, 16, 17, 18]. Malaria and fevers are also treated with leaf extracts in Northern areas of Pakistan [19]. Likewise, leaf extract is also employed for the cure of stomach, diabetes and ophthalmic diseases in Kashmir, Sudhan Gali, Pakistan [20].

The aim of the current research was to synthesise the AgNPs by a green biological route [using A. vulgaris aqueous (AVaqu) extracts] and characterisation of the synthesised nanoparticles utilising UV‐visible spectroscopy, scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT‐IR) analysis. The antibacterial and cytotoxicity effects of green synthesised nanoparticles were also evaluated via various research methods.

2 Materials and methods

2.1 Plant sampling and identification

Aerial parts of the medicinal plant were collected from Chella, Muzaffarabad, Azad Jammu and Kashmir, Pakistan during July and August 2015. The plant was identified by Prof. Dr Zahid Malik (Rtd) from the Department of Botany, University of Azad Jammu and Kashmir, Pakistan. Furthermore, the plant specimen was identified by Dr Abdul Rehman Niazi (Botanist) at M.S. Zahoor Memorial Herbarium (LAH), Department of Botany, University of the Punjab, Lahore, Pakistan and the voucher specimens were deposited. The accession number was allotted as Herbarium No. 405015.

2.2 Extract preparation

Arial parts of A. vulgaris L. were washed thoroughly 2–3 times with running tap water to remove dust and dried under shaded area for 1 to 2 weeks. The dried samples were powdered with the help of a grinder. For the extract preparation, 5.05 g of powder was added in 100 ml of distilled water and stirred at 60°C for 20 min. After boiling, the mixture was cooled and filtered with Whatman paper no. 1. The filtrate was collected.

2.3 Green synthesis of AgNPs

A. vulgaris extract was used for the synthesis of green nanoparticles (AVAgNPs) and silver nitrate (1 mM) was used as a source of metal. Synthesis of AgNPs was done according to the method as described by Zargar et al. [21] with slight modifications. For this purpose, 20 ml of A. vulgaris extract was added to 5 ml of AgNO₃ (1 mM). The mixture was heated up to 100°C until the colour changed from light green to blackish green, which indicates the preliminary conformation of AgNPs. The obtained nanoparticles were purified by centrifugation at 15,000 rpm for 30 min. The nanoparticles were washed thrice with distilled water then dried in an oven overnight. Dried nanoparticles were dissolved in dimethyl sulphoxide (DMSO) via sonication and stored at room temperature for further use.

2.4 Characterisation of AgNPs

The initial characterisation of green synthesised nanoparticles (AVAgNPs) was done using a UV–Visible spectrophotometer (SHIMADZU UV‐1601). Absorption spectra were recorded between 200 and 800 nm. The shape and size of the synthesised nanoparticles were determined using a field emission scanning electron microscope (SEM). The pellets of crude extract and AgNPs of A. vulgaris were subjected to FT‐IR spectrometric analysis (SHIMADZU FTIR‐8400S).

2.5 Antibacterial effect of AgNPs

2.5.1 Test microorganisms

Seven human clinical bacterial pathogens such as Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Staphylococcus epidermidis, Serratia marcescens, Staphylococcus aureus and Streptococcus pyogenes were used.

2.5.2 Agar well diffusion assay

The antimicrobial activity was assessed by the agar well diffusion method [22]. Nutrient broth media (oxide: CM1) were used for bacterial culture and Muller Hinton agar (oxide: CMO337) used for the antibacterial activity. The microorganisms were activated by inoculating a loop full of strain in 25 ml of nutrient broth medium and incubated at 37°C on a rotary shaker for 24 h. The overnight culture was mixed with a freshly prepared Muller Hinton agar medium at 45°C and was poured into the sterilised Petri dishes. All Petri dishes were kept at room temperature in laminar flow for solidification. In each plate, three wells of 5 mm diameter were made using a 1 ml of sterilised micropipette tip and the sterilised needle was used for the removal of agar plug. Approximately 30 µl of each AVaqu (50 mg/ml), AVAgNPs, AgNO₃, and control solvent sample (DMSO) were placed in each prepared well and placed at 37°C for 24 h. DMSO was also used as a negative control and discs of chloramphenicol (10 µg/ml) were used as positive control. Microbial growth was determined by measuring the diameter of the zone of inhibition after 24 h in millimetres [23]. The diameter of the clear zones (if >5 mm) around each well was measured with the help of a scale [24].

2.6 Spread plate count method

The spread plate technique is used to count the number of bacterial colonies in a Petri dish [25]. For the spread plate count method, all tested pathogens were treated with AVAgNPs, AVaqu, Chloramphenicol, and AgNO₃ for overnight at 37°C. Next day all treated samples (10 µl) were evenly distributed over the solidified Mueller–Hinton agar (CM 0337) and placed at 37°C overnight. After incubation, colonies were counted.

2.7 Anti‐biofilm assay

Crystal violet assay was used for anti‐biofilm activity with slight modifications [26]. Bacteria were grown in borosilicate tubes (Minitek, USA) containing 2 ml of nutrient broth medium and 30 mg/ml of AVaqu and AVAgNPs at 37°C overnight. Silver nitrate and chloramphenicol were used as controls. The negative control tubes contained nutrient broth only. After incubation, broth medium was removed and attached cells were stained by adding 125 μl of a 0.1% crystal violet. Borosilicate tubes were incubated at room temperature for 10–15 min and rinsed with water to remove excess unattached cells and dye. After staining biofilm, crystal violet was solubilised by adding 30% acetic acid and incubated at room temperature for 10–15 min. Solubilised crystal violet was quantified at 550 nm using a spectrophotometer. 30% acetic acid in water was used as the blank.

2.8 Cell viability assay

MTT (tetrazolium dye) assay was used for estimating the viability of bacterial cells [27]. For this, 0.2 mg/ml MTT (Genebase, china) was dissolved in DMSO and incubated at room temperature for 1–4 h. Bacterial cells (100 μl) were grown at 37°C in nutrient broth medium (NBM) (3 ml) overnight. Next day bacterial cells (100 μl) from an overnight culture were grown at 37°C in NBM (1 ml) for 3 h (exponentially growing cultures) by adding 100 μl to each test sample. To initiate the reduction reaction, 10 μl MTT stock solution was added to the mixture and was incubated at 37°C for 2–4 h (without shaking) with the tube cap open. Purple colour appears which indicated the formation of formazan crystals. To dissolve the crystals 500 μl of DMSO was added. The formazan crystals were dissolved at room temperature. The absorbance of each sample was measured at 570 nm using a spectrophotometer. DMSO used as a control.

2.9 Comet assay

A comet assay was performed to determine deoxyribonucleic acid (DNA) damage [28]. The comet assay (single‐cell gel electrophoresis) is a simple method for measuring DNA strand breaks in prokaryotic as well as eukaryotic cells. Bacterial cells were grown in 1 ml NBM with 100 μl of each test sample at 37°C overnight. Next day treated cultures were centrifuged at 15,000 rpm for 2 min in 1 ml Eppendorf tubes. A supernatant formed after centrifugation was removed carefully with the help of a micropipette. Bacterial cells collected in the form of pellets at the bottom were suspended in 50 μl of phosphate buffer saline solution (NaCl: 8 g/l, KCl: 0.2 g/l, phosphate buffer: 1.42 g/l, pH maintained at 7.5) and then immediately mixed with 500 μL of low melting agarose (0.3%, 0.3 g/100 ml) and spread on a microscopic slide. After the solidification of agarose, slides were immersed in lysis buffer (NaCl: 146.1 g/l, EDTA: 37.2 g/l, Trizma base: 1.2 g/l) at 4°C overnight. Next day slides were then immersed in alkaline solution (NaOH: 0.6 G/50 ml, EDTA: 3.72 g/50 ml) for 30 min. Then slides were placed in gel electrophoresis apparatus containing 10× TBE buffer (tris base: 108 g/l, boric acid: 55 g/l, EDTA: 9.3 g/l) to allow unwinding of DNA and the alkali labile damage. Next, an electrical field (100 V) was applied for 20 min to draw the negatively charged DNA toward the anode. After electrophoresis, slides were dipped in 70% ethanol (70 ml ethanol in 30 ml dH₂ O) for 5 min followed by staining with 100 μL of ethidium bromide (1× = 1 ml/9 ml dH₂ O) and then the slides were observed using a confocal microscope.

2.10 Protein kinase inhibitor assay

Streptomyces strains are used in protein kinase inhibition assay according to the procedure described by Fatima et al. [29] with minor modifications. The whole experiment was carried out under a sterile condition. An incubated Streptomyces strain was inoculated into tryptone soy broth and poured into Petri plates where the test organisms were grown. Filter paper discs were kept on the surface of prepared loan media and poured a small amount of the sample and standard on that surface. Surfactin were used as positive control while DMSO was used as negative control. Incubation period was applied 24–36 hours for target strain growth development. Formation of the bald or clear zone around the disc shows inhibition of spores and mycelia.

2.11 Brine shrimp toxicity assay

The brine shrimp lethality assay was performed by following the procedure as described by Bibi et al. [30] with minor modifications in a 96‐well polystyrene plate. Brine shrimp (Artemia salina) larvae were hatched at 37°C in a bi‐partitioned tank filled with artificial seawater. Incubation temperature was kept at 21–30°C. After two days, the hatched mature nauplii were collected in a small beaker. A stock solution of 100 mg/ml of each extract was further diluted into 200, 100, 50 and 25 µg/ml concentration for lethality test. The mentioned concentration dilution was transferred into each separated well containing 20 nauplii and sea water addition with dried yeast. The death rate of these larvae against all dilutions was observed and calculated. Negative control contains only seawater and nauplii while positive control contains standard drug doxorubicin 4 mg/ml, seawater and nauplii. The final volume is adjusted and kept under fluorescence light at room temperature for 24 h. The test was repeated three times and the number of dead nauplii was counted in each well. LD₅₀ was measured by comparing the standard drug with percentage growth inhibition.

2.12 Anthelmintic activity

Adult earthworms were collected from the region of University of Azad Jammu and Kashmir, Muzaffarabad, Pakistan for the evaluation of in vitro anthelmintic activity [31]. The earthworms were washed with distilled water to remove sand. The earthworms (3–5 cm in length and 0.1–0.2 cm in diameter) were used for the whole experiment. The anthelmintic activity of aqueous extract of A. vulgaris, AVAgNPs, AgNO₃, distilled water and normal saline solution were tested. The worms were divided into six groups containing fives earthworms of approximately equal sizes, placed in 250 ml beakers separately. Normal saline used as positive and distilled water as negative control groups. The time taken to paralyse or cause death of the individual worm was observed. Paralysis was said to occur when the worms do not revive even in normal saline. Death was ascertained when the worms lost their motility followed by fading away of their body colour. The movements of the earthworms were noticed after every 10 min.

2.13 Statistical analysis

Each experiment was repeated in triplicate and standard deviation (SD) from absolute data was calculated (http://easycalculation.com/statistics/standard‐deviation.php).

3 Results and discussions

In current research, AgNPs using AVaqu extract were synthesised, characterised and evaluated for antibacterial, in vitro cytotoxicity and DNA protection activities.

3.1 Synthesis of green nanoparticles

An aqueous extract of the medicinal plant was prepared through maceration and boiling methods. The pH of the A. vulgaris extract was 5.49. Synthesis of AVAgNPs in AVaqu extract was identified when the colour changed. The colour of A. vulgaris AgNPs (AVAgNPs) changed from light green to blackish green on heating which indicates the preliminary conformation of AgNPs [32]. The change in colour of the solution is due to the reduction of silver salt. Phytochemicals like amino acids, proteins, carbohydrates, glycosides, phenolics, steroids, and terpenoids involved in the formation of strong capping on nanoparticles. A. vulgaris rich in flavonoids provides stability to the AgNPs. According to Jha et al. [33] phytochemicals were involved in the reduction of ions and the formation of nanoparticles.

3.2 Characterisation of green nanoparticles

3.2.1 UV‐vis spectra analysis

After the reduction process, the formation of AgNPs was examined and confirmed by obtaining the respective absorption spectra. Fig. 1 a shows the absorption spectra of AgNPs obtained from the reaction of A. vulgaris extract and AgNO₃ recorded in the range of 200–800 nm. The absorbance of AVAgNPs was at a maximum of 400 nm, which indicates the synthesis of AgNPs. Our findings are consistent with Shankar et al. [34].

Fig. 1.

Characterisation of green synthesised AgNPs using A. vulgaris extract

(a) UV‐vis spectrophotometer analysis, (b) Scanning electron microscope, (c) FTIR spectra

3.2.2 Scanning electron microscopy

Advanced microscopic techniques are used generally to characterise the nanoparticles by their size, morphology and surface charge. In the current research, SEM is used to characterise the size and morphology of nanoparticles. The spherical shape of green synthesised nanoparticles was observed (Fig. 1 b). The average size of bio‐green nanoparticles was found to be 35 nm as shown in Fig. 1 b. Several other investigators have observed the absorption maxima of colloidal silver solution between 410 and 440 nm, which are assigned to the surface plasmon of various metal nanoparticles [35]. Electron microscopy techniques are very useful in ascertaining the overall shape of polymeric nanoparticles, which may determine their toxicity [36].

3.2.3 FT‐IR spectra of green synthesised nanoparticles using A. vulgari

FT‐IR is used for the identification of the functional groups of the compounds. The peaks for AVAgNPs are observed at 673.11, 1411.8, 1652.88, 1683.74, 2345.28, 2821.66, 2921.96, 2968.24, 3026.1 and 3058.89 (Fig. 1 c). All peaks appearing in the spectrum were assigned to O–H of alcohol, N–H of amines, C–H of alkanes, C=O of carboxylic acid/aldehydes or ester, N–C=O amide I bond of proteins, H–C=O: C–H stretch of aldehydes, C≡N stretch of nitriles, N–O of nitro compounds, C–N of aliphatic amines or alcohol/phenol, N–H deformation of amines, and C–C bending, respectively. These shifts in the functional groups indicate that carboxyl, hydroxyl, amide, and aldehydes may participate in the process of nanoparticle synthesis. Current results are consistent with the findings of previous results [37]. FTIR analysis confirmed that the reduction, which occurs in Ag+ ions to form AgNPs, is due to the reduction substances found in plant extract [38]. Prasad et al. [39] demonstrated that the functional groups like carboxyl (–C=O), amine (–NH) and hydroxyl (–OH), present in leaf extract are generally involved in fabrication of AgNPs. Makarov et al. [40] revealed that the proteins and peptides that exist in plant extract play an essential role in determining the nanoparticles’ shape and morphology. Amino acids such as arginine, cysteine, lysine, and methionine are capable of binding silver ions.

3.3 Antibacterial activity

Various species of Artimesia herbs such as A. absinthium (wormwood), A. dracunculus (tarragon), and A. abrotanum (southernwood) have been tested for biological activities such as anthelmintics, wounds treatment, anti‐ulcers, and anti‐tuberculosis activities [41, 42]. Various scientists reported the medicinal importance of Artimesia species such as anti‐malarial, anti‐diabetic, anti‐oxidant, anti‐pyretic, cytotoxic, anti‐microbial, analgesic, and anti‐fungal activities [41, 42, 43, 44]. In the current research, the antibacterial activity of A. vulgaris is evaluated against clinical bacterial pathogens through the agar well diffusion method. Results reveal the low sensitivity of all tested pathogens when treated with aqueous extract and green synthesised AgNPs at 30 mg/ml concentration. The range of zone of inhibition was recorded as 0.33 ± 0.57 mm to 3.0 ± 0.0 mm. Silver nitrate also had a low effect on the growth of tested pathogens. Our results are in agreement with the findings of Ahmadizadeh et al. [45]. They reported the minimum inhibitory concentration effect of A. vulgaris extract on E. coli. Ahmadizadeh et al. [45] observed that A. vulgaris extract had no effect at low concentration. On the other hand, A. vulgaris extract had no effect on the growth of P. aeruginosa at all tested concentrations.

DMSO had no effect while chloramphenicol showed the maximum inhibition of all tested pathogens. The zone of inhibition was recorded as 20.0 ± 0.0 mm to 35.0 ± 0.0 mm, respectively (Table 1).

Table 1.

Antibacterial activity of green synthesised nanoparticles using AVaqu extract (AVAgNPs), AgNO₃ and chloramphenicol

Pathogen	Test samples – zone of inhibition in mm (mean ±  SD)
	AV(aqu)	AgNO3	AVAgNPs	DMSO	Chloramphenicol (10 µg)
E. coli	3.0 ± 0.0	3.0 ± 0.0	2.0 ± 0.0	—	20.0 ± 0.0
P. aeruginosa	1.0 ± 0.0	3.0 ± 0.0	2.0 ± 0.0	—	27.0 ± 0.0
S. marcescens	0.66 ± 0.57	2.0 ± 1.0	1.66 ± 0.57	—	24.0 ± 0.0
S. pyogenes	1.0 ± 0.0	3.0 ± 0.0	1.33 ± 0.57	—	20.0 ± 0.0
K. pneumoniae	1.0 ± 0.0	2.0 ± 0.0	1.33 ± 0.57	—	35.0 ± 0.0
S. epidermidis	1.0 ± 0.0	3.0 ± 0.0	1.33 ± 0.57	—	32.0 ± 0.0
S. aureus	1.0 ± 1.0	1.33 ± 0.57	2.33 ± 0.57	—	29.0 ± 0.0

Growth inhibition recorded as (0) for no sensitivity, (<1–3 mm) for low sensitivity, (>3–6) for moderate sensitivity and (>6–35) for high sensitivity.

Methanolic extracts of A. campestris were also evaluated for antibacterial properties by Naili et al. [46]. The extract was reported to have a strong effect on S. aureus and Bacillus subtilis strains. Kasture et al. [47] have been reported that the antibacterial activity of alcoholic A. vulgaris extracts against the Staphlococcus aureus. Antimicrobial activity of four Artemisia species of Iran was also illustrated by Ramezani et al. [48]. They reported that Iranian A. scoparia had no effect on the growth of P. aeruginosa. Ahmadizadeh et al. [44] demonstrated the limited effects on S. epidermidis and E. coli are similar to our results.

The antibiotics resistance of infectious pathogens made a problematic issue in developing countries, which needs to discover new natural alternates to overcome this problem [38]. The effects of green synthesised nanoparticles were analysed through the spread plate count method against seven human clinical bacterial pathogens such as E. coli, K. pneumonia, P. aeruginosa, S. epidermidis, S. marcescens, S. aureus and S. pyogenes. A. vulgaris synthesised nanoparticles had less effect on the bacterial growth. Some plates did not show any growth its mean sample is strong enough that it did not allow bacteria to grow (Fig. 2 a).

Fig. 2.

Biological screening of green synthesised AgNPs using A. vulgaris extract

(a) Spread plate method, (b) Antibiofilm assay, (c) Cell viability assay

3.4 Biofilm inhibition effect of AVAgNPs

A. vulgaris nanoparticles AVAgNPsB show the reduced biofilm of various bacteria viz., S. aureus, S. epidermidis, P. aeruginosa, S. marcescens, K. pneumoniae, and S. pyogenes compared with AVAgNPsA (Fig. 2 b). In some cases, A. vulgaris extracts play a role in the reduction of S. marcescens and S. epidermidis biofilm. Results revealed the bactericidal effect of green synthesised nanoparticles. In conclusion, our findings suggest that AgNPs are able to prevent biofilm formation and also it may be a necessary agent in related therapies against multi‐drug resistance. Especially, in clinical pathogenic infections, biofilm plays an important role in disease development. Our findings are consistent with [49]. Biofilm inhibition of P. aeruginosa, S. aureus and E. coli was also reported by Goswami et al. [50], Park et al. [51], and Kalishwaralal et al. [52] So, the data in the present study validate that the green synthesised nanoparticles can be effectively used against these bacterial pathogens as biofilm disrupting agents.

It has also been reported that many infectious diseases related to antibiotic resistant bacteria are booming and proliferating day by day. Those microbes that form biofilms cause these infections. Those common bacteria that cause human infections and form biofilms are S. epidermidis, E. faecalis, S. viridans, S. aureus, K. pneumoniae, E. coli, and P. aeruginosa [53]. In many other studies, the anti‐biofilm activity of AgNPs was also confirmed primarily by focusing on those bacteria that show resistance to conventional antibiotics [54]. Gurunathan et al. [55] evaluated the effect of antibiotics on anti‐biofilm and antibacterial activities of AgNPs, or mixtures of both against S. pneumonia, P. aeruginosa, S. aureus and S. flexneri. They showed the strong effect on ampicillin and vancomycin and proposed that antibiotics along with AgNPs could be the best possible substitute for many bacterial infectious diseases.

A remarkable evolution, which has been occurring in nanotechnology, is the use of silver‐coated magnetic nanoparticles used against bacterial biofilms. Indeed, these engineered nanoparticles made up of the magnetic core and a silver ring showed encouraging effects [56]. Nowadays, silver is used in medical devices to protect them from biofilm activity. The biofilms from isolates of clinical P. aeruginosa were treated with AgNPs of gum arabic revealing an inhibition in the growth of bacteria and proved beneficial in catheters’ treatment [54]. The composites having AgNPs are involved in dental applications, which work against the biofilm of S. mutans [57]. Moreover, current studies advise the AgNPs in wound dressing to prevent and decrease the growth of microbes on the wound and to fasten the results of healing [58].

3.5 Cell viability assay of A. vulgaris nanoparticles

Artemisia species are used for the treatment of various diseases like cancer, diabetes, hepatitis, and inflammation as well as infections caused by fungi, bacteria and viruses [59, 60]. Results reveal that AgNPs of A. vulagris (AVAgNP) and AVaqu extract produced a significant reduction in the viability of bacterial cells compared with control pathogens (without treatment). Similarly, AgNPs showed the significant reduction of S. aureus and S. pyogenes compared with chloramphenicol (Fig. 2 c). The reduction in cell viability depends upon dose or concentration. It was concluded that both medicinal plants exhibited the antibacterial effect. Current research indicated that both AgNPs and aqueous extracts involved in the disruption of the cellular mitochondrial membrane after treatment. Our findings are in agreement with the findings of Jeyaraj et al. [61].

3.6 Genomic DNA replication inhibition

Molecular studies revealed that reactive oxygen species (ROS) damaged the sugar moiety of nucleotides in DNA [62]. If these changes remain unrepaired, they lead to disturbing the cell structure. Medicinal plants have the ability to protect DNA by scavenging ROS due to their antioxidant potential [63]. Phytochemical constituents especially vitamins, flavonoids, phenols, and tannins are known to be responsible for the free radical scavenging antioxidant activities of plants. A. vulgaris is a potential source of natural antioxidants [64]. Due to the presence of antioxidants A. vulgaris s howed DNA protection activity. Ali et al. [65] tested the DNA protection activity of Artemisia absinthium and they showed that this medicinal plant protects DNA against H₂ O₂ induced oxidative damage. The stronger DNA protective effect of ten Asteraceae plants was also shown by Chethan et al. [66]. Luo et al. [67] have reported that the aqueous extract of Artemisia species presents the anti‐microbial activity. Radford [68] reported that AgNPs killed bacteria by a highly bioactive silver ion through binding with a protein of the bacterial cell. Singh et al. [69] have suggested that AgNPs destroy the bacterial cell by ribosomes destabilisation, cell wall disruption. Comet assay also showed that AgNPs have no effects on DNA of bacterial pathogens. The DNA of bacterial pathogen is not damaged.

3.7 Cytotoxicity assay of AVAgNPs

In cytotoxicity assay, AVAgNPs showed outstanding results when compared with AVaqu extract at 100–200 μg/ml concentration (Table 2). The mortality was about 40% in the case of control sample AVaqu at 200 μg/ml concentration. The cytotoxicity effect was found to be the directly proportional to the concentration of nanoparticles. The highest mortality rate of Artemia salina was observed at 100 μg/ml concentration (AVAgNP) while 50% mortality was observed at 83.33 μg/ml concentration. Hence, the results revealed that as the concentration increases, the effect of toxicity increases. Our findings are consistent with the findings of AshaRani et al. [70]. Zubia et al. [71] reported the potent cytotoxic effect of Laurencia obtuse extract and Asparagopsis armata on cancer cell lines. Doxorubicin was used as a positive control, which indicates 80% mortality at 200 μg/ml concentration. These results recommended further for the investigation of plant's cytotoxic potential using in vitro anticancer cell lines.

Table 2.

Brine shrimp cytotoxicity assay of green synthesised AgNPs (AVAgNPs) and A. vulgaris (AVaqu) extract

Test samples	Concentration used – number of brine shrimp killed after 24 h
	200 μg/ml	100 μg/ml	50 μg/ml	25 μg/ml
AVAgNPs	100.0 ± 0.0	70.0 ± 0.0	40.0 ± 0.0	10.0 ± 0.0
AVaqu	40.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0
doxorubicin	80.0 ± 0.0	60.0 ± 0.0	40.0 ± 0.0	0.0 ± 0.0

Values (mean ± SD) are an average of three individually analysed experiments (n  = 1 × 3).

3.8 Protein kinase inhibition assay

Silver nanoparticles AVAgNPsB showed the 14.0 ± 0.0 mm zone of inhibition compared with AVaqu (11.0 ± 0.0 mm) at 50 μg/disc. It may be due to the aqueous concentration of A. vulgaris used for nanoparticle synthesis. The results of the current study suggest that extraction of phytochemical of B. ciliata and A. vulgaris could be done that serve as protein kinase inhibitors. Protein kinases involved in cell differentiation, cell proliferation, apoptosis and metabolic processes. Protein phosphorylation occurred by protein kinases and deregulation of phosphorylation causes cancer. In this respect, we can say that inhibition of protein kinases by green synthesised nanoparticles has a promising target for the treatment of cancer [72].

3.9 Anthelmintic activity

Earthworms have an anatomical and physiological resemblance to the intestinal roundworm parasites of human beings. Moreover, they are easily available and are a suitable model for screening of anthelmintic drugs [73, 74]. The shortest time required for the paralysis and death of earthworms was observed with green synthesised nanoparticles AVAgNP as 25.0 ± 0.0 min, followed by the aqueous extract of A. vulgaris, which showed paralysis and death at 55.0 ± 0.0 min of exposure. The death time for silver nitrate was highly effective which occurred within 15.0 ± 0.0 min. The distilled water had no effect even after 55.0 ± 0.0 min of exposure. The lethal effect was attributed to the blocking of glucose uptake. The presence of various phytochemical constituents such as glycosides, tannins and saponins may be responsible for the anthelmintic activity. Our results are consistent with the findings of Tripathi [75]. Further studies are required to isolate the active bioactive compounds, which are responsible for the reported activity.

4 Conclusion

The present study aims to synthesise, characterise and reveal the antibacterial, anthelmintic, cytotoxicity and DNA protection activities of AgNPs using different in vitro assays. From results, it is concluded that AgNPs exhibited antimicrobial activity against various tested pathogens. Anthelmintic activity revealed the paralytic effect while the AgNPs cause death. Silver nanoparticles could be used against various infectious agents to overcome the problem of multidrug resistant developments.