It is a well-known fact that nanotechnology emerges as one of the most important interdisciplinary fields of science with wide range of applications. It involves the synthesis and manipulation of particle structure with a dimension of less than 100 nanometers in diameter . Nanoparticles possess diverse physio-chemical properties such as optical, magnetic, catalytic properties , and biological applications in antimicrobial [3, 4], antioxidant, anti-inflammatory, antiviral, cardiac protection, wound dressing and anti-cancerous activities . Nanoparticles (NPs) result in superior chemical reactivity, biological activity, and catalytic behaviour than their own larger particles [2, 6]. They are employed in major industries like food, chemical, feed, health, space, cosmetics etc. The industries preferably employ green synthesized nanoparticles for manufacturing its products [2, 7]. Even though there are many metal NPs were investigated , silver nanoparticles (AgNPs) are considered to be the best due to the fact that the disadvantages of AgNPs are negligible as compared to its advantages .
Silver nanoparticles are included in a set of zero dimensional substances that demonstrate natural structure under its dimension extending from 1 to 100 nm . .AgNPs manifest peculiar thermal conductivity, chemical stability, and Raman scattering phenomena [11, 12]. AgNPs also possess optical, electronic, chemical photo-electrochemical, magnetic, catalytic and biological properties [13, 14] (Fig.1). This is in continuation with the application of silver metal being employed in preserving food products , and manufacturing utensils from prehistoric time onwards . Now in the 21st century, AgNPs are used in broad range of applications, such as biomedical engineering, drug delivery, food industries, antimicrobial, textile industries, agriculture, water treatment (as an antioxidant), anticancer agent, larvicides, cancer cell therapy, component of ointments etc. . Among the reported antimicrobial properties of different metal NPs, it was found that AgNPs exhibited prominent bactericidal and fungicidal activity [17, 18].
Method of synthesis of silver nanoparticles
Broadly speaking, there are Top-down and Bottom-up approaches for the synthesis of metallic nanoparticles (Fig. 2). Top down method is the method of synthesizing nanoparticles in nanoscale level by plastic deformation from large sized patterns . In bottom-up method nanoparticles are formed by the self-assembly of miniaturized atomic components [19, 20]. Although numerous methods are envisaged for silver nanoparticle synthesis, the most conventional ones are physical and chemical methods . These procedures provide productive output; that are coupled with constrains such as the utilization of harmful reagents, great running costs, more requirement of energy etc. . Some other disadvantages of these methods include energy consumption, slow synthesis and requirement of high concentration [22-24].
Nowadays green synthesis of nanoparticles is accepted widely because of its simple, non-toxic, rapid, stable nature and also less expensive . Green synthesis utilizes environmentally eco-friendly raw materials like plant extract, algae, bacteria, fungi, and enzymes . The major advantage of using plant extract includes large scale production under non aseptic condition and absence of cell culture for the production of nanoparticles . Although a considerable number of plants as well as plant parts are used for the synthesis, here we summarize the reports on various synthesis protocols and bioactivities of silver nanoparticle using plant extract of Acanthaceae.
Phytochemicals characterized from members of Acanthaceae
Acanthaceae, a family of Agiopserms covers over 4,000 plant species and about 230 genera of plants present across the globe, which largely grow in subtropical and tropical places as primary diversity centres like the South American, Indo-Malay, African (comprising Madagascar), and Mexican-Central American areas . Members of Acanthaceae family are medicinally important due to the presence of various phytochemicals like glycosides, flavonoids, phenolic compounds, naphthoquinone, benzonoids, triterpnoids etc. [42-49]; because of the presence of these phytochemicals, they show antibacterial [26, 27, 28], antifungal [29, 30], cytotoxic , anti-inflammatory , antipyretic [33, 34], antiviral [35, 36], antioxidant , hepatoprotective , insecticidal , immunomodulatory , and anti-platelet aggregation properties [40, 41].
Green Synthesis of silver nanoparticles - Acanthaceae family members
There are different methods of AgNPs synthesis protocols which allow interaction between plant extract and metal salt either by mixing, heating, incubating, stirring or shaking. This interaction facilitates the formation of nanoparticles. The major functional groups present in the Acanthaceae family members play an important role in this synthesis of AgNPs.
AgNPs having a size ranging from 10-20 nm were synthesized using Justicia glauca phytochemicals possessing functional groups like –C-O-C- of carboxylic group, O-H stretching of hydroxyl group, carbonyl group of aldehydes or ketonic group . Moreover, these nanoparticles were formed due to the major contribution from C-O-C of phenolic compounds . Dried leaf extract of Indoneesiella echioides was utilized for the creation of silver nanoparticle with a mean size consisting of ~25 nm. The production was assisted due to various functional groups present in phytochemicals revealed by FTIR analysis like the occurrence of aromatic primary amine (C–N stretch), secondary amine (N–H bend), carbonate ions, organic nitrates and aliphatic fluoro compounds (C–F stretch, C–O and C–O stretching modes), carboxylic group, C–O–C, C–OH vibrations, –C–O and –C=C stretch vibrations and Amide II band . Oxidation of aldehydes to carboxylic acid of enzymes/proteins found in plant distillate resulted in nanoparticle formation. Silver nanoparticles were formed using Nilgirianthus ciliates leaf broth having hydroxyl group, carbonyl group, alpha CH3 methyl group of aldehydes and ketones and are characterized by UV-Vis spectra, ultra high resolution SEM, FTIR, DLS .
In another method, the aqueous fresh leaf extract of Thunbergia grandiflora with alkine, amine, alkyne, alcohol and aromatic compounds were used in the formation AgNP with an average size of 2.39 nm . Fresh leaves of Justicia adhatoda yielded AgNPs of average size 20 nm and characterized by UV–visible spectra analysis as well as TEM analysis . Leaf extract of Barleria prionitis containing amine, alkane, alkene and ether groups resulted by FTIR analysis, yielded 10-20 nm sized AgNP .
Treating aqueous leaf extract of Lepidagathis cristata having functional groups like secondary amide, carbonyl group and amide resulted in the formation of AgNPs of average particle size of 30 nm. The amide group of tryptophan present in the leaves significantly contributed in the reduction of the metal ion to nanoparticle . The mixture of Justitia adathoda leaf extract composed of functional group like C-C in alkene ring and C = C of aromatic ring, ether linkage and C-N of amine and O-H of secondary alcohol resulted in the formation of nanoparticle of size 18 nm. Furthermore, presence of phenolic and aromatic compounds helped in the stabilization of NPs .
Fresh leaf extracts of Asystasia gangetica containing functional groups like alkyl halide, aromatic amine, primary amine, OH group of alcohol and phenols, CN bond of nitride, C=O bond of carbonyl group were exploited in the assembly of AgNP having a dimension of 40 – 60 nm. . Using methanolic leaf extract of Clinacanthus nutans, AgNPs were synthesized with an average size 46.4 - 53.8 nm here, carbonyl group contributed by different flavonoids involved in the formation of AgNPs .
Green synthesis of nanoparticle is the synthesis of nanoparticle using plants. Various plant parts can be used for the synthesis viz. stem, leaves, root, rhizome, flower, seed etc. Plants produce diverse bioactive secondary metabolites with different functional groups. These functional groups metal ions resulting in the generation of nanoparticles. The FTIR analysis revealed that different functional groups were associated with these processes . Phenolic compounds hold carboxyl and hydroxyl groups which can disable iron ions by chelating and further inhibiting the superoxide driven Fenton reaction. This is presumed to be the most crucial supply of reactive oxygen species (ROS). Therefore, plants with a high content of phenolic compounds are one of the best candidates for nanoparticle synthesis .
The research by Jha et al revealed that phytochemicals particularly glutathiones, polyphenols, ascorbates and metallothioneins were certainly responsible for the generation of nanoparticles . Shankar et al communicated the biosynthesis of pure metallic silver nanoparticles and they were stabilized by reducing sugars and/or terpenoids contained in the leaf broth of Azadiracta indica . Polyols, as for example, terpenoids, flavones and polysaccharides in the leaves of Cinnamomum camphora were considered as the main agents to reduce of silver as well as chloroaurate ions . Li et al reported that proteins with amine groups and vitamin C present in the extract of Cinnamomum annuum L. were accountable for the Ag nanoparticles formation and rapid precipitation . High density of highly steady silver nanoparticles were quickly synthesized using leaf extract of Datura metel which comprised of biomolecules like alkaloids, polysaccharides, amino acid, proteins/enzymes, and alcoholic compounds which could be employed as scaffolds to accelerate the formation of silver nanoparticles in solution phase . Quinol (alcoholic compound) and chlorophyll pigment resulted in the reduction of silver ions and normalization of nanoparticles . Purely natural constituents and its utilization help in the reduction and stabilization of metal nanoparticles (Fig.3) is still in exploration phase .
Some other factors responsible for the nanoparticle formation are reaction and incubation temperature. Rise in temperature boosts the speed of generation of nanoparticles. High temperature along with increased concentration of plant extract helped the formation of small sized NPs . A compacted report of silver nanoparticle synthesis of Acanthaceae is reported in Table 1.
Bioactivity of AgNPs – Antibacterial
Silver ions and silver salts were utilized for therapeutic application starting from the period of human urbanization. Silver ions and silver salts are now utilized in the form of antibacterial agent especially for wound dressing . Among all other metals, silver shows higher antimicrobial activities . It works against 650 microorganisms, including bacteria, fungi, virus, and eukaryotic microorganisms . A summary of the findings is reported in Table 2.
It was observed such that gram positive bacteria are much immune towards AgNPs compared to gram negative bacteria. Since gram negative bacteria are coated with lipopolysaccharides, they show negative charge and therefore get attached with positively charged silver ions. Gram-positive bacteria are enveloped by thick layer of peptidoglycans and linear polysaccharides which offer rigidity to the cell and thus avoid the binding of NPs to its surface. In case of gram-negative bacteria, AgNPs penetrate into the cell by forming holes in the cell wall of the bacteria [69, 70].
Bioactivity of AgNPs – Antifungal
A few reports of antifungal activities are reported on green synthesis of AgNPs employing Acanthaceace family members; for example, AgNPs synthesized using Rhinacanthus nasutus barred the growth of Aspergillus niger and Aspergillus flavus with the zone of inhibition 18.66 ± 1.52mm and 17.66 ± 1.52mm respectively . Justicia spicigera assisted AgNPs synthesis (using a concentration of 100 mg/ml) showed antifungal activity towards Macrophomania phaseolina (ZoI = 80.95 ± 1.35 mm), Alternaria alternate (ZoI = 62.12 ± 4.50 mm), Fusarium solani (ZoI = 35.60 ± 3.55 mm) Colletotrichum sp. (ZoI = 40.16 ± 2.35 nm) in PDA medium. AgNPs initiates various changes in the hyphae, cell wall and the germination pattern of fungus . The Ag+ ions released by AgNPs get attached with thiol group of cystein proteins of fungus leading to the death of fungus . Antifungal activity decreases with increase in complexity of cellular organization of organisms, which interrupt the movement of the AgNPs into the cells [73, 74].
Bioactivity of AgNPs – Biocompatibility
AgNPs synthesized from Barleria cristata were tested for its safety towards non-target organisms like Diplonychus indicus, Anisops bouvieri, and Gambusia affinis, which are mainly insect predators in ponds. Treatment resulted in negligible toxicity against organisms tested . Justicia jendaracia mediated AgNPs study revealed that they did not cause any distinct effect on soil enzyme activity, soil macronutrients, soil microbial population and plant growth parameters of Vinga mungo . Thus, the AgNPs are very safe to use; as it is less toxic to useful organisms, as well as it does not alter favourable conditions of plant growth.
Bioactivity of AgNPs – anti-inflammatory activity
There are limited reports of anti-inflammatory activities on green synthesis of AgNPs by employing Acanthaceace family members. Hemigarphis colorata mediated synthesis of AgNPs, exhibited anti-inflammatory activity or inhibition of protein denaturation through tests namely, inhibiting heat induced albumin denaturation and membrane stabilizing test of RBC. Denaturation of protein causes inflammation. AgNPs synthesized using Hemigarphis colorata plant extract showed higher protein inhibition compared to its original plant extract. Moreover, results of membrane stabilization test also demonstrated that AgNPs could show higher values in the inhibition of hemolysis. Both tests demonstrated that AgNPs could act as an effective anti-inflammatory drug .
Bioactivity of AgNPs – Anticancer
In this case also, a few reports are available, for example, AgNPs formed from Andrographis echioides demonstrated anticancer property towards the human breast adenocarcinoma cancer cell line (MCF-7) by creating free radicles like reactive oxygen species or boosting intracellular oxidative stress . AgNPs synthesized by utilizing Justicia adhatoda exhibited anticancer property towards HeLa cell lines of cancer cells . Anticancer property of AgNPs was established towards human lung adenocarcinoma cancer cells (A549) based on AgNPs derived from Indoneesiella echioides .
Bioactivity of AgNPs – Antioxidant effect
Indoneesiella echioides mediated AgNPs show antioxidant property using tests namely ABTS scavenging assay and DPPH scavenging assay . AgNPs synthesized by employing Justicia gendarussa demonstrated significant antioxidant property. They were tested for ABTS scavenging assay, DPPH scavenging assay and against nitric oxide . AgNPs formed using Andrographis serpyllifolia presented strong antioxidant activity in assays like DPPH, NO and H2O2 . AgNPs act as a natural antioxidant or efficient free radical scavenger against the free radicles which damages cell by means of oxidative stress, which in turn triggers injuries or apoptosis in organisms .
Bioactivity of AgNPs – larvicidal potential
Andrographis serpyllifolia leaf extract assisted synthesis of AgNPs showed prominent larvicidal effect on Culex quinquefasciatus. It possessed lower LC50 and LC90 values and higher mortality rate of larvae . AgNPs synthesized from Barleria cristata displayed strong larvicidal property towards Anopheles subpictus, Aedes albopictus and Culex tritaeniorhynchus  AgNPs, synthesized utilizing Nilgirianthus ciliates were experimented on Aedes aegypti, which presented higher efficiency and mortality rate. The suggested mechanism for the effective larvicidal property was due to the penetrating capability of AgNPs, which resulted in molting arrest as well as morphological deformation .
Bioactivity of AgNPs – Effect on Seed germination and growth
AgNPs prepared using Thunbergia grandiflora resulted in higher rate of germination in seeds of Vigna radiata with a concentration of 75% AgNPs. AgNPs also exhibited higher rate of shoot and root growth on treating with 25% and 50% AgNPs concentration respectively .
Synthesis of silver nanoparticles is effectively established using plants belong to Acanthaceae family. The phytochemicals present in the family members serve as both stabilizing and capping agents of the synthesized nanoparticles. They are best in their biological activities especially antibacterial action against broad spectrum of bacteria. Future studies will revolve the best mechanism of action against bacteria and the occurrence of particular phytochemicals of the family best suitable for the synthesis of nanoparticles.
CONFLICT OF INTEREST
There is no conflict of interest among the authors.