Phytochemical-assisted biosynthesis of silver nanoparticles from Ajuga bracteosa for biomedical applications

Silver nanoparticles (AgNPs) synthesized from plant extracts are widely used for the cure of many diseases from fever to cancers. Keeping in view the medicinal value of AgNPs, here we report a cost-effective phytochemical method for the biosynthesis of silver nanoparticles from Ajuga bracteosa. A. bracteosa is an important medicinal plant used to cure fever, appetite-loss, and cancer. Silver-nanoparticles were prepared from the aqueous extract of the plant. The methanolic extract of A. bracteosa (ABMF) was separated and n-hexane (ABHF) and chloroform (ABCF) fractions were obtained from the methanolic crude extract. The AgNPs were characterized by UV-Visible spectrophotometer, FTIR, XRD, and TEM. The total phenolic contents (TPC) and total flavonoid contents (TFC) in different fractions were determined and compared with AgNPs. The medicinal value of ABMF, ABHF, ABCF, and AgNPs was evaluated by antibacterial, antioxidant, anti-inflammatory, and cytotoxicity bioassays. The UV-visible spectrum showed a peak at 484 nm while FTIR results suggested strong capping of phytochemicals on AgNPs which was confirmed by a high amount of TPC and TFC. XRD analysis depicted a high degree of crystallinity and smaller size of AgNPs. TEM results showed spherical shaped AgNPs of size range 50 ± 12 nm. The biosynthesized AgNPs showed better antibacterial activity than plant extract fractions. Similarly, AgNPs have shown better antioxidant, cytotoxicity against cancer cell lines in-vitro, and anti-inflammatory activity in-vivo than a plant extract. The great medicinal value of A. bracteosa might be due to the presence of pharmacologically active phytochemicals such as diterpenoids, neo-clerodane flavonol glycosides, ergosterol, iridoid glycosides, phytoecdysones, and other polyphenols. These phytochemicals surround the silver nanoparticles during green synthesis and therefore, this capping of phytochemicals over silver nanoparticles results in enhanced biomedical applications of plant extracts.


Introduction
Metallic nanoparticles (MNPs) have emerged as potential biomedical and engineering tools for various applications including imaging [1], catalysis [2], bio-sensing [3], and drug delivery [4] due to their peculiar physicochemical and optoelectronic features. Silver nanoparticles, the most utilized MNPs, are commonly employed in the pharmaceutical industry for the manufacture of creams and ointments to prevent wounds and burns related infections [5]. Silver nanoparticles (AgNPs) are also used in diagnostics, orthopedics, drug delivery and treatment of infections, wounds, and cancer [6,7]. AgNPs are used in the development of new broadspectrum antimicrobials against a variety of pathogenic microorganisms [8]. Moreover, AgNPs have shown

Green synthesis of AgNPs
The 20 g of sterilized fine powder was poured in 200 ml of d.H 2 O and heated at 60°C-70°C for 15 min. The extract was filtered and its 100 ml was mixed with pre-warmed 400 ml of 1 mM solution of AgNO 3 with continuous stirring. The change in the color of the reaction mixture was recorded with a digital camera and quantitatively with UV-visible spectrophotometer. The reaction mixture was allowed to settle in the dark for 24 h and then centrifuged at 13 000 rpm for 20 min followed by washing once with d. H 2 O and thrice with ethanol followed by decanting supernatant and re-dispersing pellet. The washed pellet of AgNPs was dried in a desiccator with silica gel as a desiccant for more than 24 h and stored for further use.

Characterization of AgNPs 2.3.1. UV-Vis Spectroscopy
The optical properties and bio-reduction of Ag + ions into AgNPs in the reaction mixture were determined with Optizen 3220 UV double beam UV-vis spectrophotometer. For this purpose, the samples of the reaction mixture were taken at 1, 5, 10, 15, 30 and 60 min after mixing of a plant extract with 1 mM AgNO 3 solution and absorbance was recorded in the wavelength range 300-700 nm.

Fourier transmission infrared spectroscopy (FTIR)
To identify the biological agents responsible for the bio-reduction of silver ions and AgNPs stabilization, FTIR spectral analysis was determined with the Perkin-Elmer spectrum 65 FTIR spectrophotometer. The dried sample was minced with potassium bromide (KBr) pellets and analyzed with FTIR at a resolution of 4 cm −1 in the region of 4000 to 600 cm −1 .

XRD spectroscopy
The crystalline size of prepared AgNPs was carried out using x-ray diffractometer (D8 Advanced BRUKER AXS GmbH, Germany) at 40 kV and 30 mA with CuKα-radiation working between 10 and 80°of 2θ angles scanning at 2°/min.

Transmission electron microscopy (TEM)
TEM analysis was carried out for the determination of the morphology, shape, and size of AgNPs with HITACHI h-800, working at 200 kV. For the TEM-grid preparation, a drop of the reduced AgNPs solution was placed on a copper grid coated with carbon and drying under a lamp. The size of AgNPs was recorded by inbuilt software in the TEM.

Energy dispersive x-ray (EDX)
EDX is a technique used to assess the surface atomic distribution as well as the chemical elemental composition. Elemental analysis of AgNPs was assessed using the EDX detector attached to the SEM machine (Mira3-TESCAN).

2.4.
Comparative studies of AgNPs and plant extracts 2.4.1. Phytochemical analysis A. bracteosa contains a variety of phytochemicals including flavonol glycosides, neo-clerodane, diterpenoids, phytoecdysones, ergosterol, iridoid glycosides, and many other polyphenols [21]. These phytochemicals not only mediates the bioreduction of AgNPs but also adhere to these nanoparticles and improve the biomedical properties of nanoparticles. Here we quantified the polyphenols adhering to AgNPs and then compared them with polyphenolic contents of A. bracteosa extract.

Total phenolic content (TPC)
Folin-Ciocalteau reagent (FCR) process was used for the determination of TPC in plant fractions and AgNPs as reported by Ambreen et al 2019 [22]. The phenolic compounds present in samples were calculated from the calibration-curve of standard reference gallic acid. Methanolic solutions of different concentrations of 12.5, 25, 50, 100, 200, and 400 μg ml −1 of gallic acid, ABMF, ABHF, ABCF, and AgNPs were prepared. Sample solutions (0.5 ml) were mixed with 2.5 ml of FC reagent (10%) and 2.5 ml of 7.5% sodium carbonate solution and incubated at RT for 30 min. The absorbance was taken at 765 nm with UV-visible spectrophotometer. Three readings were taken for each sample and the mean value was taken. TPC was recorded as mg of gallic acid equivalents/g of sample (mgGAE/g) with the formula:  [22]. Briefly, the sample solution (1 ml) was mixed with an equal volume of AlCl 3 solution in ethanol and 5% sodium acetate solution (1.5 ml) and incubated at RT for 2.5 h. The absorbance of the reaction mixture was taken at 440 nm using a UVvis spectrophotometer. Readings of samples were recorded in triplicates and an average of three analyses was taken. Different concentrations of 12.5, 25, 50, 100, 200, and 400 μg ml −1 of rutin as standard reference were prepared to obtain a calibration curve. Total flavonoid content was measured as mg/g of rutin equivalent (RE) by using a calibration curve.
2.5. Biological evaluation of AgNPs 2.5.1. In vitro studies 2.5.1.1. Antimicrobial activity Antibacterial activity of AgNPs, ABMF, ABHF, and ABCF was tested using the disc diffusion method [23] against Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis and Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. Prepared media was added in pre-sterilized petri dishes and kept at RT to solidify. Bacterial strains were swabbed evenly on separate plates with sterile cotton swabs and incubated at 37°o vernight. The 10 mg ml −1 concentration of samples and control was used. The zone of inhibition was recorded in millimeters.

DPPH assay
The antioxidant activity of ABMF, ABHF, ABCF, and AgNPs was evaluated by DPPH scavenging activity following Hatano et al (1988) [24]. Briefly, different concentrations of the samples (50-250 μg ml −1 ) in methanol were prepared. The sample solution (1 ml) was added in 1 ml of DPPH (0.2 mM) solution in methanol. The resultant mixture was mixed robustly and placed in the dark for 30 min. The absorbance was taken at 517 nm against a blank. Then the scavenging activity was determined with the following equation: Where A control is the value of the control (having all other reagents excluding test sample) and A sample is the value of the sample.

Cytotoxic activity
Human HCT-116 and HT-29 colon cancer cell lines were selected to assess the cytotoxic effects of AgNPs. HCT-116 and HT-29 cells were cultured and maintained in DMEM media and kept at 37°C under 5% CO 2 incubation throughout the experiment. MTT assay was carried out to assess the percentage growth inhibition of HCT-116 and HT-29 cells. Colon cancer cells seeded at a density of 5000 cells/well were treated with AgNPs and crude extract of A. bracteosa at different concentrations of 125, 100, 75, 50, 25 μg ml −1 for 72 h. MTT dye was added into respective wells of 96-well plate and incubated for 4 h. Then the media was discarded and formazan crystals were diluted by adding DMSO. Finally, absorbance was recorded by a spectrometer, and the percentage viability was determined.

Anti-inflammatory activity
The anti-inflammatory activity of ABMF, ABHF, ABCF, and AgNPs was assessed using carrageenan-induced paw-edema assay according to a previously used protocol by Winter et al 1962 [26]. Briefly, six groups (n=6) of inbred rabbits of weight 500-1250 g were used in this experiment. Rabbits were fasted overnight prior to the test. The 1st, 2nd, 3rd, and 4th groups were given ABMF, ABHF, ABCF, and AgNPs respectively at a dose rate of 100 mg per kg of body weight dissolved in sodium CMC (0.1%). The animals of groups five and six were given reference drugs Indomethacin at 50 mg kg −1 and Ibuprofen at 100 mg kg −1 respectively. After 30 min of sample administration, 100 μl of 1% solution of carrageenan in PBS was administered into the sub-plantar region of the right hind paw of each test animal. The reduction in paw volume was taken through a plethysmometer at a 30 min interval for 3 h. The % protection was measured with the help of the following formula;

Statistical analysis
The data was analyzed using Graphpad prism 7.0 and the graphs were plotted. The values were expressed as Mean and Standard Error of Mean (Mean±SEM). P-value was calculated to measure the significance of the change in parameters observed for different groups.

Characterization of silver nanoparticles
In the current study, AgNPs were successfully biosynthesized using plant extract of A. bracteosa. The appearance of brownish color as shown in figure 1(a) indicated the formation of AgNPs which achieve final and mature form. The color change was due to surface plasmon resonance (SPR) and bioreduction of Ag + ions by phytochemicals [27]. The characterization of biosynthesized AgNPs was done with UV-vis spectroscopy, FTIR, XRD, EDX and TEM which confirmed spherical nanoparticles with size in the nanoscale range.

UV-vis analysis of AgNPs
The biosynthesis of AgNPs from A. bracteosa extract was observed and established with UV-visible spectroscopy. The gradual increase in the intensity of absorption peaks was observed with the passage of time. The characteristic color change was due to the excitation of the SPR in the AgNPs. In figure 1(b), the broader absorption peak at 5 min indicates that the initiation of AgNPs synthesis while a characteristic broad peak of AgNPs at 417 nm observed after 30 min indicates the maturation of AgNPs. Many previous reports observed maximum absorbance at 452, 452, 420, 427, and 475 nm for AgNPs prepared from plants Ocimum sanctum, Terminalia chebula, Piper nigrum, Syzygium cumini, and Foeniculum vulgare respectively [28]. At 60 min interval, a well-defined absorption peak appeared at 484 nm. The shifting of the SPR peak of silver nanoparticles from 417 nm to 484 nm is also known as redshift which indicates complete reduction and that the size of prepared nanoparticles increases with more reaction time. Moreover, a broad UV spectrum ranging from 350 nm to 490 nm is an indication of the poly-dispersed nature of biosynthesized AgNPs [27].

FTIR analysis of AgNPs
FTIR was carried out to determine the functional groups of biomolecules linked to the synthesized AgNPs.

Transmission electron microscopy
TEM was used to determine the size, shape, and morphology of prepared AgNPs, and results are shown in figure 3. It revealed that the majority of the biosynthesized AgNPs were spherical while some nanoparticles were found to be rough in shape and the size of AgNPs was 50±12 nm as shown in figure 3(a) and (b). Slight agglomeration was also observed. The spherical shape can be correlated with the single SPR spectrum observed in the UV-vis spectrophotometry The biosynthesis of spherical-shaped AgNPs from the leaf-extracts of Euphorbia hirta [36] and Ceratonia siliqua [37]

EDX analysis
The EDX spectrum of the biosynthesized AgNPs is shown in figure 3(c) which depicts the presence of silver as the major element. AgNPs showed a typically strong peak at 3 keV which is the characteristic of metallic silver due to surface plasmon resonance [41]. Figure 3(c) shows the quantitative information of prepared AgNPs. Moreover, figure 3(c) also shows the occurrence of other elements like C, Fe, Mg, Ca, Cl, and Na which was also observed by other researchers [42].

Comparative studies of AgNPs and plant extract 3.2.1. Phytochemicals analysis
Phenolic phytochemicals are very important secondary metabolites of plants which are involved in providing health benefits to both plants and animals. More than 8000 phenolic compounds have been discovered so far and about 4000 of these phenolic compounds are flavonoids [43,44]. Polyphenols are commonly used in health, pharmaceutical, cosmetic, and food industries as antibacterial, antifungal, antiviral, anti-inflammatory, antioxidant, anti-allergic, and anti-cancer agents [45,46]. The key biomolecules found in plants are polyphenols, flavones, sugars, terpenoids, aldehydes, ketones, carboxylic acids, and amides which are responsible for the bio-reduction of metal NPs [47].

Total phenolic contents
In the current study, polyphenols present in different fractions of plant extract of A. bracteosa and those which oxidized during nanoparticle synthesis were quantified using FC reagent, and results are expressed as gallic acid equivalents per gram of extract are shown in figure 4(a). The TPC of the various fractions of Ajuga bracteosa prepared in methanol (ABMF), n-hexane (ABHF), chloroform (ABCF) and silver nanoparticles (AgNPs) were 12.5±0.12, 15.3±0.15, 13.6±0.1 and 17.8±0.2 mgGAE/g respectively. The amount of TPC quantified from AgNPs was comparatively higher as compared to the TPC of ABMF, ABHF, and ABCF. Similar results have also been reported about the presence of a higher amount of TPC in AgNPs of crude extracts of various plants as compared to their methanolic, hexane, and chloroform fractions. For instance, Zahra et al [48] observed a higher amount of TPC content in AgNPs as compared to methanolic, hexane, and chloroform fractions of the plant used to synthesize AgNPs. According to some researchers, it is the é donating potential of polyphenols that facilitate the formation of AgNPs by bioreduction of Ag + to Ag 0 and further stabilize AgNPs suspension by  4(b)). It is clearly evident in figure 4(b) that AgNPs had a significantly higher amount of flavonoids as compared to plant extracts. Many other studies also reported that water-soluble flavonoids may involve in the reduction of silver ions indicating phytochemicals as good bio-reducers [49,51].

In vitro studies
Different parameters of biological activities like antibacterial, anticoagulant, antioxidant, anti-inflammatory, and anti-cancer of different fractions and AgNPs of Ajuga bracteosa were applied that proved AgNPs have shown the best results in comparison with frictions of the plant.

Antibacterial activity
The antibacterial activity of the ABMF, ABHF, ABCF, and AgNPs was tested for both Gram-positive (S. aureus and B. subtilis) and Gram-negative (E. coli and P. aeruginosa) bacteria. Based on the zone of inhibition produced, the AgNPs prepared from A. bracteosa extract showed good antibacterial activity against both Gram-positive and Gram-negative bacterial strains as compared to other fractions of A. bracteosa as shown in figure 5. As reported earlier, various extracts of A. bracteosa leaves, roots, and bark have shown activity against many bacterial strains. In many previous reports, a similar pattern of antibacterial activity was observed by AgNPs biosynthesized from Vitex negundo extract and Acorous calamus rhizome extract against gram-negative and gram-positive bacterial strains [27,52]. Silver nanoparticles were known as powerful antibacterial agents [53]. The AgNPs show antibacterial property by attaching to the bacterial cell membrane [54] because it is the site of the respiratory  chain, energy-transducing systems, and transport of molecules and therefore, any change in the structure of the membrane would eventually result in inhibition of bacterial growth. The AgNPs showed better antimicrobial activity because of their very large surface-area giving more room to contact the cell wall of microorganisms [55].

Antioxidant activity
The antioxidant activity of biosynthesized AgNPs was compared to plant extract preparations determined by DPPH and ABTS in vitro assays. AgNPs may possess antioxidant potential due to the existence of silver in two oxidation states Ag + and Ag 2+ or phytochemicals capped on silver nanoparticles.T All fractions of A. bracteosa showed pronounced antioxidant activity which it might be related to the presence of abundant phytoecdysteroids reported by Kayani et al 2016 [56].

DPPH assay
The methanolic, n-hexane, chloroform extracts of A. bracteosa and silver nanoparticles showed IC-50 values of 35.2, 23.3, 31, and 21 μg ml −1 respectively. AgNPs of the plant showed the best DPPH radical scavenging activity (IC50=21.6 μg ml −1 ) in comparison with other solvent fraction of plant as shown in figure 6(a). This might be due to the enhanced activity of phenols after the formation of AgNPs. Phenolic biomolecules present in plant extracts are responsible for their high antioxidant activity and act as reducing agents which in turn play a central role in the biosynthesis of silver nanoparticles [57].

ABTS assay
In the present work, ABMF, ABHF, ABCF, and AgNPs were evaluated for their ABTS radical cation scavenging activities. IC-50 values of samples range from 31 to 47 μg ml −1 . Among all extracts, the lowest IC-50 value was observed for AgNPs of A. bracteosa as shown in figure 6(b) which indicates the higher antioxidant potential of synthesized AgNPs. Many researchers have shown similar high antioxidant activities of AgNPs prepared from plant extracts with ABTS [57,58]. This is might be due to cappings of bioactive compounds such as polyphenols over AgNPs which enhanced the antioxidant of AgNPs many folds.

Cytotoxicity activity
The therapeutic potential of AgNPs for treating cancer is well known as they penetrate immediately in cancer cells causing cell lysis. Silver nanoparticles are found to be very effective cancer cell growth-inhibiting agents in many types of cancers especially lung, breast, glioblastoma, hepatic, and ovarian [59]. Here in this study, the anticancer potential of silver nanoparticles biosynthesized from A. bracteosa was tested on two colon cancer cell lines (HT-29 and HCT-116) at various dose concentrations of 25 to 125 μg ml −1 . The increase in dose concentration of silver nanoparticles and PE continuously decreased the cell viability of both kinds of cancer cells as shown in figure 7(a). However, the inhibitory effect of AgNPs and PE was more pronounced on HCT-116 as compared to HT-29. IC-50 values obtained for AgNPs and PE were (114.8±7.7 versus 196±13.9 μg ml −1 ) for HT-29 and (69.1±11.3 versus 103.6±14.5 μg ml −1 ) for HCT-116 respectively as shown in figure 7(b). These values showed that the anticancer potential of AgNPs against HCT-116 was within the clinically acceptable concentration of 100 mg l −1 [27]. Many previous studies reported the cytotoxic effect of biosynthesized AgNPs from the extract of J. dolomiaea against HeLa and MCF-7 cancer cell lines [58], from Artemisia turcomanica leaf extract against gastric cancer cell line (AGS) in a dose-and time-dependent manner [60] and from Morinda citrifolia against HeLa cell lines [61] in vitro.

Anti-inflammatory assay
Carrageenan induced Paw edema is a non-specific inflammation which is the result of various mediators [62] and a decrease in paw volume is an indication of anti-inflammatory effects. Carrageenan paw edema is highly sensitive to non-steroidal anti-inflammatory drugs (NSAIDs) and thus frequently used for assessing new antiinflammatory agents [26]. The reduction in inflammation by test samples ABMF, ABHF, ABCF, and AgNPs were measured in % and compared with reference drugs Indomethacin (50 mg kg −1 ) and Ibuprofen (100 mg kg −1 ) as shown in figure 8. The results showed the higher anti-inflammatory potential of AgNPs (89.1±2.6%) at 100 mg kg −1 as compared to standard COX-1 inhibitor Ibuprofen 69.0±3.6% and selective COX-1 and COX-2 inhibitor Indomethacin 86.7±2.4% after three hours of carrageenan induction. Moreover, all the fraction of A. bracteosa relatively showed better edema protection activity with ABMF (82.5±2.9) being the most potent followed by ABCF (71.6±3.8) and ABHF (65.7±4.0). The high anti-inflammatory activity of plant extract fractions and AgNPs might be linked with phytoecdysteroids present in A. bracteosa [56].

Conclusions
Herbal medicine's importance is well recognized in the medical field today due to multiple beneficial and limited side effects on human health. Modern and advanced medication leads are required for the cure of diseases like cancer and chronic wounds. Herbal extracts and metallic nanoparticles prepared Ajuga bracteosa could be one of the therapeutic agents for the delayed wound healing complications. Here, we evaluated the healing influence of at present well-acknowledged herbal plant A. bracteosa leaf extracts and AgNPs prepared from their leaf on different bacterial strains as well as the influence of these formulations on wounds induced artificially in the skin of rabbits. A marked degree of bactericidal effects was displayed by leaf extracts and AgNPs but AgNPs proved more lethal on both gram-positive and negative bacterial strains.