Characterization of Firmiana colorata (Roxb.) R. Br. leaf extract and its silver nanoparticles reveal their antioxidative, anti-microbial, and anti-inflammatory properties

Abstract

Nanotechnology is the integrative science in the field of physics, chemistry and biology. For the synthesis of silver nanoparticles, a simple approach was applied using Firmiana colorata (Roxb.) aqueous leaf extract. During the synthesis of this silver nanoparticle, the solution color changes from green to deep brown due to the reduction of silver. The phytocompounds present in the Firmiana colorata (Roxb.) leaf extract acts as a reducing as well as a capping agent. Identifying the presence of bioactive compounds responsible for the reduction of silver was extensively characterized by UV–Vis spectrophotometer, FTIR, SEM, and EDX. Moreover, to know the efficacy of the silver nanoparticles (AgNps) antioxidant and antimicrobial studies were evaluated against the human pathogenic bacteria. Furthermore, GC–MS analysis of the leaf extract of Firmiana colorata has been done followed by the in-silico molecular docking against the Anti-inflammatory and oxidative protein. Here within this study, a comparative evaluation was done among the Firmiana colorata (Roxb.) leaf extract and the synthesized silver nanoparticles. Results indicate that ethnomedicinally lesser known Firmiana colorata (Roxb.) and AgNps have the potency to act as anti-inflammatory, antioxidative, and antimicrobial agents.

Introduction

The use of medicinal plants in Pharmacological studies for curing any diseases predates human history. Most of the plant-based drugs, discovered a century ago are a tremendous challenge to early humans. Pharmacological studies involving medicinal plants have been the topic of several analyses in several scientific meetings all over the world. Globally, there are evidence-based studies to verify the usefulness of medicinal plants, and some of them shreds of affirmation that has provided insights into the synthesis of plant-based Phyto compounds with therapeutic applications [1]. Moreover, with the pandemic situation of covid 19, researchers were focused to develop different kinds of Phyto formulations to combat this deadly mRNA virus. However, after recovery from this disease many patients suffered from different inflammatory-related complications because of their elevated cytokine levels.

Inflammation is a complex set of interactions among soluble factors and tissues that can arise in different cell types in response to traumatic, infectious, post-ischemic, toxic, or autoimmune injuries. Most conventionally used medicines like Aspirin, Clopidogrel, Diclofenac, Enoxaparin, Ibuprofen, Naproxen, and Warfarin are available to treat these inflammatory conditions. However, these drugs have several side effects. Herbs or homemade remedies are less expensive than synthetic drugs and mostly they have very fewer side effects than synthetic medicines. Nowadays different approaches have been developed to successfully target plant-based alternative medicines to fight against different types of life-threatening diseases. One of the recently used approaches is nanotechnology. In this recent era, safe nanoparticles, and cost-effective bactericidal materials are gaining popularity in the biological field and have been used successfully as carriers of therapeutic agents, in the diagnostics of chronic diseases [2]. Plant-based synthesis of nanoparticles has been successfully employed to target inflammation.

India being a diverse nation owing to its enriched floral and faunal variations plays a crucial role in practicing Ayurveda from time immemorial. Firmiana colorata (Roxb.) belongs to the family Malvaceae, its native range is the Indian Subcontinent to China (S. Yunnan) and Sumatera, commonly known as ‘Jari–udal’ or ‘Mula’ in India, can be found in forests of the Western Ghats and Deccan of the Indian subcontinent. This species is well known to local people for its traditional medicinal value that can cure jaundice and cholera [3]. Besides it is very much effective to treat intestinal dysfunctionlike the leaf juice of Firmiana colorata is combined with the leaf juice of Duabanga grandiflora (Roxb. ex DC.) Walp. leaf juice of Chrysopogon aciculatus (Retz.) Trin., rhizome juice of Alpinia nigra (Gaertn.) Burtt, leaf juice of Mesosphaerum suaveolens (L.) Kuntze and seeds of Nigella sativa L.This combination is taken orally 3.

Despite its medicinal importance, no comprehensive work has been compiled encircling the efficacy of this plant in all proportions from the literature. Its stretchy utility as a medicine forced us to bridge the information gap in this area and to work on the medicinal, phytochemical, and pharmacological importance of this plant [4]. Here in this study, comprehensive work has been carried out to produce silver nanoparticles with leaf extract of Firmiana following a variety of techniques such as UV–Vis spectrophotometer, FTIR (Fourier transform infrared spectroscopy), SEM (scanning electron microscope), EDX (energy-dispersive X-ray spectroscopy) analysis, etc. for their characterization and comparison with freshly prepared leaf extracts. Besides, comparative analysis has also been carried out for in vitro antioxidant assay and antimicrobial assay against human pathogenic bacteria (Staphylococcus aureus, Bacillus subtilis, E. coli, and Klebsiella sp.).The local inhabitants extensively use the leaf extract of Firmiana for various ailments, therefore to identify the chemical composition of leaf extract, GC–MS (Gas Chromatography-Mass Spectroscopy) has also been employed followed by bioinformatics characterization to throw light on their efficacy against inflammatory protein and antioxidant transcription factor for the strong manifestation of in vitro results. For further information please refer schematic diagram (Fig. 1).

Fig. 1.

Diagrammatic representation of F. colorata with its various medicinal properties

Materials and methods

Collection of plant material

The leaves of the plant were collected from the University of North Bengal, Darjeeling District of West Bengal for generating silver nanoparticles and to do the other experiments. The plant sample was identified by the Plant Taxonomy and Biosystematics Laboratory in the Department of Botany, University of North Bengal.

Preparation of bio extract

Leaves were washed with running tap water three times to remove the dirt. After soaking they were crushed in a mechanical grinder. Exhaustive extraction was performed by the Soxhlet apparatus (plant material: solvent was 1:10 m/v) for 7–8 h using methanol as solvent. Then the extract was concentrated under reduced vacuum pressure at 40 °C in a rotary evaporator (Buchi Rotavapor R-3, Switzerland). Thus the obtained concentrated extract was stored at − 20ºC for further use.

Preparation of silver nanoparticles (SNP) using Firmiana colorata extract

20 mL of plant extract was added to 80 mL of 0.001 M silver solution in a drop-wise manner in a 250 mL amber bottle under room temperature (25 °C) on a hot magnetic stirrer (50 °C). After 1 h, the formation of silver particles started to appear. The color of the reaction mixture transformed spontaneously from light yellow to deep brown color, thereafter no color transformation was shown up to the end of the reaction. Afterward, the reaction mixture was allowed to cool down, and then the reaction mixture was centrifuged for 15 min at 12,000 rpm. The product thus obtained was washed three times with deionized water. Finally, a black precipitate was formed and that was dried out for 12 h at 80 °C in a hot air oven.

Characterization of silver nanoparticle

UV–Vis spectral analysis

The bioreduction of silver ions (Ag +) in an aqueous solution was monitored by periodic sampling of aliquots (0.1 ml) of the suspension and then diluting the samples with 1.9 ml of deionized water and subsequently measuring their UV–Vis spectra of the experimental diluents in 300–700 nm range operated at a resolution of 1 nm [14]. The analysis of UV–Vis spectroscopy of silver nanoparticles synthesized was carried out as a function of the time needed for bioreduction at room temperature on a model spectrophotometer.

Fourier Transform-infrared spectral (FTIR) analysis

The deep brown solution containing the nanoparticles was centrifuged at 6000 rpm for 10 min and the resulting suspension was redispersed in 15 ml of sterile distilled water. The redispersing and centrifuging process was performed thrice [15]. Thereafter, the purified suspension was completely dried at 50 °C. At last, the dried nanoparticles were analyzed for FTIR.

Scanning electron microscope (SEM) analysis

SEM analysis was conducted for studying the shape and surface morphology of synthesized nanoparticles. The film of the sample was prepared on a carbon-coated copper grid by dropping a small amount of the sample and then allowing them to dry before measurements. For the analysis of EDAX, the reduced silver was dried on a carbon-coated copper grid and performed on a HITACHI SU6600 FESEM equipped with an EDAX attachment [16, 17].

In vitro antioxidant assays

2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay

To study the free radical inhibition activity 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was performed following the method described in our previous reports with slight modifications [5]. Different concentrations of both extracts ranging from 50 to 200 μg/ml were prepared and mixed appropriately with freshly prepared DPPH solution (1 mM, diluted in 95% methanol) and kept the solution in dark for 30 min. The optical density (OD) of the solution was measured at 517 nm after 30 min using a Bio-Rad microplate reader and compared the OD with the standard ascorbic acid. The percent % inhibition was calculated using the following Eq. 1:

$$\mathrm{Percent}\mathrm{of}\mathrm{scavenging}=\frac{A0-A1}{A0}\times 100,$$

where A0 is the absorbance of the control and A1 is the absorbance in the presence of samples and standards.

Measurement of reducing power

Ferric-reducing power assay (FRP) was determined by the method of Lala et al. 2020 with slight alterations in extract doses [5]. Different concentrations ranging from 50 to 200 μg/ml of the aqueous leaf extract and synthesized silver nanoparticle (250 μl) were mixed with 0.3 M of phosphate buffer (pH 6.6) and 0.1% of potassium hexacyanoferrate, followed by incubation for 30 min at 50 °C in a water bath. After incubation, 250 μl of TCA (10%) was added to the mixture to further stop the reaction. The aqueous portion at the upper side of the reaction mixture was then transferred to another tube and mixed with 0.5 ml of double distilled water followed by 0.01% of FeCl₃ solution. The mixture was left for 20 min at room temperature (RT) and the absorbance was measured at 700 nm. Butylated hydroxytoluene i.e., BHT was used as standard.

Hydrogen peroxide scavenging assay

The hydrogen peroxide (H2O2) scavenging activity of the extracts at concentrations of 50–200 μg/ml was estimated using a hydrogen peroxide solution 7. A solution of H₂O₂ (2 mmol/L) was prepared in phosphate buffer (pH 7.4). The extracts of different concentrations were added to the hydrogen peroxide solution (0.5 mL). A mixture of phosphate buffer (2 mL) and extract (1 mL)served as the blank. The absorbance of H₂O₂ at 230 nm was determined after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide and compared to standard ascorbic acid.

Nitric oxide (NO) radical scavenging assay

Nitric oxide (NO) radical inhibition assay was done by the procedure described by the method of Sunit et al. 2013 with minor changes in the extract concentrations [6]. In short, sodium nitroprusside (10 mM), phosphate-buffered saline (pH 7.4), and various concentrations of extracts (50–200 μg/ml) were mixed to make the final volume of 6 ml. After 1.5-h incubation at 25 °C, 0.35% of sulfanilamide which was diluted in 20% of glacial acetic acid was added to 1.5 ml of the pre-incubated reaction mixture and left the reaction mixture for 6 min. Following the incubation, 1 ml of 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride (NED), was added and incubated for 40 min at 25 °C to develop the color. The percentage (%) of inhibition was calculated using Eq. (1). The absorbance was measured at 540 nm. Curcumin was used as a reference.

Iron chelation assay

The chelating activity of ferrous ions by the experimental extracts was determined with slight modification reported in the previous [7]. Various concentrations of both the samples (50–200 μg/ml) were added properly with ferrous sulfate (FeSO₄) solutions (14.5 μM) in HEPES buffer (pH 7.2) partnered by the addition of ferrozine (75 μM) to initiate the reaction. The reaction mixture was vigorously trembled and incubated for 40 min at room temperature. The ferrous ion-ferrozine complex was measured at 562 nm where EDTA was used as a standard.

Total antioxidant activity (TAA)

The total antioxidant capacity of the experimental extracts of Firmiana colorata was measured using a spectrophotometer at concentrations of 50–200 g/mL followed by the method of Bhattacharjee et al. 2021 with slight modifications. This experiment was conducted with triplicate values, and ascorbic acid was used as a positive control.

Quantification of total phenolic and flavonoid content

Quantification of total phenol content was determined by the Folin-Ciocalteu method 5. Briefly, all the experimental extracts of Firmiana colorata (100 μl) were mixed with Folin- Ciocalteu reagent (diluted 1000-fold with distilled water previously) and left for 6 min at room temperature followed by the addition of 0.06% sodium carbonate to the mixture. After 90 min of incubation at room temperature, the absorbance was measured at 740 nm. The phenolic content was determined against a gallic acid standard curve (R2 = 0.986, y = 0.007 ×  − 0.025).

Total flavonoid content was evaluated according to the aluminum chloride (AlCl3) method with modifications 5. Various extracts of Firmiana colorata (100 μl) were added to 0.5 ml of distilled water followed by the addition of 3.5% sodium nitrate (NaNo2). After 10 min of incubation at room temperature, 10% AlCl3 was incorporated and left for 5 min. The whole reaction mixture was treated with 1 mM of sodium hydroxide and diluted with distilled water. OD value was recorded at 510 nm. All the data were reported as means ± SD for 3 replications. The flavonoid content was determined against the quercetin standard curve (y = 0.207 ×  − 0.204, R2 = 0.962).

Microbiological screening

The antimicrobial activity of the methanolic extract of F. colorata and the synthesized silver nanoparticles was evaluated by the agar well diffusion method with slight modifications [5].

Culture and maintenance of microorganisms

Two Gram-positive (Staphylococcus aureus and Bacillus subtilis.) and two Gram-negative (Klebsiella sp. and E. coli) bacteria were used in this experiment. The pure cultures of bacterial samples were maintained on a nutrient agar medium [40 g/L]. Each bacterial culture was further maintained by subculturing regularly on the same nutrient agar medium and then incubated at 37 °C for 24 h. Finally, culture plates were stored at 4 °C before use in this experiment.

Screening for antibacterial activity

For the antimicrobial susceptibility test using Agar well diffusion method [20], the media was prepared with Agar (20 g/L) and MH broth (21 g/L) in a 1 L conical flask. The media prepared was then sterilized by autoclaving at 120 °C for 20 min. Petri plates were prepared by pouring 20 ml of media containing both agar and MH broth. Petri plates were allowed to stand for 30 min for solidifying the media fully. Freshly prepared bacterial inoculums which were diluted 1:1 with sterile water were then evenly spread using a sterile cotton swab upon the entire media surface31, 34. A 6 mm hole was then punched with a sterile cork borer and a 100 µl volume of plant extract and silver nanoparticle with a concentration of 2 mg/ml was pipetted into punctured wells. The plates were incubated at 37 °C for 2 days (24–48 h) and the inhibition zone diameter was taken in three different fixed directions and the average values were recorded.

Gas Chromatography-Mass Spectroscopy (GC–MS) analysis

The bioactive compounds of F. colorata leaf extract were identified using GC–MS analysis according to the standard protocol with slight modification [10].

In silico molecular docking

X-ray diffraction structure of proteinNF-κB (nuclear factor kappa-b, PDB ID: 1NFK) and FOXO (forkhead-box class O₃, PDB ID:3L2C) were obtained from the PDB database(https://www.rcsb.org/). The resolution of these proteins is 2.3 Å and 1.87 Å respectively. This resolution is good for the docking study. Protein structures were further modified by removing water and adding polar hydrogen into Auto dock vina software5. The ligands were obtained from GC–MS data. The structure of those ligands was downloaded from NCBI pub chem. (https://pubchem.ncbi.nlm.nih.gov). in sdf format. The structures of the ligands were again converted to PDB format using the SMILE online server. Finally, the docking study was done by Auto dock vina and the results were visualized in the Pymol version 1.7.4.

Statistical analysis

All qualitative data have been reported as the mean ± SD of three measurements. Statistical analysis and representation of statistical data were done using the one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test, where α < 0.001 was considered significant.

Results and discussion

UV–Vis spectroscopy

The addition of plant extract to 1 mM solution of silver nitrate changed from colorless to dark brown in about 15 min Fig. 2. The final color deepened more with an increase in time. Previous reports clarify the presence of silver nanoparticles exhibiting yellowish-brown color in solution due to the excitation of surface plasmon vibrations [15]. Previous studies showed that silver nanoparticle synthesis increases with increasing temperature. When the temperature is more than 50 °C then upon taking absorption on a UV spectrophotometer it can be seen that less time is required to synthesize silver nano than the nanoparticle synthesized at normal room temperature [19].

Fig. 2.

UV–Vis spectroscopy of Ag nanoparticle formation by reducing AgNO3 using F. colorata extract

Scanning electron microscopy(SEM) and energy-dispersive x-ray spectroscopy (EDX)

The morphology and size of particles were determined by Scanning Electron Microscopy. This shows that the particles were spherical with a range from 60 to 90 nm. The interactions such as hydrogen bonding and electrostatic interaction between the bio-organic capping molecules might be the reason for the synthesis of silver nanoparticles using F. colorata extract [23, 26, 27]. In some cases, nanoparticles were irregular in shape. One of the reasons for this incident is due to agglomeration during sample preparation Fig. 3. The qualitative and quantitative position of elements that may be concerned in the formation of silver nanoparticles was analyzed by EDX (Energy dispersive x-ray spectroscopy) analysis. The elemental profile of silver has been confirmed from the sample using plant leaf extract shown in Fig. 3. It confirms the formation of silver nanoparticles. The optical absorption peak was seen approximately at 3 keV, which is typical for the absorption of metallic silver nanocrystals due to surface Plasmon resonance, which confirms the presence of nanocrystalline elemental silver. The spectrum shows some other elements like oxygen and chlorine peak, which may be originating from the biomolecules that are bound to the surface of nanosilver particles and can be distinguished in Fig. 4.

Fig. 3.

SEM analysis of biosynthesized silver nanoparticles

Fig. 4.

EDX spectra of biosynthesized silver nanoparticle

Fourier transform infrared (FTIR) analysis

FTIR analysis was done to identify the capping, reducing and stabilizing capacity of F. colorata leaf extract. This analysis was done for both the plant extract and AgNPs Fig. 5A, B.

Fig. 5.

FTIR spectra of A F. colorata extract and B its AgNPs

The spectrum of crude leaf extract shows major peaks at 3385(–OH), 2944(–C–H), 2360(–COOH), 2034(C=C), 1643(C–O). Similar types of peaks with slight changes were observed in the spectrum of AgNPs except for the peak responsible for COOH. The COOH peak undergoes an appreciable change in the spectrum of AgNPs which indicates the possible alliance of COOH with silver ion and causes the reduction of Ag+ into AgNPs [26].

Antioxidant assay

Medicinal plants are a huge source of phytocompounds and they are often used for the treatment of numerous oxidative stress-related diseases for their effectiveness, safer consumption, low toxicity, and easy availability. In today's world, the demand for medicinal plants is continuously emerging. Simultaneously, the demand for antioxidants from natural sources has escalated many folds because of their potential property to prevent and reduce the risk of several oxidative damage [29, 30]. The antioxidant property covers a broad spectrum of the chemical phenomenon and specific antioxidant activity should not be concluded based on a single experimental model. Therefore, several in vitro antioxidants or free radical scavenging activities were carried out with our sample of interest.

2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay

The DPPH(2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay is one of the most frequently used assays of antioxidants because at a minimum concentration also the active elements can easily be detected by this test. DPPH is a free radical assay that is capable to accept an electron or hydrogen radical to become stable and reacts with a reducing agent changing the color of the solution from deep purple to pale yellow. In the present antioxidant profiling, we compared the scavenging activity of both the extracts i.e., F. colorata leaf extract and silver nanoparticles synthesized from F. colorata which were 76.2 ± 0.3 and 81.5 ± 0.6 respectively. The result showed F.colorata synthesized silver nanoparticle has the highest scavenging activity as compared to a normal plant extract and the standard ascorbic acid (65.66 ± 1.3) at the same concentration(Fig. 6A). From the above result, it is observed that both extracts are rich in antioxidants as the color of the solution decreases along with the increasing concentrations of both extracts35,36. Thus, DPPH scavenging activity by F. colorata extract and the nanoparticles which were synthesized from F. colorata leaves confirm the presence of significant antioxidants.

Fig. 6.

Antioxidant and free radical scavenging activity of F. colorata extract A DPPH scavenging activity (%) of inhibition of F. colorata extract and Nano extract were 76.2 ± 0.3 μg/ml and 81.5 ± 0.6 μg/ml respectively; B reducing the activity of F. colorata extract and Nano extract were 0.95 ± 0.1 and 1.4 ± 0.6 respectively; C H2O2 scavenging activity of F. colorata extract and Nano extract were 56.8 ± 0.1 μg/ml and 60 ± 0.2 μg/ml respectively; D Iron chelation activity of F. colorata extract and Nano extract were 32.9 ± 0.5 μg/ml and 43.8 ± 0.5 μg/ml. where α = p < 0.05 Β = p < 0.01, γ = p < 0.001, δ = non-significant when compwered to standard

Measurement of reducing power

The reducing power assay method is one of the important assays to detect antioxidant activity which is based on the principle that the substances with reduction potential react with potassium ferricyanide (Fe3+) to form potassium ferrocyanide (Fe2+). After that Fe2+ reacts with ferric chloride to form a ferric–ferrous complex that has an absorption maximum of 700 nm [19, 21]. The reducing power of both extracts was found to be increased in a dose-dependent manner i.e., at 200 μg/ml F. colorata leaf extract showed an absorbance of 0.95 whereas F. colorata silver nanoparticles showed an absorbance of 1.3 compared to the reference compound BHT which was much lower than both the extracts (Fig. 6B). The result of the reducing power assay proved that both extracts are capable to scavenge the reactive oxygen species and reactive nitrogen species.

Hydrogen peroxide scavenging assay

Hydrogen peroxide, H₂O₂ is a highly oxidizing agent, capable of inactivating enzymes by directly oxidizing the thiol(-SH) groups. In phagocytic cells, H₂O₂ mediated uncontrolled formation of HOCl, NO₂- to NO₂ conversion through the action of myeloperoxidase which causes tissue injury and initiates the pathogenesis of several diseases [13, 22]. FOX reagent method was used to perform a hydrogen peroxide (H₂O₂) scavenging assay of the plant extracts as well as the plant nano extract synthesized by silver. The results showed that both extracts are capable to scavenge H₂O₂ as compared with their reference sodium pyruvate and thus can be easily used as natural potential antioxidants. Both extracts showed the percentage of inhibition in a dose-dependent manner (Fig. 6C). At the highest concentration (200 µg/mL) F. colorata extract showed a percent of inhibition of 56.8 ± 0.1 and synthesized silver nanoparticles showed a percent of inhibition of 60 ± 0.2 whereas in the case of standard sodium pyruvate the percent of inhibition was 55.6 ± 0.2 which is less than both the experimental extract.

Nitric oxide (NO) radical scavenging assay

Nitric oxide i.e., NO is an essential bioregulatory molecule essential for several physiological processes like immune response, neural signal transmission, vasodilation, and control of blood sugar level, etc. However, an excess amount of the NO level may result in several pathological conditions, including metastasis [11]. Besides, nitric oxide has an active role as a pro-inflammatory mediator. During chronic inflammation of tissue, iNOS (a calcium-independent isoform of NOS) is generated by lipopolysaccharide and activates numerous amounts of nitric oxide. The active nitric oxide radical translocates NF-κβ and leads to the formation of chronic inflammation [24, 25]. Nitric oxide is also a signaling molecule in the immune and nervous systems and participates an essential role in the killing of microbes in the host cells. However, NO could combine with singlet oxygen to produce peroxynitrite radical, with the enormous potential to cause cellular damage. At the highest concentration (200 µg/mL) the percentage of inhibition of F. colorata extract and synthesized nanoparticles were 71 ± 0.1 and 76 ± 0.4 respectively whereas the percentage of inhibition in the case of standard curcumin was 66 ± 0.1(Fig 7A). Both extracts have shown, potent NO inhibitory activity than the standard. The NO inhibitory activity of both extracts would not only diminish the extent of reactive peroxynitrite formation but also decreases the expectancy of chronic inflammatory symptoms.

Fig. 7.

Antioxidant and free radical scavenging activity of F. colorata extract A nitric oxide scavenging activity of F. colorata extract and Nano extract were 71 ± 0.1 μg/ml and 76 ± 0.4 μg/ml respectively; B total antioxidant capacity (%) of inhibition of F. colorata extract and Nano extract were 80 ± 0.1 μg/ml and 85 ± 0.1 μg/ml respectively

Iron chelation assay

Free iron is an important element that causes upregulation of ROS as it leads to the reduction of hydrogen peroxide and produces the highly reactive OH − . Samples that have a moderate amount of chelating activity can hinder the formation of the ferrozine– Fe2 + complex (Fig. 6D). Thus the rate of color reduction is monitored to estimate the iron chelation activity of the experimental samples [10, 12, 24]. From the graph, it can be wind up that both the experimental extracts had a good source of antioxidants as the decrease in the percentage of ferrozine–Fe2 + complex along with the increase in sample concentration. At the highest concentration, synthesized nanoparticle extract (43.8 ± 0.5) shows better results than F. colorata leaf extract (32.9 ± 0.5). The reducing properties are correlated with the presence of compounds that utilize their action by breaking the free radical chain by donating an H + atom. This assay is a free radical-related process under nonenzymatic or enzymatic control in a biological system. The results of iron chelation assays suggest a decrease in dose-dependent color formation with ferrozine complex in presence of both the experimental extracts indicating their potent iron-chelating properties.

Total antioxidant activity (TAA)

Antioxidants are dynamic compounds capable of either delaying or terminating the oxidation processes which take place under the supremacy of atmospheric oxygen or ROS. Antioxidants are used for the stabilization of polymeric products, foodstuffs, cosmetics, and pharmaceutical products [14, 18]. Total antioxidant capacity/assay is an analytical method frequently determined to assess the antioxidant status of a given plant sample and can access their antioxidant response against the free radicals produced in a given system. The primary function of antioxidants is to shield the body against the destructive effects of free radicals resulting in damage.TAA was evaluated by the capacity of both the experimental extracts to diminish molybdenum (VI) to molybdenum (V). The percent of inhibition in the case of F. colorata extract was 80 ± 0.1 and synthesized nanoparticles were 85 ± 0.1 whereas standard ascorbic acid showed a % of inhibition of 72 ± 0.2, proving that F. colorata leaf extract and synthesized nanoparticles have more effective antioxidant potential than their respective standard (Fig. 7B).

Determination of total phenolic and flavonoid content

The total amount of phenolic content of the samples was 270 mg/mL and 277 mg/mL. The total amount of flavonoid content was 84 mg/mL and 118 mg/mL respectively. Plant exact and silver nanoparticles of F. colorata showed high phenolic and flavonoid content because of the phytocompounds present in them which also reveals that the samples have the significant antioxidant potential [5]. Contrary to this, the synthesized silver nanoparticles showed a good amount of phenol and flavonoid than the F. colorata leaf extract.

Microbiological screening

Quantification of antimicrobial activity using agar well diffusion method

From the antimicrobial activity, it can be concluded that both the samples (plant extract and synthesized nanoparticle) were very much effective against gram-positive bacteria with an inhibition zone of 21 mm and 17 mm, respectively, than the gram-negative bacteria Fig. 8. Although synthesized silver nanoparticles from the aqueous leaf extract of F. colorata showed more competent against this human pathogenic strains as compared to the normal plant extract.

Fig. 8.

Antimicrobial activity against A Staphylococcus sp.; B Klebsiella sp.; C E. coli; D Bacillus sp

GC MS analysis

A total of 22 bioactive compounds were detected from the GC MS analysis. GCMS result is shown in Table 1. Various bioactive compounds were detected in the sample out of which several long-chain fatty acid moieties and their derivatives were present in the sample. Hexadecanoic acid, Phytol, and Heptadecanoic acid this long-chain fatty acid that play an active role in the growth and development of plants [15]. Neophytadiene has antiproliferative, anticancerous well as antimicrobial and antifungal activity. Squalene is a triterpene and is also well-known as a natural emollient and antioxidant [5]. Vitamin E is one of the most important compounds and works as an antioxidant also involved in immune function. Stigmasterol may have an active role in hypoglycemic and thyroid-inhibiting activity [33].

Table 1.

GC–MS analysis of Firmiana colorata leaf extract

S. no	Compounds		RT	Biological activity
1	Isopropyl myristate	C17H34O2	12.6	Antimicrobial [17]
2	Neophytadiene	C20H38	12.7	Antibacterial, Rheumatism and some skin disease [18]
3	Dibutyl phthalate	C16H22O4	13.573	Antibacterial [19]
4	Hexadecanoic acid, methyl ester	C17H34O2	13.691	Decrease blood cholesterol, Anti-inflammatory [20]
5	cis-9-Hexadecenoic acid, heptyl ester	C23H44O2	14.5	Antioxidant [24]
6	9-Hexadecenoic acid, hexadecyl ester, (Z)-	C32H62O2	14.4	Antioxidant, Hypocholesterolemic [22]
7	Isopropyl palmitate	C19H38O2	14.6	No activity found
8	7-Octadecyne, 2-methyl	C19H36	15.2	No activity found
9	9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-	C19H32O2	15.3	No activity found
10	Phytol	C20H40O	15.4	Antioxidant and antimicrobial, key component for vitamin E and K [23]
11	Heptadecanoic acid	C17H34O2	15.5	Antimicrobial [24]
12	1,2-Dioctylcyclopropene	C19H36	15.9	No activity found
13	trans,trans-9,12-Octadecadienoic acid, propyl ester	C21H38O2	16.1	No activity found
14	i-Propyl 9,12,15-octadecatrienoate	C21H36O2	16.2	No activity found
15	Isopropyl stearate	C21H42O2	16.4	Skin coordinating agent [25]
16	Decanoic acid	C10H20O2	17.1	No activity found
17	Cyclohexane, 1,1'-tetradecylidenebis	C26H50	17.8	Antimicrobial [26]
18	Bis(2-ethylhexyl) phthalate	C24H38O4	19.0	Antimicrobial [27]
19	Squalene	C30H50	21.1	Skin protective, Antioxidant [28]
20	Oxirane, 2,2-dimethyl-3-(3,7,12,16,20-pentamethyl-3,7,11	C30H50O	22	Anti-microbial,anti-cancer and anti-tumor [29]
21	Vitamin E	C29H50O2	24.1	Anti-inflammatory and antioxidant [30]
22	Stigmasterol	C29H48O	26.8	Antioxidant, Antiinflamatory, antibacterial [31]

In silico molecular docking

The mechanism of the phytocompounds was further investigated through in silico molecular docking. NF-Κb and Foxo are transcription factors and are well known to exhibit the activation of the oxidative stress pathway [34]. NF-κB protein also can initiate inflammatory genes. The significant binding of the selected phytocompounds and the proteins, as well as the log P values, revealed that the phytocompounds may be effective against the oxidative as well as an inflammatory pathway. Stigmasterol and Vitamin E showed the best binding affinities with the proteins Foxo (Fig 9A, B) and NF-Κb (Fig. 10A, B)). The binding energies were − 8.3 kcal/mol, − 7 kcal/mol, − 6.7 kcal/mol and − 6.2 kcal/mol. The phytocompounds such as Stigmasterol and Vitamin E may help in the upregulating intrinsic antioxidant defense responses.

Fig. 9.

In-silico molecular docking against A FOXO- stigmasterol, B FOXO- Vit-E

Fig. 10.

In-silico molecular docking against A NF-kB- stigmasterol, B NF-kB- Vit-E

Conclusion

This research article here clearly demonstrated that excessive production of reactive oxygen species might be demolished by the leaf extracts F. colorata and F. colorata silver nanoparticles for their antioxidant properties. The eco-friendly synthesis of silver nanoparticles from AgNo3 using F. colorata leaf extract may act as a stabilizing agent at a certain temperature. Antioxidant and antimicrobial analysis shows the F. colorata and F. colorata silver nanoparticle samples are effective in treating oxidative stress and microbial infection. Moreover, the molecular docking study of the selected phytochemicals from the F. colorata sample reflected the most possible binding with intracellular targets that might protect the cells from oxidative stress. F. colorata extracts are not only rich in antioxidant properties but also capable to reduce tissue inflammation. Although our present study only emphasizes the in vitro and in silico approaches of the leaf extract further assessment with in vivo model supported by the in silico drug delivery systems might open up a new direction in nanotechnology.

Authors’ contributions

AS and CG conceived the idea. AS, ML, and SB designed the experiment. ML and SB have done the weight lab experiments. AS and SB has done the in silico works. All the authors have contributed to drafting the manuscript and approved it.

Funding

No funding was available to carry out this study.

Availability of data

The authors verify the data sustaining findings of this study are presented within the article.

Declarations

Conflict of interest

The authors do not have any conflict of interest.