Silver nanoparticles from Tabernaemontana divaricate leaf extract: mechanism of action and bio-application for photo degradation of 4-aminopyridine

Silver nanoparticles (Ag NPs) were synthesised by the reduction of Ag+ to Ag0 in the presence of enol form of flavonoids present in plant extract of Tabernaemontana divaricate (T. divaricate). Prepared Ag NPs were characterised using UV–Vis, XRD, HR-TEM with EDX and XPS techniques. XPS spectra exhibited peaks at 366 eV and 373 eV, which specified spin orbits for Ag 3d3/2, and Ag 3d5/2 that confirmed the formation of Ag NPs. Ag NPs were spherical in shape with an average size of 30 nm as revealed by HR-TEM and FE-SEM techniques. EDX studies verified the high purity of Ag NPs with silver 46.96%, carbon 16.35%, oxygen 16.22%, nitrogen 20.25% and sulphur 0.21%. LC–MS analysis of plant extract confirmed the qualitative presence of alkaloids, tannins, flavonoids, phenols, and carbohydrates. Prepared Ag NPs showed good photocatalytic activity towards degradation of 4-Amniopyridine with 61% degradation efficiency at optimum conditions in 2 h of reaction time under visible light. The ten intermediates were found within the mass number of 0–450. Ag NPs synthesised using bio-extract have also shown good inactivation against Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) bacteria due to the availability of free radicals.


Introduction
4-Aminopyridine is an aromatic compound that has an unpleasant smell and pungent odour (Lataye Dilip et al. 2006). It is a pharmaceutically active chemical utilised for the production of dyes, pesticides, explosives, and different chemical solvents (Bai et al. 2010;Chu et al. 2018;Lin et al. 2010). There is a high chance of the presence of pyridine and its derivatives (4-aminopyridine) in industrial wastewater. The US Environmental Protection Agency has categorized 4-aminopyridine as highly hazardous to the environment and individuals (USEPA) (Daware and Gogate, 2020a, b). Recent research has reported that the concentrations of 4-aminopyridine in the wastewater from different sources vary from 20 to 300 mg/L (Alonso- Davila et al. 2012;Das et al. 2010). In treated wastewater, the maximum permitted content of pyridine and its derivatives is 5 mg/L (Daware and Gogate, 2020a, b). Therefore, it is highly required to develop an efficient treatment method for industrial wastewater containing 4-aminopyridine before discharging it into water bodies.
Different treatment methods such as adsorption, electrochemical, biological, photocatalytic oxidation, and Fenton oxidation have been used for the treatment of pyridine and associated derivative compounds (Daware and Gogate, 2020a, b;Lataye et al. 2008;Mohan et al. 2004). However, these methods have drawbacks such as sludge formation (Fenton oxidation), toxic compound formation and time-consuming processes (biological methods). Literature showed that nanoparticles act as good adsorbents and photo catalysts for the removal of 4-aminopyridine (Attri et al. 2021).
Among the various metal nanoparticles, noble metal nanoparticles such as silver have shown a significant role in the field of photocatalysis, biology and medicine. Ag NPs exhibited unique optical, electrical and thermal properties.
These are used for several applications including medical, healthcare, industrial and environment purposes (Chun et al. 2009;Pryshchepa et al. 2020;Yang et al. 2018;). Nowadays, Ag NPs have been used in textile industry, wound designing and as an antibacterial agent (Cao et al. 2017;Hsu et al. 2019;Manivannan et al. 2019). Different biological methods used for the synthesis of Ag NPs were proved to be simple, dependable and non-toxic (Švecová et al. 2018;Yang et al. 2016). Recently, Seerangaraj et al. (2021) synthesised Ag NPs using plant extract of Ruellia tuberosa leaf. It was reported that nanoparticles have exhibited good photocatalytic degradation against crystal violet dye and Coomassie brilliant blue dye. Nanoparticles have also shown biomedical application against gram-negative and gram-positive bacterial pathogens. Biosynthesis of Ag NPs using Moringa oleifera plant extract was done by Chougule et al. (2021). Synthesised Ag NPs have shown 85.13% and 91.63% photocatalytic degradation of methylene blue and malachite dyes. Zamarchi and Vieira (2021) synthesised cost-effective and eco-friendly Ag NPs using Araucaria angustifolia extract and used them in the determination of paracetamol.
Tabernaemontana divaricate (T. divaricate) plant belongs to Apocynaceae and has acted as an excellent candidate for the reduction of metal reducing ions. The organs of this plant consists of several alkaloids, tannins, flavonoids, phenols and carbohydrates which are good metal reducing agents (Sivaraj et al. 2014). Shimpi and Jha (2017) revealed that bio-mediated silver nanoparticle from T. divaricata leaves extract has a great potential for the development of anticancer nano drug, which can combat life-threatening lung cancer.
In the literature, still very few investigations are available for the production of Ag NPs using T. divaricate leave extract. Degradation of 4-aminopyridine using Ag NPs synthesised from T. divaricate leaves extract has not been reported till date. In addition, the formation mechanism for the synthesis of Ag NPs using T. divaricate and the effect of pH on the formation of the Ag NPs have not been studied yet. In the present investigation T. divaricate leaves extract was used to generate Ag NPs. Adsorption and photocatalytic properties of prepared Ag NPs were evaluated under dark and visible light. Ag NPs have also been tested for antibacterial activity against gram-positive and gram-negative bacteria Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis).

Materials
Silver nitrate (99% (w/w), 169.87 g/mol), (98% (w/w) purified 4-aminopyridine with a molar mass of (94.12 g/mol)) were attained from S. D. Fine Chemical Limited (Mumbai, India). Ethanol (99.9%), sulphuric acid (98% (v/v)) were acquired from Loba Chemie Pvt. Ltd (Mumbai, India). (98%, (w/w)) refined sodium hydroxide, was purchased from Hi-Media Laboratories Pvt. Ltd (Mumbai, India). All the chemicals used for the study were of reagent grade. Clinically isolated bacterial strains of the bacteria pathogens E. coli and B. subtilis were obtained from the Microbial Type Culture Collection and Gene Bank in Punjab, India. Structure and physicochemical properties of 4-aminopyridine are shown in (Table 1).

Synthesis of Ag NPs and characterisation
Leaves of T. divaricate were taken from Dr. B.R. Ambedkar, National Institute of Technology Jalandhar, Punjab, India and then washed with distilled water to remove all the impurities and then dried at 60 °C till constant weight. Dried leaves were then grounded in powder form by mashing with a hand. Plant extract mixture was prepared by taking 100 g of powdered leaves and boiled with 500 ml of distilled water for 10 min. The solution mixture was passed from a filter paper of 11 μm pore size. The solid particles and the final solution were stored at 4 °C.
Ag NPs were synthesised under an alkaline medium at pH 10. 100 ml of AgNO 3 (1 mM) solution was heated at 70 °C and simultaneously 10 ml (T. divaricate) plant extract was added and mixed with continuous agitation on a magnetic stirrer at 320 rpm in dark. 0.1 N NaOH was added dropwise to the solution with continuous stirring to maintain the pH of the mixture up to pH 10. After 60 min of reaction, pale yellow colour of the mixture turned to brown confirming the formation of Ag NPs. After 24 h, 5 ml of the sample at different pH was collected and tested for Surface Plasmon Resonance (SPR) of Ag NPs using UV-Vis spectrophotometer. The flow chart of the process is shown in Fig. 1.
To analyse Surface Plasmon Resonance (SPR) of Ag NPs double beam UV-Vis photo spectrometer (1 cm path length) a spectrometric quartz cell (Model: UV 2600, Shimadzu, Japan) was used. Edinburgh Instruments, model FLSP-900 was used to examine the photoluminescence (PL) of Ag NPs using a fluorescence spectrometer with a xenon lamp with (400 nm) as the source of excitation. Different functional groups present in plant extract and Ag NPs were recorded using Fourier transform infrared spectroscopy (FT-IR) (Perkin Elmer, Model: RXI, Tokyo, Japan) at a resolution of 1 cm −1 using a KBr pellet and a scan range of 40,000 to 400 cm −1 . Crystalline size of Ag NPs was evaluated using X-ray diffraction (XRD) patterns by the Powder X-ray diffraction method (X'PERT-PRO PW3064, Philips Japan) with CuKα radiation having λ = 1.54 Ǻ. To analyse the element state X-ray Photoelectron Spectroscopy (XPS), VG ESCALAB MK II spectrometer containing Mg Kα excitation source (1253.6 eV) and a tron detection system was used.
High-resolution transmission electron microscopy (HR-TEM) (Hitachi, H-7500, Japan) operated at 120 kV was used to determine the particle size Ag NPs. Ag NPs were sonicated in ethanol for 60 min before being disseminated over a 200 mesh carbon-coated copper grid for HR-TEM. The elemental composition and morphology of the prepared sample were done by Energy Dispersive X-ray spectroscopy (EDX) and Field emission scanning electron microscope (FE-SEM) (Zeiss, Merlin Compact). ZEN5600 ZetaSizer Nano ZSP instrument (Malvern instruments) was used to determine the stability of the photo catalyts by measuring the zeta potential. A surface area analyzer was used to assess nitrogen absorption-desorption (Brunauer-Emmett-Teller) of samples at liquid nitrogen temperature (− 196 °C) (SMART SORB, Smart instruments Co. Pvt. Ltd., India). Before each experiment, the sample (0.4 g) was degassed for 2 h at 150 °C.
LC-MS was used to identify flavonoids and phenolic groups found in T. divaricate leaves that had interacted with metal salt (Model: Q-TOF micro, Waters, Milford, USA). For gradient HPLC, 25 mmol/L aqueous ammonium acetate buffer (pH 6.9) and methanol were employed as mobile phases. A C18 column was used for liquid chromatography (100 mm length and 2.1 mm i.d.). The mobile phase flow rate was fixed at 1.0 ml/min. The column effluent was injected into the mass spectrometer's electrospray ionisation source, and ionisation was performed in positive mode spanning the mass range of 40-1000 a.m.u.
Experimental procedure for adsorption and photocatalytic degradation of 4-aminopyridine onto Ag NPs One-hundred ml of 4-aminopyridine (30 mg/L) and Ag NPs (1 g/L) was mixed and stirred for 12 h in the dark to achieve adsorption at neutral pH 7. The adsorption experimental data were analysed using various adsorption isotherms to understand the phenomenon of 4-aminopyridine adsorption using Ag NPs. To describe the nature of adsorption, five isotherm models were used: Langmuir, Freundlich, Sips, Kobel-Carrigan, and Hill. A non-linear equation was used to fit the Langmuir adsorption isotherm (Eq. (1)) (Nanta et al. 2018).
where q e as equilibrium adsorption capacity of adsorbent in mg/g, Ce is an equilibrium concentration of a pollutant after adsorption in mg/L, Q 0 is the maximum amount of (1) q e = Q 0 bC e 1 + bC e Fig. 1 Flow chart for the preparation of Ag NPs 4-aminopyridine uptake in mg/g, and constant b refers to the adsorption bonding energy propositional to free energy and net enthalpy in L/mg. The Langmuir model assumed that monolayer adsorption occurs on a homogeneous surface and that the adsorption energy of all active sites was always equal (Trinh et al. 2020). Freundlich is a multilayer adsorption isotherm on a heterogeneous surface and all adsorption locations have distinct affinities with non-uniform adsorption distribution. The slope from 0 to 1 represented the intensity of adsorption or surface heterogeneity. A value less than 1 indicates a chemisorption process, while 1/n greater than 1 indicates cooperative adsorption (Shin et al. 2011). The non-linearized equation is employed (Eq. (2)).
where K f is a constant allied to the adsorption capacity by absorbent in mg/g and 1/n is the adsorption constant's intensity. The Sips isotherm is derived from the Langmuir and Freundlich isotherms and is used for heterogeneous adsorption systems. When n = 1, the Sips equation is equivalent to the Langmuir equation, implying a homogeneous adsorption process. The deviation from unity described heterogeneity; the Sips non-linear model equation is used (Eq. (3)).
where q ms denotes the Sips maximum adsorption capacity in mg/g, K s denotes the Sips equilibrium constant in L/mg, and ns denotes surface heterogeneity. This isotherm is commonly used to describe heterogeneous adsorbent surfaces. The non-linear Koble-Carrigan isotherm was used to represent the equilibrium adsorption data for both the Langmuir and Freundlich isotherms (Eq. (4)) (Senthil ).
The Koble-Carrigan parameters are a, b, and n. The Hill adsorption isotherm model states that individual species bind to homogeneous substrates. This isotherm assumed that adsorption was a cooperative process, with adsorbates being able to bind at a single site on the adsorbent, which can influence other binding active sites on the same adsorbent. The non-ideal competitive adsorption (NICA) isotherm was used to create the equation for this adsorption isotherm model. Hills isotherm model in non-linear form, expressed as follows where q sh is the maximum amount of 4-aminopyridine uptake in mg/g and K d is the Hill constant (mg) nh . Furthermore, in this theory if n h = 1, it implies that binding was hyperbolic or non-cooperative, if n h > 1, binding has positive cooperativity and negative cooperativity when n h < 1.

Photocatalytic degradation
For the photocatalytic assay, 100 ml of 4-aminopyridine (pH 7) and (1 g/L) Ag NPs were taken and the mixture was vigorously stirred for 12 h under the dark to attain the adsorption. Later on, the solution was subjected to (Mercury lamp of 125 W) the simulated light source for 2 h to observe photocatalytic degradation of 4-aminopyridine. After 2 h of reaction time, Ag NP residuals were filtered using filter paper (0.2 μm), and solution was analysed. The treatment was repeated for various concentrations of 4-aminopyridine from 5 to 30 mg/L, respectively.

Degradation efficiency (%)
For the photo degradation, the concentration of 4-aminopyridine was analysed using ultraviolet-vis at λ max 281 nm and degradation efficiency (%) was estimated by using Eq. (6).
where C o is the initial concentration of 4-aminopyridine before treatment and C t is concentration after reaction time, "t".

Kinetics analysis
The photo degradation kinetics of the 4-aminopyridine can be represented by the following first-order reaction kinetics where C represents 4-aminopyridine concentration, Firstorder of the reaction, k rate coefficient and 't' time. Firstorder reaction equation after integration becomes C 0 is initial 4-aminopyridine and k, reaction rate constant respectively.

Antibacterial activity
Bio-mediated synthesised Ag NPs and T. divaricate leaves extract was tested for antibacterial activity against gram-negative and gram-positive bacteria. Disc diffusion zone inhibition method was used against E. coli (gram-negative), and B. subtillis (gram-positive) bacteria to examine antibacterial activity (Hemlata Meena et al. 2020). In the disc diffusion method, pure culture of bacteria, volume 100 μl (inoculum age 24 h) was spread with an L-shape spreader on solidified nutrient agar media in petri plates after sterilization by autoclaving (Hamouda et al. 2019). Simultaneously after spreading the bacteria culture on petri plates, a solution of T. divaricate leaves extract and Ag NPs (10 μl, 40 μg/50 μl) was added to each well of 1 mm (Minz et al. 2018). The plates were incubated at 37 °C for 24 h in an aerobic incubation chamber and the inhibition zone was measured.

UV-Vis photo spectroscopy and photoluminescence of Ag NPs
UV-Vis was used to confirm the reduction of Ag + to Ag NPs using AgNO 3 solution and T. divaricate leaves extract at 70 °C. Formation Ag NPs has been optimized by varying the pH of the solution from 5 to 11 pH (Fig. 2a). The evolution of Ag NPs was estimated by observing the colour change of the solution. After the reaction of 70 min, the colour of AgNO 3 solution changed from colourless to yellowish and then turned into brown (Muthu & Priya, 2017). The colour exhibition had occurred due to the excitation of electrons in the d shell of the transition metal which affected the absorbance in the ultraviolet region (Kirubaharan et al. 2012). The synthesised Ag NPs exhibited a sharper SPR band at 415 nm, which was the characteristic wavelength of Ag NPs (Fig. 2b). No peak has been observed at lower pH i.e. (pH 5 to pH 8) as the nucleation process for the formation of Ag NPs at acidic pH was slow (Anigol et al. 2017). A sharp and broad peak was observed for Ag NPs at pH 10 and indicated the formation of poly dispersed Ag NPs that was due to tannins present in the plant extract, which interacted with the Ag + ions and capped the Ag NPs. Figure 2c shows the PL spectra of synthesised Ag NPs obtained at excitation wavelength of 400 nm. In the spectrum, Ag NPs have a significant and well-defined peak in the range of 450 nm. This emphasised that Ag NPs emit light between 400 and 700 nm which was caused by relaxation of the surface plasmon electronic motion (Vigneshwaran et al. 2007). When surface plasmon electrons absorb light at a resonant frequency, some of the absorption energy was converted to heat and some of it was radiated as PL, and recombination of sp electrons  (Parang et al. 2012;Verma and Mehata, 2016).

FT-IR
FT-IR analysis for synthesised Ag NPs and T. divaricate plant extract was done. Results were shown in Fig. 3a and b. In the spectrum of crude T. divaricate leaf extract, the peak at 3310 cm −1 was attributed to the primary amine and polyphenolic O-H group. At 1637 cm −1 a sharp peak was due to the amide I vibrations. The peaks for the proteins and other flavonoids were observed at 1408 cm −1 and 1079 cm −1 corresponded to C-C vibration of the aromatic ring and C-N stretching was a resonance of aliphatic amines respectively (Naseer et al. 2020;Noreen et al. 2020). Figure 3b showed that the FT-IR spectrum of Ag NPs has different peaks corresponding to the functional group present in plant extract capped on Ag NPs. The shift in the peaks has been observed for the Ag NPs spectrum as compared to plant extract which was due to the reduction of Ag + into Ag NPs. Ag NPs spectrum showed that -OH peak of plant extract at 3310 cm −1 was suppressed and located at 3911 cm −1 which indicated that Ag + ions were reduced (Muthu & Priya, 2017). The peak at 2362 cm −1 in plant extract was due to the N-H stretching or the C = O stretching vibrations, and shifted to 2121 cm −1 in Ag NPs showed that these groups are involved in the synthesis of Ag NPs. The peak located at 1621 cm −1 in the Ag NPs spectrum was assigned to C = O vibrations in carboxyl or C = N in the amide group (Bankar et al. 2010). Furthermore, peaks at 1408 cm −1 have also shifted to1374 cm −1 which confirmed that aliphatic and aromatic groups have taken part in the synthesis processes.

HR-TEM and FE-SEM
To analyse the particle size, shape, and morphological arrangement of Ag NPs synthesised using T. divaricate under pH-10 at 70 °C, HR-TEM and FE-SEM analyses were done. The images of Ag NPs showed that nanoparticles were monodispersed and spherical in shape with an average particle size of 30 nm ( Fig. 4a and b). In another study, Ruíz-Baltazar et al. (2018) synthesised Ag NPs using Cynara cardunculus leaf extract. The prepared Ag NPs exhibited semi-spherical morphology with a particle size of less than 45 nm. Muthu and Priya (2017) revealed that the surface morphology of Ag NPs synthesised using Cassia auriculata flower extract. The particles were found of spherical and triangle in shapes with an average size of 35 nm. In the present study, Ag NPs have shown the particles size in the range of 30 nm which showed that T. divaricate extract was a better reducing and capping agent as compared with another plant extract. Figure 4c shows the XRD patterns of purified and dried Ag NPs prepared. XRD spectra showed four intense and sharp diffraction peaks at 38.1°, 44.4°, 64.5°, and 77.5°, which indexed to the (111), (002), (022) and (113) (Nindawat & Agrawal, 2020). Further, the crystallite size of Ag NPs from XRD results was estimated from Scherrer's equation Eq. (9) In Eq. (9) λ: 0.15406 nm is the wavelength of the radiation source and k: 0.9 is the Scherrer's constant . The estimated average crystalline size of Ag NPs calculated using Eq. (9) was 20 nm.

BET and zeta potential analysis of Ag NPs
BET analysis and N 2 adsorption/desorption isotherms were obtained to determine the surface area and the pore size of Ag NPs. The N 2 adsorption/desorption of synthesised Ag NPs are shown in Fig. 5a. BET analysis showed that biosynthesised Ag NPs exhibited surface area 3.63 m 2 /g, pore volume 0.012 cc/g and average pore radius 2.2 nm respectively. According to IUPAC classification, the isotherm obtained was of type V isotherm with H2 type hysteresis loop. Type H2 hysteresis described that the morphology of materials was not symmetric and the distribution of pore size and shape was not well defined.
Stability of Ag NPs has been examined by measuring the zeta potential using instrument (ZEN5600 Zeta Sizer) and results are shown in Fig. 5b. It was found that with the (9) d = k cos increase in pH of Ag NPs, zeta potential increased. At pH 7, zeta potential increased to − 17 mV and indicated the stability of nanoparticles. Jebril et al. (2020) also observed that the zeta potential value of Ag NPs was negative (− 13.1 mV) which emphasised the stability and good dispersion of Ag NPs.

EDX
EDX analysis of Ag NPs showed strong signals at 3 keV, which is a characteristic peak of metallic silver nanocrystals (Fig. 6a). It was observed that Ag NPs using T. divaricate leaf extract were in line with previously reported data of EDX spectra for Ag NPs (P. Singh et al. 2016). The signals for other elements such as carbon, nitrogen, oxygen, and sulphur were also found in the spectrum. Furthermore, elemental mapping of Ag NPs was done and results showed the dispersal of Ag NPs with 46.96% of purity, carbon 16.35% in the form of CaCO 3 , which was due to the plant extract, oxygen 16.35%, nitrogen 20.25%, and sulphur 0.21% (Fig. 6b). The elements predicted in mapping were due to the capping stabilization by the plant extract.

X-ray photoelectron spectroscopy (XPS)
The XPS analysis showed the analysis of the composition and oxidation state of elements present in the Ag NPs. XPS spectrum for Ag NPs revealed indicating the existence of Ag, C, and O (Fig. 7a). The atomic concentration and binding energy of the identified elements in the XPS spectrum are shown in Table 2. The presence of Ag metal was specified by the strong signal of Ag 3d in the range (~ 365 eV to 374 eV) (Ajitha et al. 2015). Figure 7b showed high-resolution Ag 3d characterised by two peaks situated at 366 eV and 373 eV, which appeared due to the spin-orbital splitting which was affirmed to Ag 3d 5/2 and Ag 3d 3/2 core levels (Ruíz-Baltazar et al. 2018; Hu et al. 2014). Figure 7c represents the peak of O 1s with the binding energy of 529 eV and 530 eV respectively, which was due to the oxygen present in the carboxyl group (-C = O-) bound to the surface of Ag NPs (Elechiguerra et al. 2005). The high-resolution peak for C 1s spectrum with the binding energy of 283 and 284 eV was assigned to C = O, and O-C = O-of carboxyl carbon, respectively (Zhang & Feng, 2006) (Fig. 7d).

Mechanism of Ag NPs formation
T. divaricate is a shrub of the Apocynaceae family and is used as a medicinal plant. The LC-MS of plant extract was done and results are shown in Fig. 8. Analysis of the test has confirmed the qualitative presence of alkaloids, tannins, flavonoids, phenols, and carbohydrates in the aqueous extract of T. divaricata (Fig. 8). The mechanistic pathway showed that Ag NPs have formed through the reduction of Ag + to Ag 0 in the presence of enol form of flavonoids present in the T. divaricata plant extract (Scheme 1). Hence, the chelation studies of Ag + to flavonoids have been investigated in an alkaline medium only at pH 10. At higher pH, a faster rate of Ag + reduction has been noticed. The flavonoids with better Ag + reducing activity are those with a 2,3-double bond and processing both the catechol group in the β ring and the 3-hydroxyl group.

Degradation of 4-aminopyridine onto Ag NPs
Adsorption studies of 4-aminopyridine were performed before photocatalytic degradation in dark for 12 h.

Adsorption analysis
For adsorption analysis, 4-aminopyridine in the aqueous form and Ag NPs in solid form were taken, the mixture was stirred for 12 h under the dark condition to obtain adsorption at pH 9. The adsorption spectrum of 4-aminopyridine was analysed using a UV-Vis at λ max 281 nm after a different interval of time. Results emphasised that after 12 h, there was 55% of 4-aminopyridine adsorbed onto Ag NPs at pH 9 (Fig. 9a). Figure 9b revealed the plot between C 0 (initial concentration) versus Xe (the amount of 4-aminopyridine adsorbed onto Ag NPs per gram) and showed the adsorption capacity of Ag NPs at different concertation of 4-aminopyridine. Different adsorption isotherms, such as Langmuir, Hill, Freundlich, Sips, and Koble-Carrigan, were investigated to describe the nature of 4-aminopyridine adsorption on the surface of Ag NPs at constant room temperature. The nonlinear strategy was found to be more effective than the linear method in predicting optimal adsorption. On the basis of experimental data, different parameters such as the regression coefficient (R 2 ) and the sum of square error (SSE) were calculated.  The regression coefficients (R 2 ) were evaluated by using Langmuir, Freundlich, Hill, Kobel-Carrigan, and Sips models to the investigational data and results are shown in Fig. 9c. Table 3 revealed that R 2 values for Langmuir (R 2 -0.95) were more than that of Freundlich (R 2 -0.93), which gave an idea of homogeneous adsorption. To penetrate deep inside the phenomena and to confirm the nature adsorption, three variable isotherm models have been fitted to the experimental data i.e. (Sips, Kobel-Carrigan, and Hill model). The R 2 values for Sips, Kobel-Carrigan, and Hill isotherms were almost the same but higher values as compared to Langmuir and Freundlich isotherms. SSE has been observed as 0.042 and was less than unity for all the isotherms. The Sips isotherm and Kobel-Carrigan models have been incorporated from the Langmuir, Freundlich, and Hill model; therefore, a comparison was made between three-parameter models. The obtained values of n and R 2 were (n s = 2.2, R 2 = 0.97, n kc = 2.2, R 2 = 0.97 and n h = 2.2, R 2 = 0.97); from the above values, it was specified that Langmuir, Sips, Kobel-Carrigan and Hill models fit better than Freundlich model. The Q m values were determined by the Langmuir, Sip, and Hill model which were as 11.01 mg/g, 12.94 mg/g, and 12.94 mg/g, respectively, which were closer enough to the experimental value. This indicated that the main adsorption occurred on the monolayer or through a fixed number of identical sites on the Ag NPs surface.

Adsorption kinetic
The kinetic studies of the adsorption experiment were made to investigate the mechanism of adsorption. Data obtained from the adsorption experiments were fitted in the pseudofirst-order kinetic model. Pseudo-first-order kinetic model was proposed by Lagergren to describe a kinetic modelling of solid-liquid phase adsorption and the equation for this kinetic model is  where q t and q e are the amount 4-aminopyridine adsorbed at any time t (min) and at equilibrium respectively, whereas k (min −1 ) is the pseudo-first-order rate constant. A graph is plotted between ln (q e -q t ) and t with the slope of k and intercept of ln q e representing the pseudo-first-order model of kinetics as shown in Fig. 10. Various parameters of pseudo-first-order kinetics were calculated for the adsorption process of 4-aminopyridine on Ag NPs and shown in Table 4. Results emphasised that pseudo-first-order model was best fitted for 20 mg/L concentration of 4-aminopyridine with R 2 value of 0.99 and slope 0.1735 Fig. 11.

Degradation efficiency (%) of 4-aminopyridine for various parameters
After adsorption, the solution mixture was kept under the visible light for 2 h. The degradation efficiency (%) of 4-aminopyridine onto Ag NPs was analysed by varying the initial concentration (4-aminopyridine), Ag NPs dose and pH.
The photocatalytic activity of Ag NPs was analysed against 4-aminopyridine. By varying the concentration of 4-aminopyridine from 5 mg/L to 30 mg/L (Ag NPs (1 g/L) and pH 7). The maximum 48% degradation efficiency has been observed for the (5 mg/L) 4-aminopyridine using Ag (11) ln q e − q t = lnq e − k t Fig. 10 Plot of pseudo first-order kinetic Ln (q e -q t ) vs time for adsorption of 4-aminopyridine at different concentrations onto Ag NPs (1.6 g/L) and pH 7 NPs (1 g/L) after exposure of 2 h. With the increase in the concentration of 4-aminopyridne from 5 to 30 mg/L, the degradation efficiency decreased (Fig. 10a). The decrease in the efficiency at higher concentrations was due to the overloading of pollutants onto the surface of nanoparticles which caused the reduction in photo degradation of reactive oxygen species .
It was observed that the degradation efficiency (%) has been increased with an increase in Ag NPs dose and the maximum efficiency obtained was 58% at catalyst dose of 1.6 g/L (concentration of 4-aminopyridine (5 mg/L, pH 7). Afterward, with the increment of Ag NPs dosage, a decrease in the removal efficiency (%) has been observed in Fig. 10b. The increase in Ag NPs dosage enhanced a number of active sites due to which a large number of 4-aminopyridine molecules get decomposed. Further increase in the dosage beyond 1.6 to 2 g/L, a decrease in the photo degradation has been perceived due to increased turbidity of the solution which prevented the entering of light photons into the solution mixture and decreased the rate of reaction (Shaikh et al. 2020;Singh & Dhaliwal, 2020).
The pH of the solution was important in photo degradation. Therefore, the pH of 4-aminopyridine has been varied from pH 3 to pH 7 (concentration of 4-aminopyridine (5 mg/L), Ag NPs (1.6 g/L) and reaction time 2 h). It was observed that with increase in pH from 3 to 7, there was an increase in the removal efficiency of 4-aminopyridine onto Ag NPs (Fig. 10c). Zeta potential studies of Ag NPs at different pH also revealed that with increase in pH, the value of zeta potential increased (Fig. 10d). At neutral pH 7, Ag NPs showed maximum value of zeta potential (− 17 mV), which might increase the degradation of 4-aminopyridine. The degradation of 4-aminopyridine onto Ag NPs was higher at neutral pH due to more stable dispersion and higher electrostatic force of attraction between Ag NPs and 4-aminopyridine (Nagar and Devra 2019;Salazar-Bryam et al. 2021).

Photo degradation kinetic
The reaction kinetics of 4-aminopyridine on to Ag NPs was studied by varying the concentration of 4-aminopyridine from 5 mg/L to 30 mg/L (Ag NPs dose 1.6 g/L, pH 7 and reaction time of 2 h).
The elementary rate equation used is as follows where t (min) represents the reaction time, C 0 (mg/L) is the initial concentration, C t is a concentration at time t of 4-aminopyridine, k 1 (min −1 ) is the first-order kinetics rate constant, respectively. Regression (R 2 ), and rate constant (k 1 ) were analysed based on first-order reaction kinetics and the results are presented in Fig. 12. It was observed that the regression was maximum at 5 mg/L concentration of 4-aminopyridine (Table 5). High regression coefficient (R 2 ) value 0.99 indicated that the data fitted well in the first-order kinetic equation. With the increase in the concentration of 4-aminopyridine, the rate constant decreased due to decrease in a number of the available active sites.

Proposed mechanism for the degradation of 4-aminopyridine using Ag NPs
LC-MS spectra were recorded to identify intermediates formed during the reaction of 4-aminopyridine onto Ag NPs. The mechanism of 4-aminopyridine degradation is proposed and supported by the daughter ions obtained in mass spectra (Fig. 13). 4-Aminopyridine has two electron donating sources one is -NH 2 group (due + R effect) and another is 'N' atom (Hesse et al. 2008). The lone pair of electrons on -NH 2 group becomes nucleophilic in nature due to + R effect and can easily affect the protons from the reaction environment. The number of intermediates was found to be ten within the mass number of 0-450. Hence, three paths are drawn to identify all products. Path # 1 shows the formation of D 1 (m/z, 127.07), D 2 (m/z, 104.4), D 3 (m/z, 234.11) and D 4 (m/z, 435.17). D 1 molecule was formed by protonation (+ H + ) followed by deammonification (-NH 2 ) due to the electrophilic effect of HO . radical. D 2 was yielded under the oxidation of D 1 product and finally converted CO 2 and H 2 O (King et al. 2012). Both the D 3 and D 4 molecules were formed from the D 1 daughter ions by the condensation reaction with the evaluation of water (Sykes et al. 1986). Path #2 shows the formation of D 5 (m/z, 248.12), D 6 (m/z, 456.14) and D 7 (m/z, 455.19) molecules. D 5 molecule was formed by hydroxylation followed by dehydration reaction (Scheme 2 (a)) and was converted to D 6 molecule through condensation by elimination of water molecules. Hence four pyridine moieties are linked by an ether (-O-) linkage. D 7 molecule was yielded by substitution reaction of -NH 2 group by HO . radical under the pH-7. Now, an intermediate of molar mass 111.02 (m/z) (path#3) originated under de-ammonification (-NH 3 ) of 4-aminopyridine was coupled to D 8 (m/z, 248.87) molecules by dehydration followed by hydroxylation reaction through an intermediate of m/z, 188.16. D 9 (m/z, 266.19) molecule was originated from D 8 by hydroxylation followed by protonation. Finally, D 10 (m/z, 146.26) was formed 3, 4 dihydroxy pyridine (Scheme 2b) by the coupling with another molecule of 4-aminopyridine (Hesse et al. 2008).

Antibacterial assay
The antibacterial test was conducted for T. divaricate leaves extract, and bio mediated Ag NPs synthesised using T.  . 14a and b) as compared to the gram-positive B. subtillis 12 mm ( Fig. 14c and d). In the case of T. divaricate leaves extract, a minor inhibition zone was observed against both the pathogenic bacteria. The reason for the antibacterial activity of Ag NPs was that nanoparticles get attached to the surface of the cell membrane disturbing the permeability and respiration functions of the cell (Ramesh et al. 2015).

Stability and reusability test of Ag NPs
The stability and reusability of Ag NPs synthesised using T. divaricate were evaluated to ensure its efficiency. The Scheme 2 (a) & (b) Proposed mechanism for the degardation of 4-Aminopyridine using Ag NPs stability of Ag NPs was analysed using a UV-Vis spectrophotometer for the different time intervals. Ag NPs have shown sharp UV-Vis peaks for 30 days, which confirmed that synthesised Ag NPs were stable and could not be oxidised (Fig. 15a). Furthermore, the reusability of Ag NPs was tested for eight successive degradation experiments of 4-aminopyridine (20 mg/L, pH 7, Ag NPs -1.6 g/L), and results are shown in Fig. 15b. After each cycle, Ag NPs were collected from the reaction mixture, washed and dried at 80 °C for further experimentation. Ag NPs have shown degradation efficiency in all the cycles and exhibited 61% degradation of 4-aminopyridine after 1st cycle, and further, it declined to 30% in the last cycle. The drop in degradation efficiency was due to the gathering of 4-aminopyridine molecules onto the poisoning sites of Ag NPs.

Comparison with other studies
In the present study, environmental friendly, Ag NPs have been synthesised using T. divaricate leaves extract. Synthesised nanoparticles were characterised using UV-Vis, XRD, HR-TEM and XPS analysis. To the best of our knowledge, there is a gap in the literature for mechanistic pathway reduction of Ag + using T. divaricate. There is no study available in the literature on adsorption and photo degradation of 4-aminopyridine using Ag NPs. A comparison of bio mediated Ag NPs synthesised from different plant extract in studies in literature with the present study is shown in Table 6. It has been confirmed that Ag NPs synthesised from T. divaricate extract have showed effective antibacterial and photocatalytic activity as compared to other studies. In this study, 61% degradation of 4-aminopyridine has been achieved using Ag NPs and showed inhibition for E. coli (35 mm), B. subtillis (12 mm).

Conclusion
1. Ag NPs were procured from T. divaricate leaves extract. UV-Vis spectrum showed a sharp and broad peak at pH 10 that indicated the formation of poly dispersed Ag NPs. The particles of Ag NPs were found of spherical and triangle in shapes with an average size of 35 nm. Mechanistic pathway has been proposed for the reduction of Ag + to Ag 0 in the presence of enol form of flavonoids present in the T. divaricata plant extract at pH 10. 2. Ag NPs were found to be a good adsorption and degradtion catalyst for the removal of 4-aminopyridine from aqueous solution. Studies showed 55% adsorption and 61% photocatalytic degradation efficiency of 4-aminopyridine under the optimum conditions (4-aminopyri-Scheme 2 (continued) dine: (5 mg/L), pH-7 and Ag NPs: (1.6 g/L) within 2 h of reaction time. 3. The degradation of 4-aminopyridine onto Ag NPs obeyed first-order reaction kinetics at different concentrations. The higher regression coefficient value (R 2 -0.99) showed that the data fitted well in first-order kinetic equation. 4. Furthermore, bio-mediated Ag NPs synthesised using T. divaricate plant extract were evaluated to be highly effective for the inactivation of E. coli growth with 35-mm inhibition zone diameter. The present study indicated that bio-mediated Ag NPs showed good photo catalytic and antibacterial activity. The study can be further enhanced for the treatment of other pharmaceutical compounds.