1 Introduction

Nanoscale materials with exceptional properties have catapulted nanotechnology into the limelight, with applications spanning electronics, medicine, textiles, and environmental protection. Because of the high surface-to-volume ratios and capacity to range in at least one dimension between 1 and 100 nm, nanoparticles are incredibly powerful materials. The particle size of nanoparticles is influenced not only by their surface area to volume ratio but also by their physical, biological, and chemical properties, which differ from those of their conventional substance [1]. Researchers have examined the antimicrobial properties of several nanomaterials, including copper, zinc, gold, and silver, against various microbiological infections. Researchers have found that silver nanoparticles have many valuable characteristics, including their potent antibacterial ability [2].

Nanoparticles can be produced artificially by a variety of methods, such as photochemical, radiation, and electrochemical ones. Still, these techniques are expensive, leave harmful residues in the environment, and pollute the environment. Because of this and their high efficiency, cheap cost, eco-friendliness, and scale-up properties, natural ingredients are increasingly being considered for the synthesis of nanoparticles [3]. By utilizing green technology for nanoparticle synthesis, we can harness the potential of this existing natural treasure in an environmentally conscious way. Therefore, this eco-friendly green synthesis method benefits the pharmaceutical sector. Also, unlike conventional methods, green nanoparticle production rarely requires a lot of money or human resources. Similar to physical or chemical methods of nanoparticle manufacturing, they pose no threat to living things and are environmentally benign [4].

Soursop or Annona muricata, belongs to Annonaceae family which comprises around 2300 species in 130 genera. At maturity, trees reach a height of 8 to 10 m, stand upright with few branches, have rough and nut-brown stems, oblong-ovate to cylindrical leaves that range in size from 14 to 16 cm in length and 5 to 7 cm in width, and bloom at various points throughout the year on multiple trunks or branches [5]. In folk medicine, the plant is used to cure a wide range of illnesses, including skin ailments, respiratory issues, bacterial and fungal infections, fever, hyperglycaemia, hypertension, and cancer [6].

Endophytes are microscopic organisms that successfully inhabit plant tissues without causing any harm to those tissues. Every vascular plant contains endophytic organisms, which defend themselves against diseases and harmful environmental conditions by secreting bioactive secondary metabolites [7]. Certain plants produce these bioactive natural chemicals due to the co-evolution between hosts and endophytes. Endophytic fungi’s physiological and ecological activities are crucial for the survival of the plants that host them and it is important to understand the diversity of symbiotic bacteria, their host or habitat despite their relative abundance as these endophytes possess significant therapeutic properties [8]. There is a common belief that medicinal plants contain endophytic fungi that produce helpful compounds.

The present investigation seeks to produce and describe silver nanoparticles (Colleto-AgNPs) through an aqueous endophytic extract of Colletotrichum siamense Prihast from A. muricata leaves. This research expands upon previous work that used the Quality-by-design methodology to examine the therapeutic potential of Colleto-AgNPs through in-vitro tests for antibacterial, antioxidant, and antidiabetic effects and the impact of process variables on its synthesis [9].

2 Materials and methods

2.1 Isolation and Cultivation of the Endophyte

Following our previous reports [8], the fungal endophyte was extracted from mature, healthy, and fresh A. muricata leaves. Its identification was verified through micro and macromorphological analysis at the Agharkar Research Institute in Pune, Maharashtra, India, affiliated with the National Fungal Culture Collection of India (NFCCI) (Supplementary File). The endophytic fungal culture that had been purified was kept in a deep freezer at -18 °C in cryovials containing Sabouraud dextrose broth that had been supplemented with 15% glycerol (v/v) [10, 11].

2.2 Synthesis of Silver Nanoparticles (AgNPs)

Sabouraud Dextrose Broth (SDB) was used to cultivate the isolated fungus for three days at 27⁰C [8]. After centrifugation at 4500 rpm for 15 min, the broth was filtered through Whatman No. 1 filter paper to get a cell-free filtrate. The cell-free fungal extract was combined with 100 mL of sterile distilled water in volumes of 2 mL, 5 mL, and 8 mL, respectively, to produce the aqueous fungal extract, with the relative volumes depending on the percentage of extract needed and used for Colleto-AgNPs synthesis.

Following the experimental design [12, 13], a defined pH was used to add a determined amount of aqueous fungal extract [14] to a specified amount of 1 mM silver nitrate solution [15]. The mixture was allowed to react at room temperature for a predetermined reaction period. From yellow to dark brown, the color of the reaction mixture changed [12]. A 20 min centrifugation run at 10,000 rpm separated the solids from the liquid. On two or three occasions, this centrifugation process was carried out by adding a tiny quantity of distilled water to flush out any residual contaminants. Overnight drying at 90 °C was subsequently performed on the sediment.

2.3 Statistical Experimental Design

Researchers used the Box Behnken design (BBD) to study how different formulation variables affected the size and wavelength of Colleto-AgNPs particles [12, 16]. We used Design-Expert® (Version 11, Stat-Ease Inc., Minneapolis, USA) to generate 27 experimental runs. We used analysis of variance (ANOVA) (F Value) to examine the effect of independent variables on the responses (Y1: particle size and Y2: wavelength) at three levels, specifically X1: reductant concentration, X2: AgNO3: reducant, X3: reaction time, and X4: reaction pH (Table 1). The built model was analyzed using Fisher’s F-test and Analysis of variance (ANOVA). The coefficient of determination, R2, expressed the model equation’s fit quality. The fitted model’s correlation with the responses was then demonstrated using contour and surface plots. Contour plots and response surfaces were used to better understand the relationship and interaction between the causes and responses that were explored.

Table 1 Levels of variables chosen for the experimental BBD
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2.4 One Factor-at-a-time (OFAT) Analysis

Using OFAT, we were able to establish the ideal values for the parameters used to create silver nanoparticles. The pH, reaction time, concentration of the reductant (AgNO3), and concentration of the reductant were among these parameters.

2.5 Characterization of Nanoparticles

The synthesis of Colleto-AgNPs was confirmed by analyzing the reaction mixture’s absorption spectra with a UV-visible spectrophotometer (ThermoFisher Scientific, Orion AquaMate 8000 UV-Vis v1.007 2W2W029302, USA) over a wavelength range of 200–600 nm. A distinctive surface plasmon resonance (SPR) band in the 420–570 nm region indicated the production of AgNPs [14]. The Fourier-transform infrared spectroscopy (FTIR) was used to identify the functional groups in the produced nanoparticles, whereas the particle size and zeta potential of synthesized nanoparticles were assessed using a particle size and zeta potential analyzer (Model: Zen 3600, Malvern Instruments, Malvern, UK). An angular range of 20° < 2θ > 80° was used for the powder X-ray diffraction (XRD) analysis of the produced Colleto-AgNPs. Energy dispersive X-ray microscopy (EDAX) was used by a scanning electron microscope (SEM) to examine the microstructure of the silver nanoparticles. A TEM analysis revealed the nanoparticles’ intricate surface texture. The antibacterial activity of Colleto-AgNPs is strongly correlated with their size and shape [17].

2.6 Brine Shrimp Lethality Bioassay

In order to determine whether the synthesised nanoparticles were cytotoxic, we tested them on Artemia salina Leach eggs [12, 16]. In brief, the initial step was to create three distinct concentrations (in geometric series) of Colleto-AgNPs in seawater: 1000, 100, and 10 ppm. Afterwards, 10 out of 11 shrimps were given 3 mL of each test solution. The assay was conducted in triplicate, and the control tubes contained seawater in an equivalent amount. The number of survivors was counted and the LC50 was determined after 24 h.

2.7 In-vitro Biological Assays

2.7.1 Antibacterial Assay

Silver has a lengthy history of medicinal usage in many parts of the world. Research has shown that nanosized particles of silver have enhanced antibacterial activity when contrasted with bulk silver. The antibacterial efficiency of the Colleto-AgNPs produced was investigated using the agar well diffusion method [16, 18]. Gram-positive bacteria such as S. aureus and L. monocytogenes, as well as gram-negative bacteria such as E. coli and S. typhimurium, were procured from the National Collection of Industrial Microorganisms (NCIM) in Pune, India.

The broth dilution procedure assessed each bacterium’s minimum inhibitory concentration (MIC) before the zone of inhibition tests were conducted using Colleto-AgNPs solution as the test medication [19]. Individual microwells were filled with 50 µL of Mueller-Hinton (MH) broth and Colleto-AgNPs at several concentrations. Additionally, bacterial inoculums containing 106 CFU/mL of L. monocytogenes, S. aureus, E. coli, and S. typhimurium were introduced and inoculated at 37ºC. Following a 24 h period of rest, 20 µL of p-iodonitrotetrazolium dye (0.5 mg/mL) was introduced and left to incubate at 37 ºC for 30 min. Streptomycin was the antibiotic of choice in both experiments.

For agar well diffusion method, the subcultures of the organisms were grown onto nutrient agar slants at 35 °C from pure cultures. A cork borer was used to make four wells of 6mm into each PCA (Plate Count Agar) plate. The spread plate method infected these plates with their respective bacterial suspensions, which were produced in normal saline. A micropipette was used to pour 20 µL of test drug solution (varying concentrations) into each well of all the bacterium plates. Variegated concentrations of streptomycin were applied to a separate set of plates that had been inoculated with the four bacteria (the positive control group). The plates were left to incubate at 30 ± 2 °C for 24 h, after which the inhibition zones were measured.

2.7.2 Antidiabetic Activity

2.7.2.1 α- Amylase Enzyme Inhibitory Assay

The α-amylase enzyme inhibitory activity of Colleto-AgNPs was determined using the methodology described by Amalraj et al. [20]. The following amounts of Colleto-AgNPs were used: 50, 100, 150, 200, and 250 µg/mL. 100 µL of each concentration was combined with a starch solution (1%) that contained 20 mM phosphate buffer saline (pH 6.9) and 6 mM sodium chloride. The mixture was pre-incubated at 25ºC for 10 min followed by incubation under the same conditions after adding 100 µL of pancreatic α-amylase enzyme (0.5 mg/mL). Further, 200 µL of dinitrosalicylic acid was added and incubated at 100 ºC for 5 min to terminate the hydrolytic reaction that had been started. Acarbose served as a positive control to measure absorbance at 540 nm, and the combination was further diluted with deionized water when the samples reached room temperature. The antidiabetic ability (α-amylase enzyme) of the Colleto-AgNPs was calculated with the following equation,

$$\eqalign{& {\rm{Inhibition}}\left( \% \right) = \cr & {{{\rm{Absorbance}}{\mkern 1mu} {\rm{of}}{\mkern 1mu} {\rm{Acarbose}} - {\rm{Absorbance}}{\mkern 1mu} {\rm{of}}{\mkern 1mu} Colleto - {\rm{AgNPs}}} \over {{\rm{Absorbance}}{\mkern 1mu} {\rm{of}}{\mkern 1mu} {\rm{Acarbose}}}} \cr & \times 100 \cr}$$
2.7.2.2 α- Glucosidase Enzyme Inhibitory Assay

The method of Halarenkar et al. [21] followed in conducting the α-glucosidase enzyme inhibitory assay was used to assess the ability of Colleto-AgNPs to inhibit the enzyme. A mixture of 50 µL of Colleto-AgNPs solution in different concentrations (50, 100, 150, 200, and 250 µg/mL) and 50 µL of p-nitrophenyl-α-D glucopyranoside dissolved in phosphate buffer was prepared using sodium phosphate buffer (100 mM pH 6.9). The mixture was then incubated at 37 ºC for 5 min. Each microwell was then supplemented with 100 µL of phosphate buffer containing α-glucosidase enzyme (0.1 U/mL). We used acarbose, a typical enzyme inhibitor, to quantify the absorbance at 405 nm after 30 min of resting. The antidiabetic ability (α- glucosidase enzyme) of the Colleto-AgNPs was calculated with the following equation,

$$\eqalign{& {\rm{Inhibition}}\left( \% \right) = \cr & {{{\rm{Absorbance}}{\mkern 1mu} {\rm{of}}{\mkern 1mu} {\rm{Acarbose}} - {\rm{Absorbance}}{\mkern 1mu} {\rm{of}}{\mkern 1mu} Colleto - {\rm{AgNPs}}} \over {{\rm{Absorbance}}{\mkern 1mu} {\rm{of}}{\mkern 1mu} {\rm{Acarbose}}}} \cr & \times 100 \cr}$$

2.7.3 Antioxidant Activity

We used the previously described approach of Raj et al. [22] to determine the DPPH radical scavenging capability of Colleto-AgNPs. An equal volume of DPPH solution (0.1 mM) in methanol was added to 100 µL of Colleto-AgNPs from various concentrations (50, 100, 150, 200, and 250 µg/mL). The mixture was incubated for 30 min at room temperature, and the absorbance was recorded at 517 nm. Ascorbic acid was used as the standard antioxidant medication, and the DPPH radical scavenging activity was measured as IC50 µg/mL. The antiradical ability of the Colleto-AgNPs was calculated with the following equation,

$$\eqalign{& {\rm{Inhibition}}\left( \% \right) = \cr & {{{\rm{Absorbance}}{\mkern 1mu} {\rm{of}}{\mkern 1mu} {\rm{Ascorbic}}{\mkern 1mu} {\rm{acid}} - {\rm{Absorbance}}{\mkern 1mu} {\rm{of}}{\mkern 1mu} Colleto - {\rm{AgNPs}}} \over {{\rm{Absorbance}}{\mkern 1mu} {\rm{of}}{\mkern 1mu} {\rm{Ascorbic}}{\mkern 1mu} {\rm{acid}}}} \cr & \times 100 \cr}$$

2.8 Statistical Analysis

The collected results were presented as mean ± standard deviation. The IC50 value for enzyme inhibitory (α-amylase and α-glucosidase enzyme) and antioxidant (DPPH) experiments was obtained using non-linear regression analysis. By applying the findings to one-way ANOVA (Analysis of Variance) using Duncan’s test at the 5% (%) significance level, the statistical difference or significance of the reported data was ascertained. Statistical significance was determined for reported values with P < 0.05, and STATISTICA 12.5 software was utilised.

3 Results and Discussion

The current investigation found that the reducing agent in the endophytic fungus C. siamense aqueous extract turned a dark brown color when it came into touch with the AgNO3 solution. Enzymes such as reductase, nitrate reductase, and hydrogenase are being considered as potential pathways for the bio-reduction of silver. Figure S1 illustrates that the color of solution changes from colorless to pale yellow to dark brown as the reaction progresses, which is a visual signal of Colleto-AgNPs synthesis. When surface plasmon vibration is excited, a color shift occurs, which means silver nanoparticles are in the solution. These findings can be confirmed if the surface absorption band is located at about 420 nm, as predicted from UV-visible spectra [12]. A reducing agent is required for the bio-reduction of silver to occur; this agent is reported to have reduced Ag+ in an AgNO3 solution to Ag0 on the nanoscale, as shown in Eq. 1 [12];

$${Ag}^{+}+ {e}^{-} \to {Ag}_{0}$$
(1)

3.1 Optimization of Silver Nanoparticles

The Box Behnken Design (BBD) experiment was used to find the optimal parameters for synthesizing Colleto-AgNPs. The concentration of the reductant, reaction pH, duration of the reaction, and the ratio of AgNO3 to reductant are the variables examined in this investigation. One possible route for AgNPs synthesis is enzymatic reduction. The nitrate reductase enzyme is commonly considered the mechanism, acting as a reducing agent. This study examined the relationship between nanoparticle formation and reductant concentration, among other parameters. The effects of independent factors on the response variables were examined using BBD. These variables were reductant concentration (X1), AgNO3: reductant (X2), reaction time (X3), and reaction pH (X4). The experimental runs for BBD were 27 in total, and Table 2 shows the resulting matrix of variables with examined levels and practical responses, including particle size (Y1) and wavelength (Y2).

The values for Y1 and Y2 responses spanned from 95.32 to 119.64 nm and 401 to 430 nm, respectively, as outlined in Table 2. The Quadratic model emerged as the optimal fit for both Y1 (R2: 0.9518 and PRESS: 408.02) and Y2 (R2: 0.8137 and PRESS: 1814.94). This suggests that the chosen model is adept at predicting 95.18% and 81.37% variations in the dependent variables Y1 and Y2, respectively. Further affirmation of the model’s significance was sought through ANOVA, as detailed in Table S1. The response Y1 and Y2 values ranged between 97.45 and 144.45 and 406–431 nm, respectively (Table 2). The Quadratic model was best fit for both Y1 (R2 0.9438 and PRESS: 1412.36) and Y2 (0.8616 and PRESS: 1666.14). Based on this, it can be corroborated that the selected model can accurately predict 94.38 and 86.16% changes in dependent variables Y1 and Y2, respectively. ANOVA was used further to determine the model’s significance (Table S1).

F values of 14.39 and 8.49 for responses Y1 and Y2 corroborate the model’s significance. Predicted R2 values of 0.70 and 0.57 for Y1 and Y2 did not differ by more than 0.2 from adjusted R2 of 0.88 and 0.76, respectively. This confirms reasonable agreement between the two values. Plotting observed and predicted values verified the significance of both quadratic models (Figure S2). The relationship between independent and dependent variables can be studied using the following equations,

$$\eqalign{& {Y_1} = + 140.28 + 3.74{X_1} + 2.29{X_2} + 0.8183 {X_3} \cr & \,\,\,\,\,\,\,\,\,\, + 7.71 {X_4} - 2.86 {X_1}{X_2} - 0.9625 \cr & {X_1}{X_3} + 7.30{\rm{ }}{X_1}{X_4} + 0.1125 \cr & {X_2}{X_3} - 0.6100 {X_2}{X_4} + 7.69{X_3}{X_4} - 13.23{\rm{ }}{X_1}^2 \cr & \,\,\,\,\,\,\,\,\,\,\,\,\,\, - 21.71{X_2}^2 - 15.07{\rm{ }}{X_3}^2 - 8.43{\rm{ }}{X_4}^{2 } \cr}$$
(2)
$$\eqalign{& {Y_2} = {\rm{ }} + 435.33 - 0.3333{X_1} - 0.2500{X_2} \cr & - 0.8333{X_3} + 8.08{X_4} + 1.0000{X_2}{X_3} + 0.5000{X_2}{X_4} \cr & + 1.50{X_3}{X_4} - 18.17{X_1}^2 - 15.04{X_2}^2 - 14.67{X_3}^2 - 8.79{X_4}^2 \cr}$$
(3)
Table 2 BBD matrix runs with results of response variables (Independent and dependent variables)
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X1 showed a significant positive effect on Y1, while X4 showed substantial positive impacts on Y1 and Y2. Terms X1 × 4 and X3 × 4 exhibited significant positive effects on Y1. The quadratic term of each factor (X1 to X4) significantly negatively affected both responses. 3D response surface plots were used to validate these results further (Fig. 1).

Two-factor interactions (X1 × 4 and X3 × 4) were further confirmed by the crossed lines observed in interaction plots (Figures S2 and S3). Figure S4 shows model diagnosis plots, which include a normal and residual versus run plot. On critically observing these plots, it can be inferred that residuals followed a straight line, signifying an even distribution of errors. The residuals versus run plot similarly demonstrated a random dispersion.

Fig. 1
figure 1

3D response surface plots showing the effect of variables on response Y1 (A-E) and Y2 (F)

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3.2 Model Validation

Software suggested that optimized values, i.e., 5.079% of reductant concentration, 1:1.23 ratio of AgNO3: Reductant with a reaction time of 1.15 h and pH value of 8.77, will produce optimized formulation. The region of optimized formulation is depicted in Figure S5. The predicted particle size and wavelength values were 105.86 nm and 413.5 nm, respectively. Predicted and actual results were in close agreement, indicating the model’s validity.

3.3 Optical Characterization by UV-visible Absorption

Fig. 2
figure 2

UV-visible spectra of Colleto-AgNPs at reductant concentration:- 7.664%, AgNO3 : Reductant ratio :- 1:3.122, reaction time :- 3.271 h, and reaction pH :- 10, showing an absorption peak at 420 nm

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One simple and effective way to verify the creation of nanoparticles is by using UV-visible absorption spectroscopy. The UV-visible absorption spectra of the optimized batch of AgNPs, as per the Box Behnken Design, are displayed in Fig. 2. A wide absorption band from 300 to 500 nm and a maximum at 420 nm in the spectra point to the presence of Colleto-AgNPs.

3.4 Microscopic Analysis by SEM with EDAX and TEM Studies

In Fig. 3, the morphological examination results of the produced Colleto-AgNPs are shown in both the SEM examinations (Fig. 3A) and the EDAX spectra (Fig. 3B). Both the TEM and SAED analyses indicated that the nanoparticles had a spherical form (Fig. 3C and D). Applying EDAX analysis improves the identification of the minerals in the synthesized nanoparticles. The produced silver nanoparticles were discovered to include several minerals, including calcium, silver, carbon, and oxygen, according to EDAX analysis. The pre-analysis sample treatment is responsible for the presence of gold and palladium in the results.

Fig. 3
figure 3

(A) SEM, (B) EDAX, (C) TEM (D) SAED (E) XRD analysis of Colleto-AgNPs

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3.5 Structural Analysis

The XRD pattern of the finished Colleto-AgNPs in the 2Ɵ range of 20⁰-80⁰ is displayed in Fig. 3E. The Colleto-AgNPs exhibited diffraction peaks at 38.1⁰, 44.35⁰, 64.5⁰, and 77.48⁰, as recorded by the XRD peaks. A strong peak at 38.10⁰ diffraction suggested that the silver crystal was perpendicular to the plane. A mean particle size of 16.16 nm was determined for the produced nanoparticles using the following formula:

$$D = {\rm{ }}0.9\lambda /\beta cos\theta$$

where D is the crystalline size, λ is the X-ray wavelength (CuKa / 1.541862 A), β is the full width at half maximum, and Ɵ is Bragg’s angle.

3.6 Particle Size Analysis

The average particle size was found to be 98.48 nm (Fig. 4A), and the polydispersity index (PDI) was 0.198 with a zeta potential of -4.92 mV (Fig. 4B). Particle characteristics shown in Fig. 4 include a mobility of -1.626e-004 cm2/Vs, a Doppler shift of 12.84 Hz, a base frequency of 127.3 Hz, and a conductivity of 0.3243 mS/cm.

Fig. 4
figure 4

(A) Particle size and (B) Zeta potential analysis of the synthesized Colleto-AgNPs

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3.7 Cytotoxicity Study by Brine Shrimp Lethality Bioassay

The cytotoxic effect of synthesized Colleto-AgNPs was investigated using a brine shrimp lethality experiment with varying nanoparticle doses for 24 h. Results revealed that brine shrimps were highly susceptible to Colleto-AgNPs, even at lower doses. Since the LC50 falls within the range of 100 to 1000 ppm, the Colleto-AgNPs were determined to have an LC50 value of 312.51 ± 32.54 and could exhibit some biological activity. KCrO7 solution (positive control) showed LC50 < 10 µg/mL, whereas no deaths were observed in seawater (negative control). Our results were in agreement with the cytotoxic efficacy of nanoparticles reported by Gawas et al. [16].

3.8 In-vitro Antibacterial Activity

Microbial diseases pose serious health threats, and the emergence of multidrug-resistant microbes necessitates the development of new effective techniques. Nanotechnology offers a novel approach to combat these diseases by utilizing the antimicrobial properties of nanomaterials, which interact easily with microbial cells to exhibit their mechanisms [23]. AgNPs are considered an essential type of metallic nanoparticles due to their antibacterial properties and their ability to form silver salts (Ag) by reduction, and they are unique in having an increased surface area to volume ratio [24]. These properties make them suitable in diverse areas like biomedicine, catalysis, pharmaceuticals, and biosensors. Silver has a long history of being used as an antimicrobial agent in treating Neisseria gonorrhoeae [25]. Compared to Ag, the usage of AgNPs has increased due to its substantial surface area. Green synthesized AgNPs have proven their efficacy as an efficient antioxidant, antibacterial, anticancer, antidiabetic, photocatalytic, and mosquitocidal agent [26].

Table 3 Minimum inhibitory concentration (MIC) of Colleto-AgNPs
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The study adopted the microdilution method to determine the minimum inhibitory concentration (MIC) of Colleto-AgNPs and was reported to be highly effective in suppressing E. coli, L. monocytogenes, S. aureus, and S. typhimurium at the dosage of 23.43, 46.87, 187.5 and > 750 µg/mL (Table 3). The study found that E. coli was slightly resistant to Colleto-AgNPs and the standard antibiotic, streptomycin, as it required a high concentration of 187.5 and 11.7 µg/mL, respectively. The leaf extract of A. muricata required an increased amount of > 750, 375, 187.5, and 93.75 µg/mL to inhibit or kill S. typhimurium, E. coli, L. monocytogenes, and S. aureus. While at > 750, 750, 375, and 187.5 µg/mL concentrations, the fungal extract of C. siamense inhibited S. typhimurium, E. coli, L. monocytogenes, and S. aureus.

In the disc diffusion method, at a concentration of 750 µg/mL, Colleto-AgNPs exhibited the strongest inhibitory action against L. monocytogenes (23.98 mm), and then against S. aureus (22.9 mm), E. coli (20.23 mm) and S. typhimurium (17.76 mm). From Table 4, it is evident that the conventional antibiotic streptomycin exhibited the highest zone of inhibition by inhibiting the growth of L. monocytogenes (25.02 mm), followed by S. typhimurium (23.52 mm), S. aureus (23.34 mm), and E. coli (22.21 mm). Accordingly, the fungal endophyte synthesized silver nanoparticles can be considered an appropriate broad-spectrum antimicrobial as they have exhibited better antibacterial activity against the studied microorganisms even at a low 125 µg/mL concentration.

At the high concentration, Colleto-AgNPs activity was similar to streptomycin in inhibiting the growth of S. aureus and L. monocytogenes. The study also estimated the antibacterial activity of A. muricata leaf extract and C. siamense (endophyte) extract. The zone of inhibition produced by A. muricata leaf extract ranged from 17.9 mm to 22.13 mm, and the zones produced by C. siamense (endophyte) extract ranged between 14.12 mm and 19.23 mm. The antibacterial activity of these extracts was less compared to the activity of Colleto-AgNPs and streptomycin (Table 4). In the present study, the Colleto-AgNPs have expressed noteworthy antibacterial activity against S. typhimurium, L. monocytogenes, and E. coli.

Table 4 Antibacterial activity of Colleto-AgNPs against pathogenic bacteria
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According to NajeerAhamed et al. [27] the AgNPs synthesized using Colletotrichum gloeosporioides have shown reduced antibacterial activity by forming inhibitory zones of diameter 14 and 15 mm against E. coli (ATCC 25,922) at the concentration of 25 and 50 µg/mL and against MDR 1 and MDR 2 strains of E. coli, the zone of inhibition formed by 25 and 50 µg/mL of C. gloeosporioides mediated AgNPs was in the range between 12 and 15 mm [23]. The AgNPs synthesized from Penicillium isolated from the leaves of Curcuma longa have shown their antibacterial property by forming zone of inhibition of 14 mm and 13 mm against S. typhimurium and E. coli at the dosage of 80 µg/mL [28].

The silver nanoparticles synthesized from the aqueous extracts of Nigella sativa revealed very poor bacterial inhibitory activity even at higher concentration with the inhibition zones of 16.45, 17.38, and 14.35 mm against the bacterial strains E. coli, S. aureus and S. typhimurium, respectively [29]. The difference in the antibacterial activity exhibited by Colleto-AgNPs is due to the structural variation among the gram-positive and gram-negative bacteria. The AgNPs exhibit their antibacterial activity by disrupting the cell walls and membranes, altering the cell membrane’s permeability, impairing proteins, inhibiting DNA replication, and increasing the production of reactive oxygen species, which results in oxidative stress [30].

The nanoaparticle also causes denaturation and dysfunctioning of ribosomes and mitochondria. It is fascinating that these AgNPs release or produce toxic silver ions to bacterial organisms, disrupting the physiological processes and damaging the cell components (Fig. 5) [24]. The increased surface-to-volume ratio nature of AgNPs also influences their antibacterial activity. Compared with the previously synthesized AgNPs, the antibacterial efficacy of AgNPs (Colleto-AgNPs) synthesized using Colletotrichum siamense is remarkable.

Fig. 5
figure 5

The possible antibacterial mechanism exhibited by AgNPs include disruption of cell wall and cell membrane (1), denaturation of ribosomes (2), disruption of DNA replication (3), denaturation of RNA (4) and proteins (5), generation of ROS (6), mitochondrial dysfunction (7) and disruption of electron transport chain (8)

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3.9 In-vitro Antidiabetic Activity

Diabetes is a multifactorial disease characterized by elevated blood glucose levels, aberrant pancreatic insulin production, or insulin resistance in peripheral tissues [31]. The pancreatic α-amylase and intestinal α-glucosidase enzymes, which break down carbs, are known to have a role in the development of diabetes by elevating postprandial glucose levels. The kidney and liver are negatively impacted by miglitol, acarbose, and voglibose that inhibit the activity of α-amylase and α-glucosidase enzymes. These enzymes, located in brush borders of intestinal absorptive cells, hydrolyses the non-absorbable polysaccharides and oligosaccharides into absorbable monosaccharides (Fig. 6) [32]. Hence, finding robust α-amylase and α-glucosidase inhibitors in diabetes patients that are safe and derived from natural sources is of utmost importance. The effect of Colleto-AgNPs on blood sugar levels was assessed in this study using enzyme inhibition assays of α-amylase and α-glucosidase.

In the current investigation, the standard drug used was acarbose, which partially required a IC50 of 25.07 ± 0.21 µg/mL concentration to inhibit the 50% α-amylase enzyme concentration (Table 5). The study evaluated the enzyme inhibitory property of A. muricata leaf extract, C. siamense (endophyte) fungal extract, and Colleto-AgNPs on comparison with acarbose under various concentrations (50, 100, 150, 200, and 250 µg/mL). Compared to the α-amylase enzyme inhibition activity of A. muricata leaf extract and the fungal extract of C. siamense, the activity of the nanoparticle synthesized (Colleto-AgNPs) using C. siamense was superior with IC50 of 30.57 ± 0.17 µg/mL, while the former exhibited activity with the IC50 of 51.19 ± 0.82 and 47.43 ± 0.71 µg/mL.

Fig. 6
figure 6

Possible antidiabetic mechanism of AgNPs through the breakdown of polysaccahrides, oligosaccharides, and disaccharides that inhibits the activity of α-amylase and α-glucosidase enzymes

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Like the results of α-amylase enzyme inhibitory activity, finer results were demonstrated by Colleto-AgNPs against α-glucosidase enzyme with an IC50 of 32.30 ± 1.23 µg/mL. The individual extracts of A. muricata leaves and C. siamense endophytic fungus extract required IC50 of 49.08 ± 0.09 and 44.19 ± 0.52 µg/mL to cause partial inhibition of α-glucosidase enzyme. The standard drug acarbose showed remarkable activity with IC50 of 24.16 ± 0.44 µg/mL. Ishnava et al. [32] evaluated the α-amylase inhibition activity of AgNPs synthesized using Enicostema axillare leaf extract. This nanoparticle has revealed good inhibition of about 70% with a least 10 µg/mL concentration. The AgNPs synthesized with Hybanthus enneaspermus had less α-amylase and α-glucosidase enzyme inhibitory activity compared to the results of Colleto-AgNPs, IC50 exhibited by AgNPs was 41.27 ± 0.11 and 42.87 ± 0.44 µg/mL, respectively [33].

Table 5 In-vitro antidiabetic and antioxidant activity of A. muricata leaf extract, C. siamense extract, and Colleto-AgNPs
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3.10 In-vitro Antioxidant Activity

The reactive molecules comprising reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the byproducts of a biological system. These reactive molecules or species are also considered signaling molecules [34]. The molecular network interaction established by these ROS and RNS between the signaling pathways of ROS and RNS plays a vital role in regulating stress responses. Human body contain non-enzymatic antioxidants and enzymatic natural antioxidants to balance the reactive molecules. However, an increased population of these free radicals destroys the antioxidant defence system in humans, which eventually results in oxidative stress by damaging the DNA, proteins, and lipids and alters the cell structure [7] (Fig. 7). In adverse conditions, they induce brain injury, cardiovascular and neurodegenerative diseases, and sepsis [35].

Fig. 7
figure 7

Possible antioxidant mechanism of AgNPs by inhibiting the dysfunction of mitochondria (1), reducing the oxidative stress (2), inhibiting DNA fragmentation (3) and stress on endoplasmic reticulum (4), and apoptosis (5) caused by ROS and RNS

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The present study estimated the free radical scavenging activity of Colleto-AgNPs against the DPPH radical. DPPH is a stable molecule soluble in methanol solvent and has a maximum absorption at 517 nm. The Colleto-AgNPs have displayed higher antioxidant activity by scavenging the DPPH radical with an IC50 of 26.98 ± 0.30 µg/mL, and the standardized antioxidant, ascorbic acid has exhibited IC50 of 16.14 ± 0.21 µg/mL (Table 5). The leaf and fungal extract displayed reduced antioxidant activity compared to the Colleto-AgNPs with IC50 of 38.13 ± 0.13 and 31.38 ± 0.09 µg/mL. The biosynthesized AgNPs from Astragalus flavesces leaf extracts exhibited efficient radical scavenging potential to a degree that it is recommended for its usage in food and pharmaceutical industries [36]. Similarly, our results were also higher and close to commercial standard control (IC50 16.14 ± 0.21 µg/mL) with the IC50 26.98 ± 0.30 µg/mL. A study by Geyesa et al. [24] revealed that the AgNPs synthesized using leaf extract of Verbascum sinaiticum required extremely high IC50 of 216 µg/mL to scavenge DPPH radical. With this, it can be determined that the AgNPs synthesized using C. siamense efficiently scavenged the DPPH radical.

4 Conclusion

Green synthesis has paved the way for simple, cost-effective, and non-toxic methods of producing nanoparticles for various uses. Plant extracts and microbial or fungal extracts are superior agents for reducing and stabilising noble metals in their respective ionic forms, allowing the metals to finally form nanoparticles through a stabilised colloidal system. The current work created an effective biosynthesis approach for silver nanoparticles that uses Colletotrichum siamense fungal extract as a reducing and stabilising agent. Antibacterial activity against the tested gram-positive and gram-negative bacterial strains was demonstrated by the biosynthesized Colleto-AgNPs, which exhibited a well-defined zone of inhibition. Biogenic Colleto-AgNPs have also shown promising antidiabetic activity against α-amylase and α-glucosidase enzymes. At a lower dosage, Colleto-AgNPs could scavenge DPPH free radicals. These results shed light on the potential biological uses of Colleto-AgNPs, which is very important. This study is noteworthy because no previous research has shown that C. siamense extract may be used to make nanoparticles of silver or other metal oxides. C. siamense may have future use in medicine since the results show that it is an effective reducing agent for making AgNPs. However, to ascertain the effectiveness of Colleto-AgNPs, a comprehensive investigation of experimental animal models is necessary.