Biochemical Screening, Fabrication of Green Nanoparticles and Its Antimicrobial, and Antioxidant Studies of Endophytic Fungus Phlebia Species

Abstract

Endophytes are organism dwelling totally dynamic and novel biotopes this makes them able to produce novel biochemicals that may become assets to the future. This study aims at understanding the biochemical components of the endophytic fungus Phlebia sp. synthesis of gold and silver nanoparticles from it, and the antimicrobial as well as antioxidant ability of these green synthesised nanoparticles. Aqueous fungal extract was subjected for HRLCMS analysis which revealed 34 biochemicals within the extract. Silver and gold nanoparticles were also produced from the fungal extract. UV–vis analysis revealed a peak at 450 nm for silver nanoparticle and 550 nm for gold nanoparticles. FESEM analysis confirmed the presence of these nanoparticles with its spherical shape. Both of these nanoparticles were able to produce a conspicuous zone of inhibition in the antimicrobial tests against Escherichia coli, Salmonella paratyphi. For both of the organisms under study, a concentration-dependent expansion of the zone of inhibition was discovered in the nanoparticles. However, with silver nanoparticles, a relatively high zone of inhibition and vulnerability of the organism was discovered. Four in vitro free radical scavenging assays, including the DPPH, Hydroxyl, Superoxide, and Nitric oxide radical scavenging assays, were used for antioxidant analysis. The results of every test demonstrated that green synthesised silver nanoparticles had higher activity than gold nanoparticles. All of the tests showed that silver nanoparticles were more active than gold nanoparticles with the maximum value of 86.254 ± 0.296% being discovered at the greatest concentration of superoxide radical scavenging assay.

Introduction

Since the dawn of time, the microbial population has served human needs in various fields. To find solutions to contemporary issues, however, we require innovative biotope-dwelling species because the common organism has been thoroughly investigated. Endophytes appear to be suitable since they are found in many different and highly dynamic biotopes. More than quarter million plants in the planet have the ability to host at least one million endophytes, but most of them still remain still underexplored [1]. If the current approach towards studying endophytes is altered to focus more on the characteristics of these organisms, then phytochemicals from endophytes are likely to become one of the major resources in the near future. The Ficus plant is unique because it is well-known for its phytochemicals [2–5], which have a wide range of therapeutic applications [6]. Endophytes were able to produce unique biochemicals with uncommon structures which have superior therapeutic effects since they are a population that is constantly in contact with phytochemicals in plants [7]. This will reveal the importance of the biochemical analysis of endophytes from various plants.

Due to their controllable structure, size, and unique characteristics like simple scaling up, nanoparticles have a wide range of commercial applications. There are many ways to create nanoparticles, including physical and chemical processes [8]. Physical techniques include grinding, radiolysis, evaporation, condensation, ultrasonication, and chemical synthesis, which is mostly accomplished through chemical reduction [9]. Both of these techniques are quite expensive, and its byproducts are harmful to the environment. This unpleasant synthesis and non ecofriendly nature necessitate an eco-friendly, cost-effective synthesis of these highly valuable nanoparticles. This demands for an economical, environmentally beneficial method of producing green nanoparticles. Green nanoparticles can be made from plants, algae, fungi, bacteria, and other microorganisms, but endophytes play a key role due to their ease of cultivation in lab settings and the phytoconstituents they can provide. The relevance of noble metal-mediated nanoparticles is increased because of their innocuous nature, which makes them better suited for biomedical applications [10]. Due to its abundance of phytoconstituents, the genus Ficus has unmatched tremendous medicinal potential. Various biomedical applications of the plants in this genus have been the subject of numerous research papers Still, there are few reports on endophytes, and their biochemistry, including their capacity to generate nanoparticles [11]. The growing number of immunocompromised people in the population, as well as multidrug resistant pathogenic microbes, necessitates the development of novel and effective antimicrobial agents. This is where nanoparticles come into action. Despite the fact that nanoparticle-mediated antimicrobial agents are widely investigated in the current scenario, endophytes-mediated nanoparticles are relatively rare, with a limited number of research papers [12]. This opens up new possibilities for endophyte-mediated antimicrobial nanoparticles. Endophytic fungi have the ability to produce various biochemicals, which may also act as capping and stabilizing agent for the nanoparticles generated, giving this nanoparticle the ability to quench free radicals and act as antioxidants. There are numerous reports on the effective antioxidant ability of phyto fabricated gold and silver nanoparticles [13]. To the best of our knowledge this work is the first work on the endophytic nanoparticle from the genus Ficus. This work attempts to characterise the biochemicals produced by the endophytic fungus Phlebia sp. of Ficus drupacea, including its potential to reduce gold and silver to nanoparticles and to act as antioxidants and antimicrobial agents.

Materials and Methods

AR-Grade AgNO3, HAuCl₄ Bacterial and fungal growth mediums, all chemicals for antioxidant assays were purchased from Hi media India Ltd. E. coli, S. paratyphi were purchased from MTCC (MTCC 452, MTCC 3220).

Preparation of Fungal Extract

As part of a previous study, endophytic fungus from F. drupacea fruits and leaves were isolated and pure grown in the cell and molecular biology lab of Calicut University's Botany division [14]. ITS region sequencing of fungal DNA was used for the identification. Pure cultured endophytic fungi Phlebia sp. isolated from the fruits were mass cultured in a 250 mL Erlin Mayor flask using modified potato dextrose sucrose broth. After 25 days of growth, the flasks were emptied, and the aqueous portion of the culture was separated by sieving it over filter paper Whatman No 1. This extract is used in nanoparticle production and biological analysis [12].

Biochemical Analysis

The biochemical examination of the extract is important since it could detail various biochemicals that an organism produces. In order to do this, 3 mL of the sieved extract had to be collected in an Eppendorf tube and subjected to an HR LC MS analysis.

Silver Nanoparticle Production

50 mL of sieved fungal extract is treated with 10 mL of 1 mM silver nitrate solution and placed in a preheated water bath (60 °C) for 15 min. Modified method of Tyagi et al. [15] was used in silver nano particle production.

Gold Nano Particle Production

60 mL of sieved fungal extract were taken in a 100 mL Erline Mayr flask and 5 mL of HAuCl₄ solution of 1 mM concentration was added. The solution is kept in a preheated (60 °C) water bath for five minutes. Modified procedure of Patil et al. [16] was used.

Characterization of Nanoparticles

UV–Vis Spectra Analysis

Primary analysis after the colour change indication for the production of nanoparticles were conducted by spectrophotometer analysis. Spectrophotometer (Cary 5000) was used to measure the absorbance over the wavelength range of 300–700 nm.

SEM Analysis

For FE-SEM (Scanning electron microscopic Geminisem 300, with magnification of 12×–2,000,000×, acceleration voltage 0.02–30 kV, and the Inlens BSE resolution 1.2 nm at 1 kV), examination finely powdered and oven-dried nanoparticles were used.

Antimicrobial Ability

LB agar medium was used to make agar plates for E. coli and HE agar media is used for S. paratyphi. Overnight grown bacterias were inoculated on to the specific agar plates. Nanoparticles produced using the fungal extract were powdered and dissolved in sterile distilled water and impregnated in the sterile discs [17]. These sterile discs were used in the agar plates with bacterial culture to test the antimicrobial ability of the nanoparticles.

Antioxidant Activity

DPPH Assay

DPPH assay was performed according to modified procedure of Chang et al. [18]. 1.48 mL DPPH (0.1 mM) solution was added to various concentrations of sample solutions (12.5–200 g/mL). A control with no test samples but an equivalent amount of distilled water was taken. The reaction mixture was incubated in the dark at room temperature for 20 min. The absorbance of the combination was measured at 517 nm. Ascorbic acid was taken as standard.

The values are calculated using the equation

$$\text{Percentage}\text{of}\text{inhibition}=\frac{control-test}{\mathit{control}}\times 100$$

Hydroxyl Radical Scavenging Assay

Hydroxyl radical scavenging assays were performed according to modified procedure of Chang et al. [19]. Various concentration of the sample solutions (125–2000 µg) were mixed with the reaction mixture which contain FeCl3 (100 µm), EDTA (100 μm), H₂O₂ (1.0 mM), ascorbic acid (100 μm) in KH₂PO₄—KOH buffer (20 mM pH 7.4), (2 deoxy 2 ribose (2.8 mM) and made it up to 1 mL. After 1 h of incubation 1 m of 2.8% TCA followed by 1 ml 1% aqueous TBA was added and the mixture was incubated at 90 °C for 15 min. Absorbance was measured at 532 nm after cooling. A control was maintained without adding the test solutions. Gallic acid was taken as standard

$$\text{Percentage}\text{of}\text{inhibition}=\frac{control-test}{\mathit{control}}\times 100$$

Superoxide Scavenging Assay

The assay was performed according to the modified procedure valentao et al. [20]. Different concentration of samples (125–2000 μg/mL) were taken an and added with 0.05 mL of Riboflavin solution (0.12 mM), 0.2 mL of EDTA solution [0.1 M], and 0.1 mL NBT (Nitro-blue tetrazolium) solution [1.5 mM], which were diluted to 2.6 mL using phosphate buffer [0.067 M]. A control was maintained without test solution. Gallic acid was taken as standard. Absorbance was measured in 560 nm after 5 min, followed by 30-min incubation in fluorescent light.

Values were calculated using the equation

$$\text{Percentage}\text{inhibition}=\frac{control-test}{\mathit{control}}\times 100$$

Nitric Oxide Radical Scavenging Assay

The assay was performed according to the modified procedure valentao et al. [20]. Sodium nitro prusside (50 mM) was mixed with different concentration of test sample (125–2000 μg/mL) and incubated. A control without the test compound was maintained. Incubated solution was added with Griess reagent (1% sulphanilamide, 2% phosphoric acid and 0.1% N-1-naphthyl ethylene diaminedihydrochloride). Ascorbic acid was taken as standard. Absorbance at 546 nm was measured. Values were calculated using the equation

$$\text{Percentage}\text{inhibition}=\frac{control-test}{\mathit{control}}\times 100$$

Results

Endophytic fungus Phlebia species isolated from the fruits of F. drupacea were mass cultured. After 20 days of growth the extract was harvested (Fig. 1). This fungal extract was used for the studies including biochemical analysis, gold and silver nanoparticle production and nanoparticle mediated antimicrobial studies in E. coli.

Fig. 1.

Isolated pure cultures endophytic fungus Phlebia sp. from F. drupacea fruits were mass cultured in PDA broth for fungal extract collection

Biochemical Analysis

The study on fungal extract revealed a total of 34 biochemical components through HR LCMS analysis (Fig. 2). These biochemicals were belonging to peptides, glycosides, terpenoids, sugers, carbohydrate etc. The important biochemicals include isoplumbagin, ophiopogonin b, Goyaglycoside, Physapubescin, Rodiasine, Tributyrin etc. Details of the total biochemical constituents in the fungal extracts are given in the Table 1.

Fig. 2.

Chromatogram of HRLCMS analysis done on the endophytic fungus Phlebia sp.

Table 1.

Phytochemicals revealed by HRLCMS analysis of endophytic fungal extract

S. no	Compound name	Structure	Class of Compound	RT
1	5-Methylcytidine		Methylcytidine	1.137
2	Dihydrocaffeic acid 3-O-glucuronide		Glycoside	1.198
3	Phytosulfokine b		Peptide growth factors	1.212
4	Ophiopogonin B		Steroid saponin	1.402
5	Isoplumbagin		Organic compound	1.419
6	4-hydroxy-L-threonine		Hydroxy-amino acid	1.478
7	Cynometrine		Imidazoles	1.608
8	N-Acetyl-D-fucosamine		Amino sugar	1.744
9	Lotaustralin		Cyanogenic glycoside	1.804
10	Lys-Gly		Peptide	1.891
11	Isoleucyl-Proline		Dipeptide	1.937
12	6-Deoxyfagomine		Piperidines	1.981
13	2-(4-Methyl-5-thiazolyl) ethyl octanoate		Thiazoles	2.117
14	N (6)-hydroxy-L-lysine		Amino-acid	2.231
15	Coccinin		Peptide	4.675
16	11-Hydroxyiridodial glucoside pentaacetate		Terpene glycoside	5.024
17	Glutaminyl-Arginine		Peptide	5.535
18	Tributyrin		Ester	5.604
19	Maculosin		Dipeptide	5.674
20	Tryptophyl-Alanine		Peptide	5.859
21	Phenethylamine glucuronide		N-Glycoside	5.886
22	7,8-Dihydrovomifoliol 9-[apiosyl-(1- > 6)-glucosid		Carbohydrate	5.937
23	Rodiasine		Isoquinoline	6.311
24	Adouetine Y		Cyclopeptide alkaloid	6.388
25	Physapubescin		Steroidal lactone	6.474
26	Glutaminyl-Glycine		Peptide	6.623
27	N-Heptanoylhomoserine lactone		N-acyl-amino acid	6.646
28	Hydroxy citrulline		Alpha amino acid	6.684
29	Harzianopyridone		Aromatic ketone	6.64
30	Mycinamicin IV		Mycinamicin	6.906
31	Sorbitan laurate		Ester	8.077
32	Manumycin A		Polyketide	9.811
33	Boviquinone 4		Diterpenoid	19.489
34	Goyaglycoside c		Glycoside	19.965

Silver Nanoparticles Characterization

UV–Vis Analysis

The reaction between fungal extract and silver nitrate resulted in the brick red coloration of the pale-yellow coloured reaction mixture. This was the first indication of silver nanoparticle production, this solution when subjected to UV–vis spectrometry, a peak in the wave length of 450 nm for silver nanoparticles (Fig. 3), 550 nm for gold nano particles were produced (Fig. 4). This in turn confirmed the formation and presence of silver nanoparticle in the solution analysed.

Fig. 3.

UV–vis spectrum of silver and gold nanoparticle produced

Fig. 4.

a SEM images of silver nanoparticles produced from endophytic Phlebia sp., b SEM images of gold nanoparticles produced from endophytic Phlebia sp.

FE SEM

FE SEM analysis revealed the spherical nature of both silver and gold nanoparticles. They were having a diameter of 28 and 30 nm for gold and silver nanoparticle respectively. This analysis once again confirmed the presence and spherical shape of the nanoparticles produced from endophytic Phlebia species isolated from F. drupacea ripened fruits (Fig. 5a, b).

Fig. 5.

a E. coli treated with gold nanoparticles, b E. coli treated with silver nanoparticles, c Negative control, d Positive control, e S. paratyphi treated with gold nanoparticle, f S. paratyphi treated with silver nanoparticle, g, h Negative and positive control for S. paratyphi

Antimicrobial Activities of the Nanoparticles Produced

The antimicrobial properties of silver and gold nanoparticles were investigated at various concentrations, including 100 g/mL, 500 g/mL, and 1 mg/mL. Silver and gold nanoparticles generated by the Phlebia species were discovered to have significant antibacterial efficacy against tested microbes. The investigation demonstrated a concentration dependent increase in the zone of inhibition (Fig. 5a–h). In E. coli, gold nanoparticles produced a smaller zone of inhibition than silver nanoparticles at all three test doses. S. paratyphy appears to be more sensitive to both nanoparticles, forming a larger zone of inhibition even at the lowest test dose for both gold and silver nanoparticles which is detailed in Table 2.

Table 2.

Zone inhibition produced by the organisms according to various concentrations tested

Pathogens	Zone of inhibition (in mm) according to test concentrations
	Gold nanoparticles			Silver nanoparticles
	100 µg/mL	500 µg/mL	1000 µg/mL	100 µg/mL	500 µg/mL	1000 µg/mL
E. coli	0.4 ± 0.111	2 ± 0.559	16 ± 0.5574	15 ± 0.455	20 ± 0.573	26 ± 0.512
S. paratyphi	2 ± 0.0826	8.5 ± 0.148	18.6 ± 0.417	20 ± 0.474	24 ± 0.384	29.9 ± 0.754

Antioxidant Activities

Antioxidant assays revealed the ability of both silver and gold nanoparticle from the endophytic fungus have potential ability in free radical scavenging. Percentage of inhibition was dependent on the concentration with ascending order for both silver and gold nanoparticles. Maximum inhibition percentage of 86.254 ± 0.296 was found in the highest concentration tested (2000 µg/mL) for superoxide radical with the green synthesised silver nanoparticles. IC 50 for the silver nanoparticle was 504.946 µg/mL, which was nearly comparable to that of the standard with a value of 369.015 µg/mL. Gold nanoparticle also shown its highest inhibition pattern with superoxide scavenging activity of 71.365 ± 0.6072% with an IC 50 value of 884.959 µg/mL. The lowest inhibition percentage was found with gold nanoparticles in scavenging nitric oxide radicals, with a value of 18.234 ± 0.576379 in the lowest test concentration of 125 µg/mL with an IC 50 value of 1133.455 µg/mL. Details on the inhibition patter for all the four assays performed are given in Figs. 6 and 7. In the figure AG, AU, STD are given for silver nanoparticle, gold nanoparticle, standard respectively. For both DPPH, Nitric oxide radical scavenging assay ascorbic acid was taken as standard. For superoxide radical scavenging assay and hydroxyl radical scavenging assay gallic acid was taken as the standard.

Fig. 6.

DPPH radical scavenging activity of Phlebia sp.

Fig. 7.

Free radical scavenging activity of Phlebia sp.

Among the four-assays performed, silver nanoparticle showed better quenching ability when compared to that of the gold nanoparticles. IC 50 value of silver nanoparticle showed a percentage inhibition nearly comparable to that of the standard in case of both DPPH and superoxide radical scavenging assay. Details of IC 50 values for all the assays performed are included in Table 3.

Table 3.

IC 50 values of the nanoparticles for various antioxidant assays performed

Assays	IC50 Values
	Gold nanoparticle (µg/mL)	Silver nanoparticle (µg/mL)	Standard (µg/mL)
DPPH assay	137.784	77.664	33.91
Nitric oxide scavenging assay	1133.455	429.676	156.159
Hydroxyl radical scavenging assay	1051.231	410.59	235.614
Super oxide scavenging assay	884.959	504.946	369.015

Discussions

Metabolites produced by each organism will be different and it is the reason by every individual organism remains unique. For better understanding of the chemical constituents produced by endophytic Phlebia sp. HR-LC MS analysis of its aqueous extracts were conducted and 34 chemical compounds were found. Among which some of them were very important and useful chemicals. Dihydrocaffeic acid 3-O-glucuronide is a phenolic compound which is a caffeic acid derivative. Caffeic acids are reported to have antioxidant anticancer properties [21]. Isoplumbagine is a naturally occurring quinone, with potent biochemical from this fungus which was already reported to have anti-inflammatory ant anticancer activities [22]. Ophiopogonin b is another biochemical revealed by HRLCMS screening which is found to be very effective in ROS production and there by inducing cell death in nasopharyngeal adenocarcinoma [23]. Lotaustralin which is a cyanogenic glucoside was previously reported from Lotus japonicus [24]. Maculosin was reported previously from a bacteria with incredible antioxidant activity [25]. Another biochemical Physapubescin is found to be helping in reducing the expression of hypoxia inducible factor (HIF)-2α, and this hypoxia condition in turn increases growth inhibition as well as apotopsis effect of physapubecin, this chemical is also contributing to the inhibition of angiogenesis [26]. Adouetines were the first cyclopeptide alkaloids reported in the plants and they were found to have sedative effect in mice [27]. Rodiasine is another isioqunoline found in the extract which is reported with its antimalarial activity [28]. Tributyrinis another biochemical having reports on its immense ability as an anticancerouse agent without any cytotoxicity effect to normal cell lines and so it is being considered as a pro drug with variety of researches going on it to make its easy availability and to improve its organoleptic characters [29]. Glycoside named goyaglycoside found in the extract of this fungus is already reported to have anticancerous and chemo suppressive effects [17]. Biochemical reports from the plant reveals this genus have a number of biochemicals with potential activities in various biomedical field. Antimicrobial properties of nanoparticles are well known, as they are a component of modern mechanisms for combating multidrug resistant organisms. ROS generation, membrane disintegration, ATP depletion, DNA damage, and other combined actions of nanoparticles aid in the fight against these lethal organisms [30–32]. Superior activity of silver nanoparticle throughout the experiment may be due to the difference in particle size and particle nature. Even though same extract is used for synthesising both nanoparticles, biochemicals responsible for the reduction of each particle might be different [33]. Schematic representation of the mode of action of nanoparticles are given in Fig. 8.

Fig. 8.

Schematic representation of mechanism of action of gold and silver nanoparticle on bacterial cell

Endophyte Lesiodiplodia theobromae mediated silver nanoparticles were effectively inhibiting the growth of Pseudomonas aeruginosa is a similar report to that of the current study [34]. Antimicrobial ability of gold nanoparticles from endophytic fungus Phoma sp. was reported to be very effective antimicrobial agent against phytopathogens Xanthomonas oryzae, Rshizoctonia solani [35].

The formation of nanoparticles can be characterised using a variety of methods, one of which is UV–vis analysis. Because each nanoparticle can produce a distinct peak at a specific wavelength, it is possible to confirm that the colour change in the reaction mixture was caused by the presence of the specific nanoparticles. The UV–visible analysis from the previous report on endophyte mediated silver nano particles produced a conspicuous peak 450 nm, which is same as to the results produced by endophytic Phlebia sp. silver nanoparticles [36]. Purple or violet is the typical colour of gold nanoparticles. The peaks produced in 500–600 nm wavelength range further support this [37]. Gold nanoparticles generated by endophytic Phlebia sp. formed a peak at 550 nm that is nearly identical to earlier studies [38]. FESEM examination revealed the size and shape of both the examined nanoparticles, which further helped to validate nanoparticle production.

Antioxidants are the molecules which can stabilize any reactive molecules such as free radicals especially like reactive oxygen or nitrogen species. Nano particles from gold or silver normally exist in two oxidation states such as Au⁺¹, Au⁺³ form for gold and Ag⁺¹, Ag⁺² for silver, this make them ready to accept or donate electron and there by participate in redox reaction and helps in free radical quenching [33]. Schematic diagram representing the importance of antioxidant within the body is represented in Fig. 9.

Fig. 9.

Schematic diagram representing the importance of antioxidant within the body

In the study both nanoparticles synthesised were shown to have the ability of free radical quenching. When comparing the ability of these two nanoparticles, silver was found to have superior radical quenching ability. Similar results were also reported from previous study of antioxidant assays on the leaf mediated silver and gold nanoparticle of Crassocephalum rubens [39]. In another report on the nanoparticle-based antioxidant activity of Solanum torvum fruit extract, the scientist found silver nanoparticle with better activity in all antioxidant assays other than hydroxyl radical assay. In hydroxyl radical assay the best activity was shown by the gold nanoparticle, other than which all other test reports were in accordance with the present study [33]. Green nanoparticles from Plumbago zeylanica displayed antioxidant activity, with a higher proportion of % inhibition in gold nanoparticles than in silver nanoparticles. This report contradicted with the findings of the present study [40]. All this research findings reveals the potential antioxidant ability of green synthesised silver and gold nanoparticles from various plants and other organisms.

Conclusion

The biochemical composition of the endophytic fungus Phlebia sp., which was isolated from the F. drupacea fruits, revealed 34 biochemicals, the majority of which have been reported from plants and have significant therapeutic potential. When this fungal extract was subjected to gold and silver nanoparticle production, it performed admirably in this environmentally friendly nanoparticle production. Gold and silver nanoparticles from this fungal extract with spherical shape were excellent antimicrobial agents against tested organisms and exhibited excellent antioxidant activity. Among both nanoparticles produce superior activity was revealed by green silver nanoparticles in both antimicrobial as well as antioxidant assay.

Funding

Funding was provided by University Grants Commission of India.

Data Availability

The datasets generated during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose, also there is no conflict of interest.

Ethics Approval and Consent to Participate

Not applicable.

Consent for Publication

Both the authors here by agree that there is no problem with publishing the data and work with in this journal.