INTRODUCTION:

Silver nanoparticles are nano-sized particles of silver with dimensions ranging between 1-100nm.¹ Though, frequently being described as solely ‘silver’, many of them are constituted with a major share of silver oxide owing to their large surface area ratio in comparison to solitary immensity of silver atoms. Various shapes of nanoparticles can be synthesized depending on the application criterion. The most commonly used silver nanoparticles are spherical in form; however, octagonal, diamond and thin sheet like structures are also quite frequent.¹ The extremely large external surface area permits numerous coordinate bonds with a vast number of ligands. The biological mode of silver nanoparticles synthesis has provided a platform for improved techniques in comparison to the conventional methodologies that required the usage of harmful chemical based reducing agents like sodium borohydride. The characteristic properties of silver nanoparticles relevant for human treatment are under experimental investigation in the laboratory including studies based on animal model with an assessment of its potential efficacy, biological safety concerns and biodistribution.^2-5

The Darjeeling Himalayan region is one of the richest biodiversity zones of the world with numerous plants of this area being referred to be ethnomedicinal in the context of traditional knowledge of the local people. However, laboratory based validation and appropriate documentation of these data is scanty. Herpetospermum darjeelingense (C.B.Clarke) H.Schaef. and S.S. Renner is one such representative unexplored endemic cucurbit climber stated to be utilized in veterinary treatment, specifically against bovine pyrexia besides its fruits being used as a source of vegetable.⁶The plant has been reported to be a rich source of antioxidant molecules as well. GC-MS analysis of this climber too depicted a range of bioactive metabolites validating its ethnomedicinal attributes.⁶ Previously no work based on synthesis and study of biogenic silver nano particles employing H. darjeelingense extracts has been conducted towards understanding nano-medicinal implications of this plant.

Therefore, the present research focused to bridge this knowledge gap by conducting experiments involving biogenic extract of H. darjeelingense based silver nano particles (AgNP’s) synthesis along with its characterization towards determining the extent to which antimicrobial, antioxidant and anti-lipid peroxidation efficacy is increased on account of nano particle production with its prospective potential in pharmaceutical implications.

MATERIAL AND METHODS:

Collection of plant material:

Healthy foliage leaves of H. darjeelingense was collected from ‘Happy valley-Hooker road junction’ area (27.051045°N, 88.260782°E) by skillful detachment from the climbing shoot utilizing a razor blade, being properly packed in air tight zipper bags with immediate preservation in ice stuffed insulated boxes for conveyance to the laboratory towards conducting subsequent experiments.

Preparation of sample extracts:

The collected sample leaves were washed in flowing tap water to remove surface dirt followed by rinsing in distilled water. The clean wet foliage segments were placed in petri plates and allowed to air dry with removal of left over water droplets by means of tissue paper. The dried leaves were mechanically grinded in a mortar-pestle using liquid nitrogen to obtain powdery product. An electronic balance was used to measure 3g powdery leaf mass being filled in screw capped graduated tube and high performance liquid chromatography (HPLC) grade methanol was poured cautiously to it. Methanol was used since it is referred to as an universal solvent.⁷ The solvent-powder mixture was permitted to stay for 48 hours at room temperature followed by sieving the resultant sample extract employing an ash-less filter paper. The leftover methanol in the reaction tube was made to completely dry out at gentle temperature with the final remnants being redissolved in methanol to ultimately prepare the sample concentrate.⁶ Final concentration of the sample extract was adjusted accordingly for subsequent in vitro assay as per the requisites of experimental protocols.

Synthesis of H. darjeelingense extract based silver nanoparticles (AgNP’s):

The silver nanoparticles (AgNP’s) were synthesized through reduction of silver nitrate (AgNO₃) utilizing the methanolic extract of H. darjeelingense. Experimental conditions were optimized by usage of 1ml AgNO₃ solution (1mM) being mixed to different concentration volumes (250μl – 1000μl) of sample extract (50mg/ml) with final volume adjusted to 2000μl by adding distilled water. Control solution contained every ingredient other than plant extract based silver nano formulations with final volume being made to be 2000μl through similar addition of distilled water. The reaction mixture was kept under direct sunlight in the mid-day hours from 12.00noon to 1.00PM for a time frame of 1hour. The production of a strong, persistent golden brown colour would indicate the synthesis of AgNP’s. Oakridge tubes containing the golden brown coloured solution was subjected to centrifugation at 10,000RPM set to run for 10 minutes duration with corresponding aggregated pellet being suspended in distilled water at 1mg/ml concentration ratio for characterization and further analysis.^8-11

Characterization of synthesized silver nanoparticles (AgNP’s):

Spectroscopic assessment:

The 1mg/ml strength of synthesized AgNP’s based brownish solution was investigated through spectroscopic technique with the aid of an UV-Visible spectrophotometer (Agilent Technologies-Cary 60) against a blank solution containing all reagents excepting sample extracts. A characteristic peak at around 420nm – 430nm wavelength would affirm the green synthesis of AgNP’s.

Scanning electron microscopy (SEM) analysis:

Scanning electron microscopy (SEM) was conducted to determine the surface morphology of synthesized AgNP’s. Minute quantity of the synthesized AgNP’s was adhered to the grid of a carbon-coated tape and dried out under mercury lamp for a time period of 5 minutes. 3nm Gold coating of the desiccated AgNP’s was performed employing a gold sputtering unit with the prepared sample being visualized using a SEM (JEOLJSM-IT100InTouchScope™ Scanning Electron Microscope, JEOL Solutions for Innovation, Tokyo, Japan).

Assessment of antibacterial property:

A comparative study related to antibacterial activity of methanol based H. darjeelingense extract and synthesized Silver nano formulation was conducted through well diffusion technique.^12-16 Overnight grown broth culture of two gram positive (Bacillus subtilis and Staphylococcus aureus) and two gram negative (Escherichia coli and Klebsiella pneumoniae) bacteria were used for the present assay. Mueller-Hinton (MH) agar media (Himedia Cat No. M173) was utilized for performing susceptibility tests with the prepared media being sterilized in an autoclave followed by successive plating under aseptic conditions within a laminar air flow cabinet. 100μl of respective bacterial strains were separately added to each petri plate containing MH media. Circular wells were dugout in the solidified media with aid of a sterilized steel cork-borer. Thereafter, 100μl of corresponding sample extract and varying concentration of silver nano formulations were serially poured into the dugout wells using sterilized pipette. The plates were incubated at 37ºC for an overnight duration. Reading and measurement of inhibition zone including photographs were taken after completion of 24hours incubation period.¹⁷

In vitro assay of silver nano formulations:

Assessment of antioxidant activity

Determination of free radical scavenging ability in the plant extract and synthesized silver nano formulations through DPPH induced assay technique was conducted.¹⁸300µl of methanolic H. darjeelingense foliar extract and varied concentration of Silver nano formulations were mixed to 2ml of 0.2mM DPPH (2,2-diphenyl-1-picrylhydrazyl) reagent prepared in Methanol including preparation of control solution with 0.3ml Methanol being subsequently added to 2ml of 0.2mM DPPH reagent. The reaction tubes were vigorously vortexed and incubated for 30minutes at room temperature in dark condition. An UV-Visible spectrophotometer (Agilent Technologies-Cary 60) was used to measure the absorbance value of the consequent solution at 517nm wavelength with methanolic solution of Ascorbic acid being taken as standard. The calculation of DPPH scavenging percentage was determined by the formula- DPPH quenching % = (A_Control − A_Sample) x 100/ A_Control; where A_Control signifies absorbance value of control solution and A_Sample indicates absorbance value of sample solution or silver nano formulation respectively. Milligram (mg) ascorbic acid equivalent per gram of leaf sample in respect of prospective DPPH scavenging potential was calculated accordingly in reference to prepared ascorbic acid standard curve (y= 0.876x+6.591 and R²= 0.993).

Anti-lipid peroxidation assay:

Hepatoprotective assay of methanolic H. darjeelingense sample extract and silver nano formulations were executed following standardized protocols with minor modifications.^19,20During the early morning hours goat liver was collected from slaughter houses instantaneously after slaying being ideal to be used as animal tissue derived lipid source in reference to conductance of anti-lipid peroxidation study. The experimental procedure was initiated within one hour of slaughtering towards prevention of anomalies owing to tissue degradation. The hepatic tissue was washed properly with 10g of it being measured accurately followed by homogenization of the liver in 100ml distilled water in the course of preparing goat liver homogenate. Thereafter, 2.8ml 10% goat liver homogenate, 0.1ml 50mM FeSO₄ and 0.1ml sample extract or silver nano formulations in varying concentrations were mixed thoroughly and incubated for 30minutes at 37°C for preparation of reaction mixture. Then in a test tube, 1ml of the reaction mixture was taken and to it 2ml 50% reagent solution containing a blend of 10% Trichloroacetic acid (TCA) and 0.67% Thiobarbituric acid (TBA) was vigilantly added towards stopping the reaction. The resulting solution was boiled in water bath for 60minutes being sequentially followed by centrifugation at 10,000RPM for 5minutes. Finally absorbance reading of the retrieved supernatant was taken at 535nm wavelength employing an UV-Vis spectrophotometer (Agilent Technologies-Cary 60). A blank solution was prepared containing all reagents except sample extract to serve as control besides liver homogenate with vitamin-E based aqueous extract in different concentrations (12.5 to 100mg/ml) for preparation of standard curve. Milligram (mg) per ml of vitamin-E (Tocopherol) equivalent with respect to varying strengths of silver nano formulation/sample extract was calculated apropos of the prepared Tocopherol based standard curve (y = 0.007x + 0.032 and R²= 0.997).

Employed software resources and statistical analysis:

Statistical introspection of the results obtained from antioxidant and anti-lipid peroxidation assay conducted in triplicates were performed using Microsoft Excel 2007.

RESULTS:

UV-Visible spectroscopic study:

The transition in colour of the reaction mixture from colorless to yellow and ultimately to reddish-brown was observed. Further evidence of Silver nanoparticle formation was attained through UV-Visible spectroscopic analysis with the current study exhibiting distinctive SPR bands of synthesized silver nanoparticles at wavelength of around 430nm (Fig. 1).

Fig. 1: UV-Visible spectral analysis of silver nano formulation

Scanning electron microscopic analysis:

The Scanning electron microscopic examination of biogenically manufactured Silver nanoparticle revealed a rather spherical to cubical appearance with irregular topology (Fig. 2). The constituent particle size ranged between 40–80 nm with some occasional nanoparticles being aggregated together.

Fig. 2: Scanning electron micrograph of synthesized biogenic nanoparticles

Comparative anti-microbial assay of sample extract vs. synthesized silver nanoparticles:

The methanolic sample concentrate failed to portray any zone of inhibition against all the bacterial entities, however Silver nanoparticles showed considerable inhibition zones against Gram negative bacteria (E. coli and K. pneumoniae) and one Gram positive bacteria (S. aureus). No anti-microbial activity was depicted against the other Gram positive bacteria (B. subtilis) by the silver nano formulation. Therefore, the anti-microbial effectiveness of silver nano formulations were seen to exhibit manifold increase in comparison to sample extracts.

For S. aureus, 12.5 and 25 mg/ml extract concentration containing Silver nano formulation failed to produce any inhibition of the microorganism; however, 37.5 and 50 mg/ml nanoparticle extract exhibited strong anti-microbial efficacy. E. coli too was susceptible against biogenic Silver nano particle extract of H. darjeelingense for all concentration, excepting 12.5 mg/ml concentration of Silver nano formulation. Best anti-microbial potential of green mode synthesized Silver nanoparticle extract was shown against K. pneumoniae initiating from 12.5 to 50 mg/ml Silver nano formulation (Fig. 3 and Table 1). In all cases the anti-microbial activity was directly proportional to biogenic extract concentration in the Silver nano formulation with every recorded zone of inhibition values being significantly higher than the corresponding control value.

Table 1: Antimicrobial activity of Silver nanoparticle formulation (Reading in mm) [Disc diameter = 5mm]

Name of Bacteria	Control	Extract concentration (mg/ml)
Name of Bacteria	Control	12.5	25	37.5	50
Bacillus subtilis	3	-	-	-	-
Staphylococcus aureus	4	-	-	7	9
Escherichia coli	4	-	6	7	8
Klebsiella pneumoniae	5	7	8	9	10

Anti-oxidant potential of silver nano formulation vs. sample extract:

The highest free radical scavenging action was shown by 50mg/ml silver nano formulation with minimal being exhibited by 12.5mg/ml nanoparticle concentrate. A sharp rise in anti-radical activity was observed from 12.5 mg/ml (50.119%) to 25mg/ml (83.591%) strength based nano formulation with a slow increase towards higher concentration range (Fig. 4).

50mg/ml methanolic sample extract exhibited 36.15% free radical scavenging activity corresponding to 33.743±0.01289µg/ml ascorbic acid equivalent, while 50 mg/ml extract concentrate based nano formulation showed 91.995% anti-radical activity analogous to 97.493±0.01521µg/ml ascorbic acid equivalent (Fig. 5).

Fig. 4: DPPH scavenging activity of Silver nano formulation

Fig. 5: Comparative DPPH scavenging potential of sample (control) extract vs. silver nano formulation (means with different superscripts in figure denotes significant difference with P<0.05)

Hepatoprotective activity of silver nano concentrates vs. sample extract:

A gradual increase in hepatoprotective ability from 12.5 mg/ml to 50mg/ml nanoparticle formulation was noted (Fig. 6). Highest amount of anti-lipid peroxidation activity was recorded in 50mg/ml nanoparticle concentrate corresponding to 18.128±0.03152mg/ml Tocopherol equivalent; which in comparison to plain 50 mg/ml methanolic sample concentrate corresponding to 11.7±0.06428mg/ml Tocopherol equivalent exhibits significantly higher activity (Fig. 7).

Fig. 6: Anti-lipid peroxidation activity of Silver nano formulation

Fig. 7: Comparative hepatoprotective activity of sample (control) extract vs. silver nano formulation (means with different superscripts in figure denotes significant difference with P<0.05)

DISCUSSION:

The colour transition of the reaction mixture provided visual confirmation of Silver (Ag⁺) ion reduction into nanoparticle through the action of methanolic extract of H. darjeelingense.²¹ It has been previously reported that occurrence of surface plasmon resonance (SPR) spectra in the range of 400-500nm wavelength validates the generation of nanoparticle¹⁷with observance of distinctive SPR bands corresponding to this wavelength further affirming the successful biogenic synthesis of nanoparticles.

Some nanoparticles were aggregated owing to cross-linking or evaporation of solvent during the procedure of green synthesis as portrayed via SEM visualization.¹⁷The inactivity of the Silver nano formulations in inhibiting the growth of B. subtilis may be due to occurrence of cellular level innate resistance factors. Gram-positive bacteria (S. aureus) was less susceptible against lower concentration of Silver nano formulation owing to the presence of a thick layer of peptidoglycan (PG) predominantly made up of short peptide mediated cross-linked linear polysaccharide chain, that makes the cell wall a highly rigid structure impeding Silver nanoparticle penetration through the cell wall organization.²²At higher concentration of Silver nano formulation, the binding ability of the metal onto the surface of Gram-positive bacteria increases and once penetration process gets completed, the biogenic Silver nanoparticles results in killing of the bacterial cells through interaction with Phosphorous and Sulfur containing biomolecules like DNA and protein.²³In case of Gram-negative bacteria, peptidoglycan (PG) layer forms a minor cell wall constituent which allows Silver nanoparticles to easily release Silver ions (Ag⁺) leading to cell membrane damage and bactericidal activity.^24,25This may be correlated with the result obtained in respect of anti-microbial activity against E. coli and K. pneumoniae. GC-MS analysis of methanolic H. darjeelingense extract⁶depicted a range of metabolites possessing antimicrobial properties. Inspite of being a fantastic source of these molecules, it failed to exhibit promising antibacterial attributes. Some compound in its overall chemical architecture might be interfering with its antimicrobial attributes, which also might lessen its immunological machinery against concerned pathogens that could eventually harm the plant. However, silver nano extracts of H. darjeelingense showing prospective antimicrobial activity indicates vistas of its utilization in anti-bacterial formulations owing to anticipated enhancement of metabolite based antimicrobial potential.

The silver nano formulations exhibited concentration dependent activity towards neutralizing DPPH induced free radicals. More the strength of extract in the silver nanoparticle concentrate, greater was the anti-radical quenching phenomenon. The slow increase in anti-radical activity towards higher strength of silver nano formulation (post 25mg/ml concentration) depicts attainment of saturation point in scavenging oxidative moieties. The result also clearly illustrates the increase in DPPH induced free radical quenching activity of silver nano formulations in comparison to normal plant extract. The experimental outcome thus clearly implies biogenic silver nano extracts of H. darjeelingense to possess immense potential as an anti-oxidative agent.

Considerable hepatoprotective potential was exhibited by Silver nano formulations with the nanoparticle concentrates showing anti-lipid peroxidation activity in a concentration dependent manner portraying directly proportional trend. The results evidently indicate the hepatoprotective potential of silver nano formulations of H. darjeelingense with future prospects of its usage as an anti-lipid peroxidation agent in traditional herbal medication.

CONCLUSION:

The usage of plants, microbes and fungi in biosynthesis of silver nanoparticles is paving the path for a more eco-friendly approach towards nanoparticle mediated drug targets for achieving global benefits with H. darjeelingense based silver nano formulations exhibiting promising antimicrobial, antioxidative and anti-lipid peroxidation properties. Further insights on other analogous diseases and disorders employing silver nano particles of H. darjeelingense can help to unravel the actual potential of this formulation.

ABBREVIATIONS:

AgNP’s: Silver nano particles

HPLC: High performance liquid chromatography

SEM: Scanning electron microscopy

MH: Mueller-Hinton

DPPH: 2,2-diphenyl-1-picrylhydrazyl

TBA: Thiobarbituric acid

TCA: Trichloroacetic acid

SPR: Surface plasmon resonance

PG: Peptidoglycan

ACKNOWLEDGEMENT:

Not applicable

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Received on 14.03.2023 Modified on 24.05.2023

Res. J. Pharmacognosy and Phytochem. 2023;15(4):281-287.

DOI: 10.52711/0975-4385.2023.00044