SYNTHESIS OF GOLD NANOPARTICLES USING LEAF AQUEOUS EXTRACT OF PHYLLANTHUS VIRGATUS (PHYLLANTHACEAE), ANTIMICROBIAL AND CYTOTOXIC ACTIVITY AGAINST A549 CELL LINE

SYNTHESIS OF GOLD NANOPARTICLES USING LEAF AQUEOUS EXTRACT OF PHYLLANTHUS VIRGATUS (PHYLLANTHACEAE), ANTIMICROBIAL AND CYTOTOXIC ACTIVITY AGAINST A549 CELL LINE

S. Akkulanna *, C. Nagendra, A. Srinath and A. Madhusudhana Reddy

Phytomedicine Division, Department of Botany, Sri Krishnadevaraya University, Anantapuramu, Andhra Pradesh, India.

ABSTRACT: The local tribes have used Phyllanthus virgatus for Malnutrition, Contagious infections, Inflammations (Gond Tribe), Hepatitis, Cold, Stomach-ache, Gonorrhoea, Fever, Headache, and septic ailments (Chenchus and Yanadis), etc. The gold nanoparticles have a highly specific surface area, and their unique physiochemical characteristics include catalytic activities, optimal properties, antimicrobial activity, etc. Aqueous leaf extract of Phyllanthus virgatus is used to synthesize gold Nanoparticles. The confirmation of biosynthesis of gold nanoparticles was characterized using UV Spectrophotometer, with the maximum scale observed at 551nm. SEM and EDX characterized the morphology of Gold Nanoparticles used to detect the presence of the element gold. The synthesized Nanoparticles were observed to be roughly spherical with a range of 2-50 µm. The dynamic light technique measured the hydrodynamic diameter (28.7nm) and a Zeta potential (-18.6mV). The FTIR analogue shows that the functional group assigned as=C-H,   C-C, O-H, –C=C- and –C=O capping agents. The synthesized nanoparticles have shown strong inhibitory action against pathogenic microbial Strains using the Disc diffusion method. Bacillus cereus exhibited more significant results than other microorganisms. The cytotoxicity of gold nanoparticles was studied using an MTT assay against a human lung cancer cell line (A549) at different concentrations at 24 hrs. Anticancer activity of crude aqueous leaf extract and green synthesized nanoparticle extracts exhibited different responses against the A549 cell line. P. virgatus aqueous leaf extract and the green synthesized nanoparticles exhibited cytotoxic potential properties with the IC₅₀ concentrations at 497.55 (µG/mL) and 189.97(µG/mL), respectively. The results revealed that gold nanoparticles could be an efficient therapeutic agent for lung cancer treatment.

Keywords: Gold Nanoparticles, Cell line, Phyllanthus virgatus, cytotoxicity

INTRODUCTION: The Genus Phyllanthus is well known to possess medicinally active compounds and has been used as traditional antitumor remedies throughout the world ¹ Phyllanthus species are used in Ayurvedic medicine to treat liver and kidney diseases.

Although some herbaceous Phyllanthus species resemble similar ethnomedical uses, Pharmacology activities, and chemical composition are quite different.

The secondary metabolites like alkaloids, coumarins, lignans, and terpenes contribute to antioxidant, anticancer, hepatoprotective, antidiuretic, and anti-inflammatory antiviral activities ². P. virgatus is an antiseptic and anti-inflammatory agent used by the Gond tribe, inhabited in the Eastern Ghats of India ³. P. virgatus extracts have diversified phenol compounds and exhibits high antioxidant and anti-inflammatory properties ⁴. Phyllanthus simplex Retz. synonym for Phyllanthus virgatus G.Forst.⁵ Synthesized ZnoNs with plant extract of Phyllanthus emblica exhibits potential antimicrobial activity ⁶. Synthesized and characterized Ag-Cu-Co nanoparticles with the Phyllanthus niruri extract stabilizes and capping agent for larvicidal, anti-bacterial, antioxidant and photodegradation activities⁷. Fruit extract of AgNPs of P. emblica acts as a biogenic reducing agent antioxidant and antimicrobial activity ⁸. Leaf extract with AgNPs of P. emblica shows antioxidant, anti-leishmania, and anti-inflammatory activity ⁹.

Among the metal nanoparticles, Au, Ag and Fe NPs have been widely used in medical applications. AuNPs are used in drug delivery, bioimaging and photothermal therapy ¹⁰. AgNps and AuNps are synthesized from different plant parts of families like Phyllanthaceae, Lamiaceae, Rutaceae, and Euphobiaceae, which are wide range of therapeutic uses¹¹. AuNPs have garnered the most attention among the numerous metallic nanoparticles because of the exceptional surface plasma resonance, coupled with a variety of molecules, include proteins, enzymes, antibodies etc.¹². AuNPs can also be synthesized using a variety of microbes and plants ¹³. Gold Nanoparticles has been playing vital role in biomedicine for convenient surfaces with biomolecular probes and remarkable plasma resonant optical properties ^{14, 15}. Gold Nanoparticles have a significant function in the delivery of proteins, nucleic acids, in-vivo delivery, gene therapy, targeting, etc, ¹⁶. Neither size nor shape of the AuNPs was determined to induce cytotoxicity in the human lung cancer cell line A549. Intracellular modification of Nanoparticles is determined by the cellular environment ¹⁷. Phyllanthus species were used to synthesize AgNPs, and AuNPs showed antimicrobial and antioxidant activities. The predominant secondary metabolites like stigmasterol, bergenin, chrysin, ellagitanins, rutin, galanin-8-sulfate, kaempferol-8-sulfonate, (structures shown below) etc., recorded in Phyllanthus virgatus ^{18, 19, 20. 21}.

Phyllanthus sps. found to contain alkaloid securinine and euginole (terpenoids) as a principal role in the bioreduction of AgNo₃ to HAuCl₄. The hydrometholic extract of P. virgatus exerted greater antioxidant and cytotoxic effects on human hepatoma HepG2 cells than P. amarus extract ²². Green synthesis of gold nanoparticles using diverse plant extracts has been reported ^{23, 24, 25} P. virgatus showed a stronger cytotoxic effect than P. amarus ²⁶. The anisotropic gold and spherical silver nanoparticles were synthesized by reducing aqueous choloroauric acid and silver nitrate solution with the extract of Phyllanthin at room temperature. The reduction rate of HAuCl₄ is greater than the AgNo₃ at the same amount of phyllanthin extract ²⁷. The present study focused on green synthesis of gold Nanoparticles using P. virgatus leaf extract and its antimicrobial activity. The human lung cancer cell line (A549) was assessed with different concentrations.

MATERIAL METHODS:

Collection of Plant Material: The plant was collected from the fields of Kalasamudram, Anantapuramu district, Andhra Pradesh, India. The Phyllanthus virgatus was identified, and a voucher specimen (No.50202) was housed at Sri Krishnadevaraya University herbarium SKU. P. virgatus leaves were washed with tap water, followed by distilled water to remove dust and other contaminants, and then they were allowed to shade dry at room temperature for a day.

Preparation of Leaf Extract: 10 g of dried leaves were weighed, and 100 ml of double distilled water was added and boiled for 30 min at 60˚C. After cooling, the extract was filtered using Whatman no.1 filter paper and stored at 4°C for further use.

Preparation of 1mM HAucl4 Solution: An accurate concentration of 1mM of Gold Nanoparticles prepared by dissolving 0.03639 g HAucl₄ in 100 ml of double distilled water and stored in amber colour bottle to prevent auto-oxidation of Gold particles

Green Synthesis of Phyllanthus virgatus Leaf Nanoparticles: 2ml of P. virgatus leaf extract was added to 200 ml of Aucl4 (1mM) Solution and exposed to the sunlight for half an hour. Leaf extract acted as reducing agent and the stabilised solution leading to the formation of nanoparticles.

Analysis of Bioreduced Gold Nanoparticles:

UV-Vis Spectroscopy: Synthesis of Gold nanoparticles was confirmed through a UV-Visible Spectrophotometer. The absorption was scanned at 470-700nm.

SEM analysis of Gold Nanoparticles: SEM images investigated the Morphology and size of the Gold Nanoparticles. Thin films of the sample were prepared on a carbon-coated copper grid by just dropping a very small amount of the sample on the grid.

EDAX: The presence of elemental gold was determined, and the samples were dried at room temperature. Then, the sample composition of the synthesized NPs was analyzed.

Particle size: Particle Size analysis of NPs were carried out on HORIBA SZ-100 Laser light Scattering analyzer with the following measurement parameters: Refractive index fluid – 1.330, Angle – 15.00, average count rate- 5.2 kcps with run completed 3 times.

Zeta Potential: The Zeta potential of gold NPs was carried out on HORIBA SZ-100 Laser light scattering analyzer with the following measurement parameters, Conductivity -0.279 ms/cm, Dispersive medium viscosity-0.892mPa.s, electrode voltage-3.3 Vat 25.2˚C measurement was completed thrice. The magnitude of the zeta potential is predictive of the colloidal stability. Nanoparticles with Zeta Potential values greater than +25 mV or less than -25 mV typically have high degrees of stability. In the case of a combined electrostatic and steric stabilization, a minimum zeta potential of ± 20 mV is desirable ²⁸.

Fourier Transform Infrared (FTIR) Studies: Fourier transform infrared spectroscopy (FTIR) measurements were performed using Bomem MB-3000 (make: Canada) spectrophotometer equipped with KBr disc method. Each sample was finely grounded with KBr to prepare the pellets under a hydraulic pressure of 400 kg and spectra were scanned between 4000to 400 cm^-1.

Antimicrobial Activity: In-vitro activity of synthesized Nanoparticles was determined using disc diffusion method ²⁹. The test microorganisms were obtained from the Microbial type culture collection center, Institute of Microbial Technology (IMTECH), Chandigarh, India. Bacterial and Fungal strains. Approximately 20 ml of molten and cooled media was poured in sterilized Petridishes. The plates were left over of 30 min. 30-50 µl of the stock solutions were applied to each sterilized filter paper discs of 5mm. Discs were dried and preserved for antimicrobial study. The plates containing the test organism and AuNPs were incubated at 37ºC for 24-30 hrs. The plates were examined to measure the zone of inhibition at different concentrations (20µl, 30µl and 40µl).

Anticancer Studies: Nanoparticles and crude form of drug obtained from leaf extract were added separately into 200 µl cell suspension (24 hours incubated cells) containing 96 well plates at required cell density (20,000 cells per well),  by different concentrations. The culture plates were incubated for 24 hrs at 37°C in a 5% CO₂ atmosphere. After incubation period, spent media was removed, and MTT reagent with a final concentration of 0.5mg/ml of total volume was wrapped on the plate with aluminum foil to avoid exposure to light. The plates were incubated for 3 hours, and then MTT reagent was replaced by 100 µl solubilizing solution (DSMO) for dissolution purposes in a gyratory shaker.

MTT to insoluble Formosan crystals, which upon dissolution into an appropriate solvent exhibits purple colour, the intensity of color proportional to the number of viable cells and measured with spectrophotometrically or an ELISA reader at 570nm and 630nm wavelength. The IC₅₀ value was determined using a linear regression equation i.e., Y =Mx+C Here, Y = 50, M and C values were derived from the viability graph.

RESULTS AND DISCUSSION:

UV-Vis Spectroscopy: Synthesized Gold nanoparticles were confirmed through a UV-visible spectrophotometer. The absorption was recorded at 551nm. Gold loses its yellow color when dispersed in the form of gold nanoparticles in solution and adopts a ruby-red color. The intensity of the peak steadily increases with the density of the Nanoparticles with respect to time. Fig. 1 & Fig. 2.

FIG. 1: (A) PHYLLANTHUS VIRGATUS LEAF EXTRACT (B) 1MM AQUEOUS HAUCL₄ AND (C) AUNPS FIGURES                  

FIG. 2: UV-SPECTRUM

  SEM and EDAX: The present experimental investigation reports the green synthesis of nanoparticles using P. virgatus leaf aqueous extract. The formation of ruby-red colour conforms to the reduction of Auto AuNps. The synthesized Nanoparticles were observed to be roughly spherical with a range of 2-50µm.

FIG. 3: SEM

Fig. 3 The surface peaks were observed for carbon, oxygen and chlorine, suggesting the presence of biomolecules at the surface of AuNps or its proximity. The significant peak at 2 Kev is characteristic for the element Gold Fig. 4.

FIG. 4: EDAX SPECTRA OF NANOPARTICLES SYNTHESISED FROM PHYLLANTHUS VIRGATUS

Particle Size and Zeta Potential Analyser: Laser diffraction particle size analyzer provides the details about the particle's nature, such as monodispersed, dispersed, and polydispersed.

The results revealed that nanoparticles show monodisperse at 0.146 indexing and various sizes of Nanoparticles ranging with an effective diameter around 28.7 nm, and the Average zeta potential was -18.6Mv Fig. 5 & Fig. 6.

  FIG. 5: ZETA POTENTIAL                                        

FIG. 6: PARTICLE SIZE

Fourier Transforms Infrared Spectroscopy: The FTIR spectrum of gold NPs, as shown in Fig. 3, reveals that the gold NPs show a broad peak at 3421 cm-1 is due to the presence of O-H stretching frequency, the peak at 1595 cm-1 indicates the presence of C=O stretching frequency, the peak 1387 cm-1 indicates the presence of C=C stretching frequency and the peak 1127 cm-1 corresponds  C-O stretching frequency, the peaks at 1068 cm-1 indicates C-C stretching, the peak at  cm-1indicates the presence of =C-H bending frequency. The stretching frequencies indicate that amino acid residues and protein peptides can effectively bind to the surface of metals, thereby functioning as a coating agent on the surface of the nanoparticles (preventing agglomeration) and serving as a reducing agent. The results suggest that the biological molecules can synthesize AuNPs in an aqueous medium Fig. 7.

FIG. 7: FTIR SPECTRA OF NANOPARTICLES SYNTHESISED FROM PHYLLANTHUS VIRGATUS

Antimicrobial Activity Study: In-vitro activity of gold nanoparticles was tested against Staphylococcus aureus (7443), Salmonella enteric (98), Pseudomonas aeruginosa (7296), Bacillus subtilis (1133) Klebsiella pneumonie (7028), Candida albicans (854), Bacillus cereus (1272), Micrococcus liteus (2470), Salmonella typhe (3224), Fusarium oxysporum and  Escherichia coli (1668) by disc diffusion method and the results were tabulated. The gold nanoparticles exhibit significant antimicrobial activity against all tested microorganisms. The maximum zone of inhibition was found against Bacillus cereus, moderate in Salmonella enterica, and no effect on fungal strains (Fusarium oxysporum and Candida albicans). Comparing with P. virgatus aqueous extract, synthesized Gold Nanoparticles have shown better results against test microorganisms. Zone of Inhibition was increased with increasing the concentration of AuNps but in Salmonella enterica at 20 and 30 µg/ml concentration. Only Bacillus subtilis didn’t show any results on Plant extract, but P. virgatus Nps showed significant results in increasing the concentration of NPs Fig. 8 & Table 1.

FIG. 8: ZONES OF INHIBITION OF (A) HAUCL4 (B) PHYLLANTHUS VIRGATUS AQUEOUS EXTRACT  (C) 20 µL  OF AUNPS (D) 30 µL AUNPS (E) 40 µL AUNPS

TABLE 1: ZONES OF INHIBITION OF (A) HAUCL4   (B) PHYLLANTHUS VIRGATUS AQUEOUS EXTRACT  (C) 20 µL  OF AUNPS D) 30 µL AUNPSE) 40 µLAUNPS

Percentage of Cell Viability: Different concentrations of aqueous leaf extracts of P. virgatus were subjected to determine the percentage of cell viability study using A549 at 48 hrs. All were found to be concentration dependent, while a decreased in cell viability was observed in the treatment of aqueous leaf extract of P. virgatus Fig. 9 Table 2 & Graph 1.

FIG. 9: PHOTOGRAPHS OF A549 CELLS AT 48 HRS (A) STANDARD (B) CONTROL (C) 25µG/ML (D) 50µG/ML (E) 100 µG/ML (F) 200µG/ML AND (G) 400µG/ML

TABLE 2 & GRAPH 1: ABSORBANCE READINGS AT 570NM IN ELISA PLATE READER OF THE  P. VIRGATUS LEAF EXTRACT AGAINST THE A549 CELL LINE

 Cytotoxicity Study of the P. virgatus aqueous Leaf Extracts and its Nanoparticles against A549 Cell Line: The observations were statistically analyzed, which revealed that data of cell cytotoxicity study by ELISA reader against A549 cells, P. virgatus aqueous leaf extract and its Nanoparticles showing cytotoxic potential properties with the IC₅₀ Concentrations at 497.55 (µG/mL) and 189.97 (µG/mL) compared to the Standard Drug, Cisplatin with IC₅₀ concentration at 15µM used for the study. The results strongly reveal that the Nanoparticles synthesized with the leaves of P. virgatus have possible therapeutic potential against human lung cancer cell lines (A549) based on the dosage of the drug after the incubation period of 24 hours Fig. 10 Table 3 & Graph 2.

TABLE 3 & GRAPH 2: ABSORBANCE READINGS AT 570NM IN ELISA PLATE READER OF THE PV NPS AGAINST THE A549 CELL LINE

FIG. 10: CYTOTOXICITY STUDY OF THE P. VIRGATUS AQUEOUS LEAF EXTRACTS AND ITS NANOPARTICLES AGAINST A549 CELL   LINE

CONCLUSION: Phyllanthus virgatus leaf extract was found suitable for green synthesis of AuNPs. The reduction of gold ions by the leaf extract forms stable Nanoparticles with spherical and ranging from 2-50µm. The concentrations of leaf extract and metal ions play an important role in the green synthesis of AuNPs. The spectroscopic characterization using UV-Vis, SEM, EDX and Particle size analyzer was useful in proving their minute characteristics like size, shape and, composition, etc. FTIR evidenced the formation and stability of the bio-synthesized AuNPs which can be studied further to understand the chemical and molecular interactions which could be responsible for Nanoparticles synthesis. The presence of Phenol compounds acts as a reducing and capping agent for the preparation of NPs. AuNps cannot affect fungal strains, but they promise inhibition against test bacterial strains. Prepared nanoparticles could be used to make medicinal devices and to treat human lung cancer.

ACKNOWLEDGEMENT: The authors are grateful to the Department of Biotechnology, New Delhi, for financial assistance and thankful to the Andhra Pradesh Forestry for permission and help during field exploration trips.

CONFLICTS OF INTEREST: The authors declare that there is no conflict of Interest

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    How to cite this article:

    Akkulanna S, Nagendra C, Srinath A and Reddy AM: Synthesis of gold nanoparticles using leaf aqueous extract of Phyllanthus virgatus (Phyllanthaceae), antimicrobial and cytotoxic activity against a549 cell line. Int J Pharm Sci & Res 2024; 15(10): 3026-34. doi: 10.13040/IJPSR.0975-8232.15(10).3026-34.

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