Objective: To study the antibacterial activity of Tabernaemontana divaricata (Apocynaceae) secondary metabolites capped silver nanoparticles(SNPs) and gold nanoparticles (AuNPs).Methods: In the present investigation, SNPs and AuNPs were synthesized using an aqueous extract of T. divaricata leaves. Leaf aqueous extractwas mixed with 1 mM silver nitrate and chloroauric acid for the biosynthesis of nanoparticles, and the same was analyzed using ultraviolet-visible(UV-Vis) spectrophotometer and particle size analyzer. The antibacterial activity of SNPs and AuNPs was determined against various bacterial culturesincluding laboratory isolates using the agar well diffusion method.Results: The results recorded from UV-Vis spectrum supported the biosynthesis and characterization of SNPs and AuNPs. The SNPs when comparedto AuNPs, showed the highest antibacterial activity against Gram-positive and Gram-negative bacteria.Conclusion: The present study envisions on the biosynthesis of SNPs from T. divaricata plant which are emerging as antibacterial therapy in modernmedical applications.Keywords: Antibacterial activity, Gold nanoparticles, Silver nanoparticles, Tabernaemontana divaricata

A, KHUSRO

M, Purushothaman

Raj Jp, Preetam

Sg, Panicker

English

Innovare Academic Sciences: E-Journals

Vol 9, Issue 1, 2016 ISSN - 0974-2441ANTIBACTERIAL ACTIVITY OF TABERNAEMONTANA DIVARICATA (APOCYNACEAE) SECONDARY METABOLITES CAPPED SILVER AND GOLD NANOPARTICLESPURUSHOTHAMAN M1, PREETAM RAJ JP1, KHUSRO A1, PANICKER SG1,2*1Department of Plant Biology and Biotechnology, PG Biotechnology, Loyola College, Chennai - 600 034, Tamil Nadu, India. 2Helen Keller Research Centre, Loyola College, Chennai - 600034, Tamil Nadu, India. Email: sgpi1061@yahoo.comReceived: 04 August 2015, Revised and Accepted: 29 August 2015ABSTRACTObjective: To study the antibacterial activity of Tabernaemontana divaricata (Apocynaceae) secondary metabolites capped silver nanoparticles (SNPs) and gold nanoparticles (AuNPs).Methods: In the present investigation, SNPs and AuNPs were synthesized using an aqueous extract of T. divaricata leaves. Leaf aqueous extract was mixed with 1 mM silver nitrate and chloroauric acid for the biosynthesis of nanoparticles, and the same was analyzed using ultraviolet-visible (UV-Vis) spectrophotometer and particle size analyzer. The antibacterial activity of SNPs and AuNPs was determined against various bacterial cultures including laboratory isolates using the agar well diffusion method.Results: The results recorded from UV-Vis spectrum supported the biosynthesis and characterization of SNPs and AuNPs. The SNPs when compared to AuNPs, showed the highest antibacterial activity against Gram-positive and Gram-negative bacteria.Conclusion: The present study envisions on the biosynthesis of SNPs from T. divaricata plant which are emerging as antibacterial therapy in modern medical applications.Keywords: Antibacterial activity, Gold nanoparticles, Silver nanoparticles, Tabernaemontana divaricata.INTRODUCTIONMedicinal plants have numerous applications in the field of medical sciences because most of the prescribed drugs are obtained from herbs, trees, and shrubs. Plants are well-known as a potential source of modern medicine [1]. Tabernaemontana divaricata, commonly known as pinwheel flower belongs to the family Apocynaceae, is a beautifully shaped evergreen shrub which blooms in spring but flowers may appear sporadically throughout the year and distributed throughout Bangladesh and other parts of the South East Asia. The phytochemistry, alkaloids and non-alkaloid constituents such as terpenoids, steroids, flavonoids, phenyl propanoids, phenolic acids and enzymes from the leaves, stems, and roots have been reported. In folklore practice, it is used to treat fever and diarrhea. The plant is also used as a tonic to the brain, liver, and spleen. It is reported that plant extract possesses antinociceptive, antioxidant, anti-inflammatory, and reversible acetylcholinesterase inhibition activities [2-4]. The flower juice can be mixed with oil and used as eye drops. The milky juice of the leaves along with oil is applied over the forehead for pain in the eyes. The roots are ground along with water and given internally for intestinal worms. Nanobiotechnology is an emerging field that applies the nanoscale principle and techniques to understand and transform bio systems (living and non-living). Nanoparticles have expressed significant advances owing to a wide range of applications in the field of biomedical, sensors, and antimicrobials. Synthesis of nanoparticles from plant extract is advantageous because they are of low cost, fast, efficient and generally lead to the formation of crystalline nanoparticles with a variety of shapes and sizes. In view of the medicinal applications of this plant, the present study was designed to investigate the antimicrobial activity of silver nanoparticles (SNPs) and gold nanoparticles (AuNPs) synthesized from T. divaricata to examine the pharmacological basis of the use of the plant in folk medicine for the treatment of infectious diseases.METHODSPreparation of the extractFlowers of T. divaricata were washed thoroughly with autoclaved distilled water and dried in shade for a week and ground using a mixer to coarse powder. The powder was used for preparing the aqueous extract. 1 g of leaf powder was boiled in 10 ml of deionized water for 10 minutes. It was cooled and filtered through Whatman No. 1 filter paper, and the filtrate was stored at 4°C until further use.Synthesis of SNPsSilver nitrate (AgNO3) of analytical grade (AR) was purchased from Merck (India). To synthesize SNPs, 1 ml of the aqueous extract of T. divaricata flower was added to 100 ml of 1 mM AgNO3 solution in 150 ml glass beaker. Then the beaker was incubated for 24 hrs at room temperature on a magnetic stirrer in the dark place for the reduction of SNPs. The color change from light yellow to dark orange indicates the formation of SNPs. An initial setup was also maintained as flower extract without the addition of AgNO3.Synthesis of AuNPChloroauric acid was purchased from Sigma–Aldrich Chemicals. To synthesize AuNPs, 1 ml of the aqueous extract of T. divaricata flower was added to 100 ml of 1 mM gold chloride solution in 150 ml glass beaker. This mixture was kept for 24 hrs at room temperature on a magnetic stirrer at 100 rpm. The color change from light yellow to purple indicates the formation of AuNPs. An initial setup was also maintained as flower extract without the addition of gold chloride.Particle size analysis and zeta potential determinationThe particle sizes were determined using particle size analyzer (Malvern Zetasizer nanosizer). Particle sizes were calculated based on measuring the time dependent fluctuation of scattering of laser light by the nanoparticles.Test microorganisms of interestBacillus subtilis (MTCC 441), Enterococcus faecalis (ATCC 29212), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (MTCC 3615), Escherichia coli (ATCC 25922), Proteus vulgaris (MTCC 1771), Vibrio cholera (ATCC 14035), Bacillus licheniformis strain 018, Bacillus tequilensis strain ARMATI, and B. subtilis strain AK were used for antibacterial assay.Research ArticleAsian J Pharm Clin Res, Vol 9, Issue 1, 2016, 98-100 Purushothaman et al. 99Antibacterial activityAntibacterial activity was carried out using the well diffusion method on Mueller Hinton Agar (MHA) plates. The test cultures were swabbed on the top of the solidified media and allowed to dry for 10 minutes. The aqueous extract of T. divaricata flowers, synthesized T. divaricata SNPs and AuNps were used at two different concentrations (100 µl and 150 µl per well). The wells were loaded and left for 30 minutes at room temperature for compound diffusion. AgNO3 and gold chloride solutions were used as control. The plates were incubated for 24 hrs at 37°C, and the zone of inhibition was measured in millimeters (mm).Minimum inhibitory concentration (MIC)MIC of SNPs and AuNPs were determined against B. tequilensis strain ARMATI, B. subtilis strain AK, B. subtilis, S. aureus, B. licheniformis strain 018, and V. cholera. The bacterial suspension was prepared, and 100 µl of MHA broth was added to the microtitre plate and incorporated with different concentration (500, 250, 125, 62.5, 31.25, 15.625, 7.81, 3.90, 1.95, 0.976, 0.48, 0.24 µl) of SNP and AuNPs. The microtitre plate was incubated at 37°C for 24 hrs.RESULTS AND DISCUSSIONSynthesisNanotechnology has become a very active and vital research topic which is rapidly developing in industrial sectors and spreading to almost every field of science and engineering. Nanostructured inorganic, organic, and biological materials may have existed in nature since the evolution of life started on earth. Rapid synthesis and excellent reducing capping of SNPs and AuNPs through the plant extract mediated process has a time-saving advantage over microbial synthesis and the laborious and lengthy procedures involved with the mare the environmental friendly. SNPs and AuNPs biosynthesis may open new process in pharmaceutical chemistry, microbial biotechnology, biomedical and material science, nanotechnology, and biotechnology fields. In the present study, the green synthesis of SNPs through plant extracts were carried out. The appearances of yellowish-orange in the reaction vessels suggest the formation of SNPs (Fig. 1). It is well-known that SNPs exhibit yellowish-brown in aqueous solution due to excitation of surface plasmon vibrations in SNPs [5]. AgNO3 is used as reducing agent as silver has distinctive properties such as good conductivity, catalytic and chemical stability. The aqueous silver ions, when exposed to herbal extracts, were reduced in solution, there by leading to the formation of silver hydrosol. The time duration of the change in color varies from plant to plant. In the present study, T. divaricata synthesized SNPs after 24 hrs of reaction. The extracellular synthesis of AuNPs occurred during the exposure of T. divaricata flower extract to 1 mM gold chloride solution (Fig. 2). The complete reduction of gold ions was observed within 1 hr. The color change in the reaction mixture was observed during the incubation period because the formation of AuNPs is able to produce the particular color in the reaction mixtures due to their specific properties. The appearance of dark purple is a clear indication of the formation of AuNPs.Antibacterial activity by Agar well diffusion assayThe antibacterial effects of biologically synthesized SNPs and AuNPs have been investigated against Gram-positive and Gram-negative strains; E. faecalis, S. aureus, Staphylococcus epidermis, E. coli, B. tequilensis strain ARMATI, B. subtilis strain AK, B. subtilis, P. vulgaris, V. cholera and B. licheniformis strain 018. A clear zone of growth inhibition was observed against strain ARMATI, B. subtilis, S. aureus, V. cholera and strain 018. It confirms the antibacterial activity of biologically synthesized nanoparticles. The highest zone of inhibition was observed in the well loaded with SNPs, and less zone of inhibition was observed in the well loaded with AuNPs. On the other hand, there Fig. 1: Optical photograph of synthesized silver nanoparticles and zeta size distribution of synthesized silver nanoparticlesFig. 2: Optical photograph of synthesized gold nanoparticles and zeta size distribution of synthesized gold nanoparticlesTable 1: Antibacterial activity of synthesized nanoparticlesS. No. Bacterial culture Zone of inhibition (mm)Flower extract AgNO3 SNP Gold chloride Gold NP01 S. epidermis - 6 - - -02 E. coli - - - - -03 B. tequilensis strain ARMATI - 5 5 - -04 P. vulgaris - - - - -05 B. subtilis strain AK - 5 - - -06 B. subtilis - 5 4 - -07 E. faecalis - - - - -08 S. aureus - 5 5 - -09 V. cholera - 3 3 - 310 B. licheniformis strain 018 - 5 5 - 6V. cholera: Vibrio cholera, S. epidermis: Staphylococcus epidermis¸ E. coli: Escherichia coli, B. tequilensis: Bacillus tequilensis, P. vulgaris: Proteus vulgaris, S. aureus: Staphylococcus aureus, B. subtilis: Bacillus subtilis, E. faecalis: Enterococcus faecalis, B. licheniformis: Bacillus licheniformis, SNPs: Silver nanoparticles, AgNO3: Silver nitrateAsian J Pharm Clin Res, Vol 9, Issue 1, 2016, 98-100 Purushothaman et al. 100was no zone of inhibition observed in the well loaded with flower extract (Table 1).In the present investigation, nanoparticles show higher inhibition against the Gram-positive S. aureus (5 mm) compared to other Gram-negative strain V. cholera (3 mm), employed in this antibacterial assay. Savithramma and Rao [5] demonstrated the antibacterial effect of SNPs and the growth of Pseudomonas and Rhizopus species were inhibited maximum by the SNPs synthesized from leaf extract of Svensonia hyderobadensis, indicating that the SNPs may have an important advantage over conventional antibiotics. AuNPs also have potential activity against microbial pathogens, and it mainly depends on the size and shape of the particles. Consequently, the interaction between Gram-positive bacteria and SNPs were certainly stronger than that of Gram-negative bacteria. The cell wall of gram-negative bacteria consists of an outer membrane composed of lipid, protein, and lipopolysaccharides which act as a barrier and provide effective protection against the antibacterial agent. However, the cell wall of the Gram-positive bacteria lacks an outer membrane [6].MIC of biologically synthesized SNPs and AuNPsThe synthesized SNPs and AuNPs were effective in inhibiting the bacterial growth. The MIC was checked against Gram-positive (B. tequilensis strain ARMATI, B. subtilis strain AK, B. subtilis, B. licheniformis strain 018, and S. aureus) and Gram-negative (V. cholera) Table 2: MIC of synthesized SNPs and gold nanoparticlesS. No. Nanoparticles Bacterial cultures MIC (µg)1. SNPs B. tequilensis strain ARMATI 62.52. B. subtilis strain AK 31.253. B. subtilis 15.634. S. aureus 7.815. V. cholera 7.816. B. licheniformis strain 018 3.97. Gold nanoparticles V. cholera 2508. B. licheniformis strain 018 125SNPs: Silver nanoparticles, MIC: Minimum inhibitory concentration, V. cholera: Vibrio cholera, B. tequilensis: Bacillus tequilensis, S. aureus: Staphylococcus aureus, B. licheniformis: Bacillus licheniformisbacteria. The SNPs and AuNPs were used in different concentration such as 500, 250, 125, 62.5, 31.25, 15.63, 7.8, 3.9, 1.95, 0.976, 0.48, 0.24 µl in order to determine the MIC. The SNPs showed MIC value of 62.5 µl for strain ARMATI, 31.25 µl for strain AK, 15.63 µl for B. subtilis, 7.81 µl for V. cholera, 3.9 µl for strain 018, and 7.81 µl for S. aureus. The AuNPs showed MIC value of 250 µl for V. cholera and 125 µl for strain 018 (Table 2).CONCLUSIONFreely water soluble terpenoids, steroids, alkaloids, and proteins of T. divaricata are responsible for the biosynthesis and stability of spherical shaped nanoparticles in an aqueous medium. The SNPs showed the highest antibacterial activity against both Gram-positive and Gram-negative bacteria. The AuNPs were not found to be a potential bactericidal agent. The SNPs showed the MIC concentration ranging from 62.5 to 3.9 µg against bacteria. The AuNPs showed the MIC concentration of 250 µg and 125 µg against V. cholera and B. licheniformis strain 018, respectively.REFERENCES1. Devaraj P, Arun R, Aarti C, Kumari P. Synthesis and characterization of silver nanoparticles using Tabernaemontana divaricata and its cytotoxic activity against MCF-7 cell line. Int J Pharm Pharm Sci 2014;6:86-90.2. Pushpa B, Latha KP, Vaidya VP, Shruthi A, Shweatha A. Phytochemical analysis and antimicrobial evaluation of leaves extract of Tabernaemontana coronaria. J Chem Pharm Res 2012;4:3731-3.3. Gopinath SM, Suneetha TB, Anand S. Evaluation of antibacterial activity of Tabernaemontana divaricata leaves against the causative organisms of Bovine mastitis. Int J Res Phytochem Pharmacol 2011;1:211-3.4. Poornima K, Krishna R, Gopalakrishnan K. Toxicological evaluation of ethanolic extract of Tabernaemontana coronaria. Asian J Trop Dis 2012;2:679-84.5. Savithramma N, Rao LM. Antimicrobial activity of silver nanoparticles synthesized by using medicinal plants. Int J Chem Technol Res 2011;3(3):1394-402.6. Xu C, Lin X, Ren H, Zhang Y, Wang S, Peng X. Analysis of outer membrane proteome of Escherichia coli related to resistance to ampicillin and tetracycline. Proteomics 2006;6(2):462-73.

ANTIBACTERIAL ACTIVITY OF TABERNAEMONTANA DIVARICATA (APOCYNACEAE) SECONDARY METABOLITES CAPPED SILVER AND GOLD NANOPARTICLES

https://innovareacademics.in/journals/index.php/ajpcr/article/download/8313/3732/33440

ANTIBACTERIAL ACTIVITY OF TABERNAEMONTANA DIVARICATA (APOCYNACEAE) SECONDARY METABOLITES CAPPED SILVER AND GOLD NANOPARTICLES

Abstract

Similar works

Full text

Available Versions

Innovare Academic Sciences: E-Journals