Plant Generated Silver Nanoparticles and their Antibacterial Effect in Combination with Levofloxacin and Amikacin

Introduction

Over the past three decades, advanced technology and research had enabled nanotechnology to become one of the most active areas of research in modern science. This field basically deals with atoms or molecules that ranges between 1 to 100 nm in size. The manipulation of such atoms or molecules give rise to a better or completely new properties based on their size, shape, distribution and morphology of both its individual atoms/molecules and their corresponding bulk. [1]. These enhanced properties have enabled nanotechnology to gain attention and novel application in various fields such as health care, cosmetics, biomedical, food and feed, drug delivery,  gene delivery, space industries, electronics and many other areas.

Among all metallic, organic and inorganic nanomaterials, metallic nanoparticles had been the most promising type as it contains large surface area to volume ratio that enables it to have a great antibacterial effect like silver, gold, copper, palladium, platinum and ruthenium. Among various metallic nanoparticles, silver nanoparticles had been gaining attention for its unique shape and size that enhances its properties such as effective anti-bacterial agent with lower toxicity, anti-fungal, anti-inflammatory, anti-angionesis and anti-platelet activity [2,3,4].

Generally, there are three ways to synthesize the nanoparticles to cater the demand of nanoparticles which are physical, chemical and biological method [5, 6]. Among all the three methods, biological approach has emerged as a viable option. Usage of various biological resources such as plants, bacteria and fungi had shown to be a simpler , cost effective and most importantly environmentally friendly as there is no need to use high temperature, high pressure and harmful chemicals in the process[7,8,9].

In this study, H. Sabdariffa plant leaves were used for the synthesis of silver nanoparticles and characterization was carried out by UV-Visible spectroscopy, TEM to confirm size, shape and distribution of nanoparticles. These silver nanoparticle were analysed for its antibacterial effect against various pathogens and compared with antibiotics were studied.

Materials and Method

Collection of plant

H.Sabdariffa plant was bought from Tiang Nombor 9, Kampung, Rengit, Johor from a local who plant them commercially and was authenticated by botanist in Ipoh Malaysia.

Preparation of extract

The plant was washed with distil  water to remove dirt and residue. Leaves are separated from the stem and left to dry under the sun for a week. The dried leaves are grinded using commercial grinder into powder form. The powder is transferred into 500 ml beaker.  4 grams of the powder form was weighed and dissolved in 100ml of distilled water in a 250 ml beaker and  boiled for 10 minutes. The aqueous solution was filtered twice using filter paper to remove all the residue. The filtrate was  kept in refrigerator for further use.

Synthesis of silver nanoparticles

The cell filtrate was taken out and kept as such for some time.1mM silver was added dropwise and shaked it continuously. After that conical flask was covered with aluminium foil and put on the orbital shaker for 48 hours. Colour change is observed from dark yellow to light yellow which indicated formation of silver nanoparticles.

Characterization of AgNPs

The silver nanoparticle synthesized was subjected  to JASCO V -650  UV-VIS spectrophotometric analysis  in order to determine the absorbance (lambda max) range from 300 to 600 nm .TEM analysis was used to determine the shape. size and dispersity of nanoparticles. For TEM (JEOL JEM) analysis sample was prepared by diluting the nanoparticle solution with distil water. One drop of nanoparticle was put on carbon coated grid and subjected for TEM analysis

Antibacterial Assay

The nutrient agar media was used for the growth of bacteria and each agar plate was labelled and swabbed with selected pathogens (Bacillus cereus, Escherichia coli, and Salmonella enterica) to check the antibacterial effect of nanoparticles by using disc diffusion method. Each disc was impregnated with 40µg nanoparticles and compared  with levofloxacin and amikacin.  The petri dish is kept in incubator for overnight at 37 °C.

Result and Discussion

The aqueous extract of H.sabdariffa was used for the synthesis of silver nanoparticles. When the  aqueous extract was challenged with silver nitrate colour change from dark brown into  light brown colour Fig 1[10].

The reduction of silver ion into silver nanoparticles is confirmed by UV-Vis spectroscopy which is the common technique used for qualitative analysis of nanoparticles. It measures the surface plasmon resonance (SPR) peaks. Unique optical properties of nanoparticles are due to the property of SPR which is the collective oscillation of the conduction electrons in resonance with the wavelength of irradiated light. (Bindhu M.R. and Umadevi M., 2013)[11, 12]

The UV-vis spectroscopy graph showed an absorbance peak at 422.15 nm which is specific for silver nanoparticles. Previous studies suggested spherical AgNPs have SPR peak between 410 to 450 nm Fig2 [11,12].

Figure 1: Colour change of extract after addition of silver nitrate.  Click here to View figure

 

Figure 2: UV-visible spectrum of Silver nanoparticles synthesized from Hibiscus sabdariffa  Click here to View figure

 

Transmission Electron Microscopy (TEM) is used to measure shape, size, distribution and morphology. TEM  showed the AgNPs obtained are predominantly spherical in shape, well distributed with size ranges from 19 nm to 50 nm with an average of 30 mm. Fig 3.

Figure 3: TEM analysis showing the morphology of synthesized silver nanoparticles from H. sabdariffa extract  Click here to View figure

 

The antibacterial activity of biosynthesized silver nanoparticles were tested against four different human pathogens ie Salmonella enterica, Escherichia coli Staphylococcus aureus and Bacillus cereus respectively using  disc diffusion method. These nanoparticles were evaluate alone and also in combined form with amikacin and levofloxacin.Each blank disc was impregnated with 40 µg of nanoparticles whereas for antibiotics each disc was impregnated with 20µg nanoparticles in order to find out the synergistic effect. These silver nanoparticles showed good antibacterial activity but also works better in combination with antibiotic and enhances their antibiotic efficacy. Among all  the four pathogens S. enterica, S. aureus, and E. coli, showed the highest reading (40 mm)  in combination with  Levofloxacin where as  for synergistic effect with Amikacin, S. enterica and E. coli (35 mm) gave the highest showed highest zone of inhibiton as shown in table 1.Fig 4 showed the graphical representation of combined effect of silver nanoparticles with amikacin and levofloxacin.These silver nanoparticles have the efficacy to enter inside the  cell and causing the cellular damage  by inactivating the essential enzymes of the cell hence causing the cell death by generating the free radicals that plays  active role for antibacterial effect[13,14,15.].

Table 1: Zone of inhibition (mm) of synthesized nanoparticles in combination with antibiotics. Am – Amikacin, Levo – Levofloxacin

Pathogenns	AgNPs	Am	Am+ AgNPs	Levo	Levo+ AgNPs
Salmonella enterica	15	19	24	35	40
Bacillus cereus	16	21	27	21	26
Staphylococcus aureus	16	25	28	34	40
Escherichia coli	16	23	28	35	40

 

Figure 4: Bar chart showing the effect of synthesized nanoparticles, antibiotic and antibiotic in combination with synthesized silver nanoparticles against human pathogens.  Click here to View figure

 

Conclusion

Hibiscus Sabdariffa acts as a good source for the biosynthesis of silver nanoparticles and it showed good antibacterial activity against various pathogens. The synthesized silver nanoparticles also showed excellent synergistic combined effect when combined with levofloxacin and amikacin.

Acknowledgement

Author would thanks Centre of Research and innovative universiti Kuala Lumpur for providing financial assistance to carry out this study.

Conflict of Interest

There is no conflict of interest among authors

References

1. Ahmed.S.; and Ikram.S.; Silver Nanomedicine & Nanotechnology.;2015,6:309.
2. Ahmed, S .; Journal of Radiation Research and Applied Sciences,; 2016,  9, 1-7.
   CrossRef
3. Kalashwaralal, K,; Deepak, V,; Pandian, K,; Kottaisamy, M,; Manikanth, SB, ;Karthikeyan, B,; Gurunathan, S.; Coll Surf B: Biointerface. 2010, 77, 257-62.6
4. Monali, G,; Kesherwani, J,; Ingle, A,; Gade, A,; Rai, M.; Nanomedicine: Nanotechnology, Biology and Medicine.; 2009, 5, 382-86
5. Roco, C,; Nanopart Res,; 2005, 7, 707-12.
   CrossRef
6. Raveendran, P,; Fu, J,; Wallen. SL,; Virender, K,; Sharma, ;Rai A,;  Chem Soc,; 2003,125, 139- 40.
7. Goodsell, DS,; Wiley Liss. Hoboken. 2004.
8. Ingle, A,; Rai, M,; Gade, A,; Bawaskar, M,;. J Nanopart Res,; 2009, 11, 2079-85
   CrossRef
9. Monali, G,; Kesherwani, J,; Ingle, A,; Gade, A,; Rai, M,;Nanomedicine: Nanotechnology, Biology and Medicine.; 2009, 5, 382-86.
10. Sastry, M, ;Ahmad, A,; Islam Khan, M,; Kumar, R,;  Current Science,; 2003, 85, 162-70.
11. Mulvaney, P,;. Langmuir,; 1996, 12, 788 – 800.
    CrossRef
12. Chauhan, R., ; Reddy, A,; Abraham,; J, Applied Nanoscience,; 2015, 5, (1) 63-71.
    CrossRef
13. Yang, H,; Liu, C,; Yang, D,; Zhang, H,;  Xi, Z; Journal of Applied Toxicology,;2009, 29(1), 69–78.
    CrossRef
14. Pal, S,; Tak, Y, K.,; Song, J,M,; Applied & Environmental Microbiology,; 2007, 73(6), 1712–1720.
    CrossRef
15. Rajan, A,; Cherian, E, ; Baskar, G,; International Journal of Modern Science and Technology,; 2016, 1(2) 52-57

---

This work is licensed under a Creative Commons Attribution 4.0 International License.