GREEN SYNTHESIS OF SILVER NANOPARTICLES USING STINGLESS BEE HONEY AND

STUDY OF ITS ANTIMICROBIAL AND HEPATOPROTECTIVE EFFECT

BACHELOR OF SCIENCE IN
BIOTECHNOLOGY

Date: 1ST Chaitra, 2078

 Content
 Background
 Introduction
 Hypothesis
 Objectives
 Literature review
 Materials and methodology
 Expected results
 Budget allocation
 Work Plan
 References
 Background
Liver disease accounts for approximately 2 million deaths per year worldwide, 1
million due to complications of cirrhosis and 1million due to viral hepatitis and
hepatocellular carcinoma

Honey has a good therapeutic effect in terms of improving the antioxidant enzyme level
and regulate the cascade of hepatic enzymes pathway.

Green Synthesis of Silver Nanoparticles(AgNPs) from different honey samples produced

by different species exhibit unique antifungal activity, antibacterial action on cells,
antioxidant, anticancerous, anti-inflammatory and anti-obesity.

AgNPs have promising applications in many areas such as biological sensors, drug and
gene delivery, antimicrobial protection, biomedical application, health care, cosmetics,
food, feed, photo-electrochemical application. 
Introduction
Stingless bee is a small bee from the species of Trigona or Meliponine (also
known as puttka in Nepal) and has a high medicinal benefit than other bee
species which had been alleged by the traditional medical practitioner

The stingless bee fauna of Nepal is a single species of Tetragonula Moure, which
researcher have tentatively identified as Tetragonula iridipennis (Smith).

Green synthesis of AgNPs from honey is the simple, environmental benign

method that use Honey as both reducing and stabilizing agent in the presence of
AgNO3 as precursor.

Proteins in honey seemed to be the capping agent which stabilized the

nanoparticles while possible sugars (sucorse,fructose) acted as the reducing
agent. Fig.: Stingless bee
Hypothesis
Stingless Bee Honey(SBH) found in Nepal have antimicrobial and hepatoprotective
effect .

 SBH could be used in formulating AgNPs and their enhanced antimicrobial activity
against microbes as well as hepatoprotective effect in invivo model can be studied.
Objectives
Synthesis of AgNPs using SBH and determine the antimicrobial properties of these
nanoparticles

To determine the antioxidant potential, cytotoxicity assay of AgNPs.

To analyze the hepatoprotective effect of these synthesized AgNPs in mouse.

 Literature Review

 By adjusting the pH of the aqueous solution containing metal ions and honey, nanoparticles of
various size could be obtained at room temperature.

 Crystalline monodispersed and nearly spherical silver nanoparticles of size ∼4 nm were synthesized
at ambient conditions using aqueous solution of AgNO3 and honey at a pH of 8.5
• The colloid obtained at a pH of 9 was found to be spherical in shape and the sizes of the
nanoparticles ranged from 25-70nm

• The synthesized AgNPs from honey showed efficient antibacterial activities against the
tested Gram-negative and Gram-positive bacterial strains.
• This study reveals that natural honey has a remarkable protective effect against CCl4 -induced
mice liver toxicity at antioxidant enzyme, histological and protein expression level.

• The findings of this study clearly demonstrate that natural honey has a good therapeutic effect
in terms of improving the antioxidant enzyme level and decreases the hepatic functions
enzymes towards reference range in CCl4 -induced liver toxicity.
Carbon tetrachloride (CCl4) is a xenobiotic released into water as waste from several
industries, thereby leading to hepatotoxicity when living organisms are exposed to
it.

• Carbon tetrachloride is used as a hepatotoxin to induce liver damage in mice.

• The biochemical and histological parameters signify that AgNPs were effective in
protection against CCl4-injured mice liver.
Materials required
Materials:
 UV spectrophotometer, pH meter, Centrifuge, autoclave, Compound Microscope, microplate reader,
 All the glassware including sample collection bottle, pipette, measuring cylinder, volumetric flask,
conical flask, test tube, petri dish, Syringe etc.

Chemical Required:
 AgNO3, NaOH, HCl, deionized water,
 All the chemical required for microbial culture media, control antibiotics, buffers,distilled water,
Reagents for biochemical tests(DPPH, CCL4, Olive oil) .
 Hepatoprotective drug(Silymarin)

Other Requirement:
 Gram positive and Gram negative bacterial strains, Swiss albino mice
 Methodology Chart
Green Synthesis of AgNPs using SBH

Identification and characterization

• UV–Vis spectroscopy
• Fourier transform infrared spectroscopy
(FTIR)
• X-ray diffractometry (XRD)
• Scanning and transmission electron
microscopy (SEM, TEM)
 

Antimicrobial Antioxidant In-vivo Hepatoprotective Study

• DPPH Free radical scavenging
• Agar well diffusion assay • Assessement of liver function test
• Broth Microdilution Method Cytotoxicity • Liver Histopathology
• Brine Shrimps Lethality Bioassay

Statistical Analysis
 Methodology and Analysis
Green Synthesis of AgNPs using SBH
Stock solution is prepared by dissolving 1mM sliver nitrate (AgNO3;) and volume made up to
250 ml with distilled water.
10 g of honey was dissolved in 90 ml of deionized water. 10 ml of SBH samples was added to 90
ml of 1mM AgNO3 solution and allowed to react at room temperature
The reduction of Ag ions by SBH at different pH and different incubation time is monitored
periodically by UV-Visible spectrophotometer(300-700nm) .The characteristics absorption peaks
will help us for the further analysis of suitable honey solutions containing Ag nanoparticle at
optimal pH ,concentration,incubation time,etc.
Further Characterization is done through FTIR, XRD, SEM ,etc
Determination of Antioxidant Potential of AgNPs
 DPPH scavenging activity% = (A1 –A2 / A1) *100
A1 is the control absorbance and A2 is the sample absorbance.Change
in absorbance was measured at 517nm
The IC50 value (mg/mL), a minimum concentration of sample that
inhibits 50% DPPH radical is reported

Antimicrobial Activity
Agar well diffusion method

Microorganism Nanoparticle Blank Standard Drug Measurement

(AgNPs) of Zone of
synthesized Inhibition(mm)
Bacteria, Fungi At Different Antibiotics and
Concentration Antifungal
In vivo Hepatoprotective Study
15 Swiss albino mice divided in 5 Group. Each group consisted of 3 mice.

Experimental Design

Group I I Group II I Group IIII Group IV I Group V

Normal CCl4 intoxicated CCl4 intoxicated

NormalI Treated with CCl4 intoxicated Treated with Treated with
AgNPs (Control) SBH drived Standard Drug
AgNPs (Silymarin)

Biochemicalparameters
Liver function test will be performed by analyzing Albumin, Alkaline Phosphatase, Bilirubin
(Total, Direct, Indirect), Total Protein, SGPT, SGOT ,albumin to globulin ratio in above mentioned
groups .
Statistics
Results obtained were expressed as mean ± SD
Expected Outcomes
Synthesis, identification and characterization of Ag nanoparticles from stingless honey
bee honey found in Nepal .

Establishing interrelationship between Ag nanoparticles and their potential

antimicrobial ,antioxidant and ultimately hepatoprotective effect (in vivo model )
 Budget

S.N. Particulars Expected Amount

1 Sample collection 10,000

2 Media and chemicals 20,000

3 Characterization of AgNPs 20,000

4 Invivo Hepatoprotective Analysis 20,000

5 Miscellaneous 5,000

6 Total 75,000
 Work Plan
Work list  February March  April May June  July
 
           
Proposal writing 
           
Literature review
           
Sample collection 
           
Green synthesis of AgNPs from SBH
and its characterization  
           
Antimicrobial Antioxidant and
Cytotoxicity assessment of AgNPs
           
Hepatoprotective effects of AgNPs in
mice
           
Data analysis 
           
Report writing
           
Report
 References
• Chet P. Bhata, Victor H. Gonzalez , and Deborah R. Smith Traditional uses and relative cultural importance of Tetragonula iridipennis (Smith)
(Hymenoptera: Apidae: Meliponini) in Nepal
• Daizy Philips(2009) , Honey mediated green synthesis of gold nanoparticles https://doi.org/10.1016/j.saa.2009.12.058
• Saleh A. Almatroodi,Therapeutic Implication of Honey against Chronic CarbonTetrachloride-Induced Liver Injury via Enhancing Antioxidant
Potential and Maintenance of Liver Tissue Architecture,2021
• Biswa, R., Sarkar, A. and Subba, S.K. 2017.Ethnomedicinal uses of honey of stingless beeby Nepali community of Darjeeling foothills of West
Bengal, India, p. 648–653. India:NISCAIR-CSIR.
• H. Haiza, A. Azizan, Aizat Hazwan Mohidin, D.S.C. Halin, Green synthesis of silver nanoparticles using local honey,2013
• Ghosh, S., Derle, A., Ahire, M., More, P., Jagtap, S., et al., 2013. Phytochemical analysis and free radical scavenging activity of medicinal plants
Gnidiaglauca and Dioscoreabulbifera e82529 PLoS ONE
• P. Prema, Chemical Mediated Synthesis of Silver Nanoparticles and its Potential Antibacterial Application 2011
• Suvro S, Honey- The natural sweetener become a promising alternative therapeutic: a review, South Indian J BiolSci, 2015, 1(2), 103-114.
• Tuksitha, Lalita & Chen, Yi-Lin & Chen, Yi-Ling & Wong, Kie-Yiong & Peng, Chi- Chung. (2018). Antioxidant and antibacterial capacity of
stingless bee honey from Borneo (Sarawak). Journal of Asia-Pacific Entomology. 21. 10.1016/j.aspen.2018.03.007.
• Zhang, Hongru & Jacob, Joe Antony & Jiang, Ziyu & Xu, Senlei & Sun, Ke & Zhong, Zehao & V, Nithya & Achiraman, Shanmugam. (2019).
Hepatoprotective effect of silver nanoparticles synthesized using aqueous leaf extract of Rhizophora apiculata. International Journal of
Nanomedicine. Volume 14. 3517-3524. 10.2147/IJN.S198895.
• Zhang, Hongru; Jacob, Joe Antony; Jiang, Ziyu; Xu, Senlei; Sun, Ke; Zhong, Zehao; Varadharaju, Nithya; Shanmugam, Achiraman
(2019).Hepatoprotective effect of silver nanoparticles synthesized using aqueous leaf extract of Rhizophora apiculata. International Journal of
Nanomedicine, Volume 14(), 3517–3524. doi:10.2147/IJN.S198895