INTRODUCTION:
Over a past few decades, metallic nanoparticles have drawn immense interest in the field of research and modern science because of their unique physico-chemical properties, small size, high surface area to volume ratio, tunable pore size and surface plasmon behavior (Iravani et al., 2014; Kalyani et al., 2019). Nanotechnology in general deals with, designing, synthesis, characterization and utilities of materials in its nano scale or having dimensions in 1-100 nm range (Kouvaris et al., 2012; Shekhawat et al., 2014). These nano-structured materials often show remarkable difference in the physical, chemical and biological properties compared to the same material at macro-scale. and thus proving to be alternative solutions to environmental and technological challenges in the field of catalysis, biomedical, solar energy conversion, water treatment, agriculture fields, polymer science, mechanical engineering, optical and electronic fields (Pal et al., 2011; Kalyani et al., 2019; Shah et al., 2015; Vidya et al., 2013).
Recently, the plant based metallic nanoparticles gained very popularity, because of their less expansiveness, non-toxic, non-hazardous, more stable and environmentally benign nature in comparison to physical and chemical methods based synthesized nanoparticles (Sangeetha et al., 2011; Anbuvannan et al., 2015; Gondwal and Joshi, 2018 and Sharmila et al., 2019). Further phytochemicals (flavonoids, alkaloids, polyphenolic acid, proteins, essential oils and saponins) present in the plant extracts acts as reducing, capping and stabilizing agent in these green synthesis (Babu et al., 2015; Azizi et al., 2017 and Shama et al., 2021).
Zinc oxide nanoparticles have drawn the attention of many researchers because of their several applications in photo catalysis, electronics, communications, UV-protection cosmetics, sensors, water purifiers, biological and medicinal industries (Sharma et al., 2010; Kandwal et al., 2019b). Further, Zinc oxide nanoparticles are suitable for thin coating applications because they exhibit good antibacterial and antifungal activities even at lower concentrations (Setyawati et al., 2013; Azizi et al., 2016 and Supraja et al., 2016).
Ajuga bracteosa a member of Lamiaceae is, diffused, softly villous perennial herb found distributed at an elevation of 2000 m and sub-Himalayan tract. It is widely distributed in Indian Himalayan region from Kashmir to Uttarakhand and in mountains of Nepal, China, Afghanistan and western Asia.
The herb isused in wide spectra of diseases i.e., herbis used in the treatment of malarial fever, body pains, gout, hepatitis, diabetes, hypertension, blood purifier, cooling agent, diarrhea and other digestive related problems (Hamayun et al., 2006; Pal et al., 2011; Kumar et al., 2013; Qing et al., 2017 and Ahmad et al., 2018). Besides this, plant also exhibits several pharmacological activities such as antifungal (Ganaieet al., 2018), antibacterial (Vohra and Kaur, 2011), antioxidant, anticoagulant, antidepressant, anti-inflammatory (Kayaniet al., 2016; Palet al., 2011), antiviral activity against HCV (Yousafet al., 2018), inhibitory activity against cholinesterase and anti-cancer (Qing et al., 2017; Riazet al., 2004).
Several phytochemicals like alkaloids, essential oils, withanolides and terpenoids has been reported from different herb (Shad et al., 2016; Hussainet al., 2012; Verma et al., 2002; Riazet al., 2004; Vohra and Kaur, 2011) coupled with no report has been texted regarding the antimicrobial activity of zinc oxide nanoparticles for this plant, motivated us to workout the green synthesis and antimicrobial activity of zinc oxide nanoparticles from this herb.
MATERIAL AND METHODS:
Collection and authentication of plant materials:
Fresh and healthy leaves of Ajuga bracteosa was collected from Nagdev forest range, Pauri Garhwal, Uttarakhand and authenticated from University of Jammu and its accession no. HBJU 16002 was collected.
Preparation of Ajuga bracteosa leaf extract:
The collected leaves were thoroughly washed with tap water followed by distilled water in order to remove any of adhesive dirt. Leaves were then shade dry for 15 days. Dried leaves were than mashed with mortar-pestle and 5 g of finely powdered leave was taken with 100 ml double distilled water in 250 ml Erlenmeyer conical flask and heated at 65 °C for 25 minutes. Then, the extract was allowed to cool at room temperature and filtered using Whatmanfilter paper no. 1.
Synthesis of ZnO nanoparticles:
Ajuga
bracteosa leaf extract was taken and heated on magnetic stirrerat
60
for 15 minutes in a 250 ml Erlenmeyer flask, and then 1:1
of 100 mM zinc nitrate solution (aqueous) was mixed drop by drop to it with continuous stirring.
Change in colour of the solution from dark red to brownish, indicated the
synthesis of ZnO NPs. The solution of the reaction mixture was allowed to cool
at room temperature, followed by its centrifugation for 15 minutes at 6000 rpm
and washing with distilled water followed with ethanol to remove un-reacted
material if any. The obtained pellets were than dried in the oven at 60 for 10 hrs followed by mashing in
mortar-pestle to get fine and uniform powdered grayish black ZnO nanoparticles and stored for the characterization of
ZnO nanoparticles and antimicrobial activities.
Characterization:
Initially,
at regular intervals formation of zinc oxide nanoparticles was preliminary
monitored by using Elite-double beam UV-visible spectrophotometer. Then, ZnO
nanoparticles was subjected to X-ray diffraction (XRD) analysis (X'PERT-PRO
Diffractometer, PANalytical; CuKα radiation, λmax = 1.54
Å) and its spectra was reported in the range of 2
from 0
to 70. Fourier transform infrared spectroscopy (Spectrophotometer
Perkin Elmer Model RZX) analysis ranging from 4000-500 cm-1 was to
recognize the phytochemicals present in the plant extract accountable for
capping and stabilizing the nanoparticles. Energy Dispersive X-Ray (EDX) was
performed for determining the elemental composition of synthesized
nanoparticles. Transmission electron microscope (JEOL JEM 1400) analysis was
used to determine the surface morphology of ZnO nanoparticles.
Antimicrobial activity:
The zinc oxide nanoparticles synthesized from Ajuga bracteosa leaf extract were tested for antimicrobial activity by Agar well diffusion method against five bacterial and two fungal strain i.e., Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumonia, Escherichia coli, Pseudomonas aeruginosa, Aspergillus fumigates and Trichoderma viride. Muller Hinton agar medium and Potato dextrose agar/broth of Hi Media Pvt. Bombay, India were used for antibacterial and antifungal test respectively.
RESULT AND DISCUSSION:
The synthesis of phytomediated metallic nanoparticles was confirmed by visual markers i.e., change in colour of the reaction mixture followed by their preliminary characterization by UV visible spectrum and a broad absorption peak at λmax = 349 nm confirmed the presence of ZnO nanoparticles in the solution and its calculated energy band gap was around 3.56eV (Figure 1)
Figure. 1. UV-visible spectrum of synthesized ZnO nanoparticles
XRD analysis
The
XRD spectra showed the crystalline nature of ZnO nanoparticles and the average
crystallite size was calculated using Debye-Scherrer’s equation (Das et al.,
2010; Aziziet al., 2017; Karthiket al., 2014). XRD spectra of
synthesized nanoparticles showed sharp peaks at 2 = 32.00° ,
34.08° , 36.46° , 47.72°
, 56.79° ,
63.01° and 68.25° which are indexed to (hkl) (100), (002),
(101), (102), (110), (103) and (201) lattice
planes respectively (Table-1) of hexagonal wurtzile structure of nano crystals
(Figure 2).
Table-1: Peak list for average size calculation of nanoparticles
|
2θ (°) |
hkl |
FWHM left |
d-spacing (Å) |
Rel. int. (%) |
|
32.00 |
110 |
0.70 |
2.79 |
58.91 |
|
34.62 |
002 |
0.35 |
2.59 |
75.54 |
|
36.46 |
101 |
0.56 |
2.46 |
100 |
|
47.72 |
102 |
1.04 |
1.90 |
15.60 |
|
56.79 |
110 |
0.65 |
1.62 |
35.42 |
|
63.01 |
103 |
0.71 |
1.47 |
28.76 |
|
68.25 |
201 |
1.47 |
1.37 |
21.33 |
Where θ is the Bragg's diffraction angle, FWHM is full width at half maxima, (h,k,l) are miller indices and d-spacing is interplanar distance.
Figure 2. XRD spectra of synthesized ZnO nanoparticles
TEM analysis:
TEM analysis was used to study the surface morphology of ZnO nano structures (Figure 3). These results confirmed that ZnO nanoparticles were hexagonal in shape with their average size less than 27 nm.
Figure 3. Transmission electron micro- image of ZnO nanoparticles
EDX analysis:
EDX was carried out for the composition of nanoparticles. Figure 4 shows sharp signal at 1 keV and 0.5 keV corresponding to Zn and O respectively, thus confirming the formation of ZnO NPs. The other signals that were noticed in the spectra may be attributed due to the bioactive components present in plant extract.
Figure 4. EDX pattern of ZnO nanoparticles
FTIR analysis:
FTIR spectra of nanoparticles showed several peaks which confirm the presence of different functional groups which may responsible for the reducing, capping and stabilizing of nanoparticles. FTIR peaks in Figure 5 at 3430.6cm-1 corresponds to the stretching vibrations of hydroxyl group of polyols. Peak at 1622.7cm-1 may be ascribed to C=O stretching vibrations of extensively conjugated systems. The peak at 1495.1cm-1 may be attributed to C=C aromatic ring stretching. The peak at 1384.1cm-1 may be represented to C-O stretching of ArOH. Peaks below 526.7 cm-1 region may be attributed to the ZnO nanoparticles.
Figure 5. FTIR spectrum of ZnO nanoparticles
Antimicrobial activity:
Agar well diffusion method was used to perform the antimicrobial assay and the zone of diameter of inhibition (mm) was measured for all tested organisms i.e., Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumonia, Escherichia coli and Pseudomonas aeruginosa, Aspergillus fumigatus and Trichoderma viride (Table 2 and Table 3). Sodium chloride solution was used as positive control. In the present study and it was observed that saline have no effect on bacterial growth and gives no zone of inhibition, while the synthesized ZnO nanoparticles showed antimicrobial potential against all test organisms. The maximum zone of inhibition was reported for Klebsiella pneumonia 25.00 mm and Aspergillus fumigates 12.00 mm (bacteria and fungus respectively) and minimum zone of inhibition was reported for Streptococcus pneumonia 9.00 mm and Trichoderma viride 11.00 mm (bacteria and fungus respectively). Thus from the results, it was confirmed that the synthesized nanoparticles have potential antibacterial and antifungal activity.
Senthilkumar et al., (2014) working on antimicrobial activity of zinc oxide nanoparticles using Camellia sinensis against four bacterial strains reported maximum zone of inhibition in Klebsiella pneumonia, supporting the present study. Besides this, number of other worker also reported the antibacterial and antifungal activity of plant based ZnO nanoparticles (Sharma et al., 2010; Rajivet al., 2013). The possible mechanism for the antibacterial activity of synthesized ZnO nanoparticles in the present study may involve the absorption of the nanoparticles by the host where these nanoparticles starts the generation of free radical which interrupts the basic metabolism and some time may cause damage or alteration to genome of the host and results in the death of the host organism (Stoimenov et al., 2002; Xie et al., 2011; Purohit et al., 2020).
The present work created the uniformly distributed, crystalline, hexagonal in shape ZnO nanoparticles with their average size less than 27 nm using plant extract and the reports of Sangeethaet et al., (2011) and Anbuvannanet et al., (2015) support the present findings.XRD spectra of synthesized nanoparticles showed sharp peaksindexed to lattice planes indicating hexagonalwurtzilestructure ofZnOnano crystals, and the same results were reported by Kandwalet et al., (2020), Khajuriaet et al., (2019b). EDX and FTIR (Aziziet al., 2017; Sharmilaet al., 2019) spectra show the prominent peaks of various functional groups of phytochemicals which suggested that ZnO NPs were synthesized with the help of hydroxyl (O-H) and amino (NH2) groups (peak above 3050 cm-1), carbonyl (C=O) groups (peak above 1600 cm-1), unsaturated carbons (peaks between 1400-1600 cm-1) etc. Thus, these phytochemicals may be responsible for the synthesis and stability of ZnO NPs through the electrostatic interaction and capping of metallic NPs. The finding for EDX and FTIR studies are in correlation with the existing literature (Kandwal et al., 2019a and Khajuria et al., 2020 and Gondwal and Joshi, 2018; Fu and fu, 2015) which further support the successful synthesis of ZnO nanoparticle in the present work.
CONCLUSION:
It can be concluded that green synthesis of ZnO nano particles using Ajuga bracteosa leaf extract is successful rapid, energy saving, environmentally benign and cost effective method. In the present study hexagonally shaped, crystalline ZnO nanoparticles with their average size less than27 nm were synthesized. It is observed that bioactive components of leaf extract of Ajuga bracteosa may be responsible for reducing, capping and stabilizing the zinc oxide nanoparticles. The Zinc oxide nanoparticles showed good antimicrobial activity against tested microorganism and thus can serve as potential nano drugs in various industrial and biomedical applications.
ACKNOWLEDGEMENTS:
The second author grateful acknowledged the financial support from Council of Scientific and Industrial Research in the form of CSIR fellowship.
CONFLICT OF INTEREST:
Theauthors declare that there is no conflict of interests regarding the publication of this article.
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Received on 20.07.2021 Modified on 04.08.2021
Accepted on 14.08.2021 ©Asian Pharma Press All Right Reserved
Asian J. Pharm. Ana. 2021; 11(4):275-280.
DOI:10.52711/2231-5675.2021.00047