1. INTRODUCTION:
Future research field includes nanoparticles as the main concern of scientists as the nanoparticles have specific site of action, and the small size give enhancement to the permeability of immune response through the membrane of the cells[1] and the small size increases the surface to volume ratio and the gradational effects on the particles are also reduced[2] nanoparticles size ranges from 1nm to 900nm, and size range between 1-100nm have higher microbial activity[3]. Since ancient time herbs and medicinal plants are used by humans for their therapeutic effects. Plants are an excellent source for pharmaceuticals and health care products[4].
Artemisia vulgaris belongs to Asteraceae family commonly known as Mugwort and Afsantin[5] having traditional use as herbal medicine for treating problems related to stomach and menstrual problems[6]. Traditionally it is used as an antiseptic, diuretic, analgesic, antipyretic, diaphoretic, anthelmintic, hypoglycemic, antispasmodic, larvicide, expectorant and tonic. This species is also an effective remedy to treat colic, depression, asthma, rheumatism cancer, dyspepsia, hepatosis, epilepsy, cough, diarrhea, headache, hemorrhage and inflammation[7]. Additionally, anti-nociceptive[8] and hepato-protective[9] properties have been reported in this plant species.
They have received increasing interest because of their medicinal applications, especially with regards to flavonoids[10]. They are used in gastric diseases, in malaria, as antifungal, anthelmintic, sedative and emmenagogue agents[11]. Artemisia vulgaris L. (Mugwort) is a tall aromatic perennial herb, which grows in the hilly districts of India in areas up to 2400m elevation. In traditional medicine, this plant is widely used for the treatment of diabetes and extracts of the whole plant are used for epilepsy and in combination for psychoneurosis, depression, irritability, insomnia, anxiety, and stress[12]. Infusion of the leaves is given as a vermifuge. Mugwort is commonly used in traditional European medicine as a choleretic and for amenorrhoea and dysmenorrheal[13]. In herbal medicine, aerial parts of A. vulgaris are being used as an anthelmintic, an antiseptic, an antispasmodic, and a tonic for vital organs and for various disorders including hepatosis[14]. In various studies, A. vulgaris showed antibacterial activity and showed efficacy in the correction of breech presentation[15]. Its crude extract has been used as an antimalarial agent for thousands of years, and it was found that artemisinin extracted from A. vulgaris had antitumor activity[16].
There has been increase in the oral candidiasis reported in the last year, candial species were found to be associated mostly with oral infections[17], although Candida forms the oral commensal microflora[18,19], Candida species can grow in extreme environment conditions, Candida species with reduced susceptibility to antibiotics, and spreading issues concerning the safety of chemical preservatives and drugs have prompted researchers to study antimicrobial agents from natural resources[20]. More than 35,000 plants species are being used in various human cultures for medical purposes[21]. Vitamin Bx or Vitamin H which is in general PABA is essential for some bacteria’s and fungus for their growth in synthesis of folic acid[22]. There is a wide availability of clinically useful anti-bacterial and the anti-fungal analogues researches in pharmacological and medical field provides new approaches in combating human diseases[23]
The need of new anti-microbial agents is justified because more microorganisms are being resistance to the present drugs available in the market. Worldwide researchers are trying to synthesize new drugs with better pharmacokinetic and dynamic properties with less adverse effects[24] Thus, the development of new agents with potent antibacterial and antifungal activities and fewer adverse effects is urgently desired[25] and the WHO has fact listed that still 80% of population relay on plants for medicine[26] and Indians have a traditional connection with medicinal plants as its part of the cultural traits and used since ancient times[27]
The present study deals with the formulation and characterization of silver nanoparticles using aqueous leaves extract of Artemisia vulgaris and their antifungal activity.
2. MATERIALS AND METHODS:
2.1. Collection of plant material:
The aerial part of Artemisia vulgaris Linn were collected from Thanjavur, Orthanadu, Thirumangalakkottai east. The plant specimens were identified authentically by Botanical Survey of India, Southern Regional Centre, and Coimbatore.
2.2. Preparation of leaf broth:
The collected fresh leaves of Artemisia vulgaris. Linn were washed thoroughly with deionized water for 3 times. The leaves were grinded using an electric motor.
The broth solution was prepared by taking 10g of grinded leaves with 100ml of distilled water in a 250ml of Erlenmeyer flask and the mixture is boiled at 60oC for 5minutes. Cool the broth solution at room temperature and the clear solution were decanted.
2.3. Preparation of Silver nanoparticles:
The 12ml of this broth solution was added to 88ml of 1 mM aqueous AgNO3 solution. Then the solution was heated at 60oC for 5minutes. The resulting solution became brown in color and the extract was filtered through Millipore hydrophilic filter (0.22µm) and used for further experiments[1].
2.4 Characterization:
2.4.1 UV-VIS spectroscopy:
UV-VIS spectroscopic studies were carried out on a Shimadzu UV-VIS double beam spectrophotometer over a wavelength range of 200-400 nm.
2.4.2 Zeta Sizer and Zeta Potential:
The average particle size and zeta potential of the prepared AgNPs was evaluated with the help of Malvern Zeta-sizer nano ZS90 at 25.1oC after suitable dilution with distilled water.
2.4.3 SEM Studies:
Scanning electron microscopy analysis is used to study the morphology and size of the nanoparticles. Surface morphology was determined by the SEM. In this, the formulations were analyzed at different magnifications 16000X and 65000X.
2.4.4 Minimum Inhibitory Concentration:
The MIC of solutions for selected microbes was determined using the micro dilution growth susceptibility test. A serial dilution of the dispersion of Ag ions and the dispersion of silver nanoparticles were prepared within a desired range (10-109).
2.4.5 Well Diffusion Method:
The agar plate is made by using Sabouraud dextrose agar medium and it is solidified. The culture is poured and spread on the medium and the wells are made then different concentration of the AgNPs are made and poured in the wells and incubated at 37oC for 24hrs.
3. RESULT AND DISCUSSION:
Initially the prepared nanoparticles were confirmed by observing the solution was colour changed from pale yellowish green to brown colour. The colour change in the reaction mixture indicated the formation of silver nanoparticles (AgNPs). During the visual observation Artemisia vulgaris L. is mixed with silver nitrate showed colour changed from pale yellowish green to brown colour due to the reduction of silver ion; which is indicated the formation of silver nanoparticles. This colour arises due to excitation of surface plasmon vibration in AgNPs. They are the surface-active molecules that play an awfully necessary role in reducing and stabilizing process of silver nanoparticles.
Fig 1: preparation of silver nano particles
3.1. Characterization of silver nanoparticles (AgNPs):
3.1.1. UV visible spectra:
The preliminary characterization of AgNPs was monitored by UV-VIS absorbance spectra analysis. The maximum absorbance study peak appears at the range of 200 to 400nm.
Fig 2: UV spectra of Artemisia vulgaris L.
This range from 200 to 400nm is indicates very small nanoparticles. The formation of AgNPs was observed which is established in the absorbance intensity.
3.1.2. Zeta sizer:
The zeta sizer of the prepared silver nanoparticles (AgNPs) is illustrated below,
Fig 3: zeta sizer of Artemisia vulgaris L.
From this result, the average size of the AgNPs is around 104.5 nm. The morphology and size of the particles were determined. The prepared AgNPs ranges from 100 to 110nm.
3.1.3. Zeta potential:
The zeta potential of the prepared AgNPs is illustrated below,
Fig 4: zeta potential of Artemisia vulgaris L.
The range should be in -25 to +25mV. The average negative potential value is -18.5 mV with high colloidal stability. From the literature, it is evident that AgNPs having zeta potential having stable for a long period.
3.1.4. Scanning Electron Microscopy (SEM) analysis:
Scanning electron microscopy analysis is also to know the absorbance of nanoparticles. In this study, the reducing potential of Artemisia vulgaris leaves extract was investigated for synthesizing silver nanoparticles without the addition of any external reducing or capping agent. SEM analysis confirmed the morphology of the synthesized nanoparticles.
Fig 5: SEM analysis 16,000 X magnification
Fig 6: SEM analysis 65,000 X magnification
3.2.1. Minimum inhibitory concentration:
The antifungal activity of the silver nanoparticles (AgNPs) prepared by the modified process was evaluated for pathogenic Candida albicans by means of determination of the minimum inhibitory concentration (MIC) and the time dependency of Candida growth inhibition was determined.
Fig 7: MIC showing the inhibition
Table 1: MIC of Artemisia vulgaris AgNPs
|
Dilution |
Growth |
|
101 |
- |
|
102 |
- |
|
103 |
- |
|
104 |
- |
|
105 |
+ |
|
106 |
+ |
|
107 |
+ |
|
108 |
+ |
|
109 |
+ |
+ represents Fungal growth
- represents No Fungal growth
3.2.2. Zone of Inhibition:
The result of the anti-fungal activity of the prepared AgNPs against C. albicans showed good zone of inhibition compared to the A.vulgaris leaves broth. The results are as follows:
Table 2: Zone of inhibition of C. albicans by Artemisia vulgaris Ag NPs
|
Samples |
Zone of Inhibition (mm) |
|
Control (sterile water) |
Nil |
|
Standard (100µg/ml) |
37 |
|
A.vulgaris leaves broth |
23 |
|
A.vulgaris AgNPs |
30 |
Fig 8: Zone of inhibition of C. albicans
4. CONCLUSION:
Silver nanoparticles have received considerable attention in Nano medicine. It have become a leading research field, scientist squares measured concerned in synthesizing safe, effective and most cheaper and less toxic drugs to combat disease like diabetes, cancer, epilepsy, etc. Their small size gives them an edge where as evading the immune response and also give the ability to cross relatively impermeable membrane. The silver nanoparticles are vigorously involved in the anti-microbial activity against a lot of disease causing food born and water born pathogenic microorganisms. Green synthesis of nanoparticles was more preferred than the chemical synthesis since it is involved in the reduction of metal using hazardous chemicals.
The rapid biological synthesis of silver nanoparticles using aqueous leaves broth of Artemisia vulgaris L provide eco-friendly, simple and efficient route for synthesis of nanoparticles. Hence, silver nanoparticles hold great promise for reaching the goal of anti microbial activity and have attracted wide attention of researchers.
The formed silver nanoparticles were determined by the colour change from yellowish green to brown. Initial studies include characterisation of prepared silver nanoparticles by UV-VIS Spectrophotometry between 200-400 nm was determined. The particle size and stability of synthesized silver nanoparticles were analysed by zeta seizer and zeta potential. The average size of silver nanoparticles was obtained at 104.5 nm. The silver nanoparticles show good stability at -18.5 mV by zeta potential. SEM analysis showed spherical shaped nanoparticles.
The microbial assay of A.vulgaris AgNPs was compared with standard Fluconazole (100 µg/ml) solution. The zone of inhibition in case of nanoparticles was comparatively greater than the Artemisia leaves broth. The lowest concentration of the inhibiting agent that completely inhibits fungal growth was examined visually by checking the fungal turbidity of the tubes. The MIC was found to be 104 dilution. Thus the silver nanoparticles showed promising effect on treatment of fungal infection.
5. CONFLICT OF INTEREST:
No conflict of interest.
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Received on 12.04.2019 Modified on 05.05.2019
Accepted on 31.05.2019 © RJPT All right reserved
Research J. Pharm. and Tech. 2019; 12(10):4822-4826.
DOI: 10.5958/0974-360X.2019.00834.5