www.ietdl.org Published in IET Nanobiotechnology Received on 2nd March 2013 Revised on 18th July 2013 Accepted on 24th July 2013 doi: 10.1049/iet-nbt.2013.0015
ISSN 1751-8741
Lawsonia inermis-mediated synthesis of silver nanoparticles: activity against human pathogenic fungi and bacteria with special reference to formulation of an antimicrobial nanogel Arpita Gupta1, Shital R. Bonde1, Swapnil Gaikwad1, Avinash Ingle1, Aniket K. Gade2, Mahendra Rai1,3 1
Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati 444 602, Maharashtra, India Department of Biology, Utah State University, Logan, UT 84322-5305, USA 3 Laboratório de Química Biológica, Instituto de Química – Unicamp Cidade Universitária ‘Zefferino Vaz’, Barão Geraldo-CEP 13083-970, Caixa Postal 6150 Campinas, SP, Brazil E-mail: [email protected] 2
Abstract: Lawsonia inermis mediated synthesis of silver nanoparticles (Ag-NPs) and its efficacy against Candida albicans, Microsporum canis, Propioniabacterium acne and Trichophyton mentagrophytes is reported. A two-step mechanism has been proposed for bioreduction and formation of an intermediate complex leading to the synthesis of capped nanoparticles was developed. In addition, antimicrobial gel for M. canis and T. mentagrophytes was also formulated. Ag-NPs were synthesized by challenging the leaft extract of L. inermis with 1 mM AgNO3. The Ag-NPs were characterized by Ultraviolet-Visible (UV–Vis) spectrophotometer and Fourier transform infrared spectroscopy (FTIR). Transmission electron microscopy (TEM), nanoparticle tracking and analysis sytem (NTA) and zeta potential was measured to detect the size of Ag-NPs. The antimicrobial activity of Ag-NPs was evaluated by disc diffusion method against the test organisms. Thus these Ag-NPs may prove as a better candidate drug due to their biogenic nature. Moreover, Ag-NPs may be an answer to the drug-resistant microorganisms.
1
Introduction
Medicinal plants have been used by human beings to combat diseases, from the dawn of civilisation [1]. Herbal medicines are in great demand in the developed as well as developing countries for primary healthcare because of their wide biological activities, higher safety margins and lesser costs [2]. L. inermis (Henna) is a dwarf shrub and is well-known worldwide for its cosmetic use because of unique active principles that are present in the leaves. It has a diverse range of bioactive molecules, making them rich sources of different types of medicines. Henna is believed to give healthy and beautiful hair and also reduce body temperature in cases of high fever. L. inermis is grown in dry tropical and subtropical zones, including North Africa, India, Sri Lanka and the Middle East [3]. This plant showed strong fungicidal as well as anti-inflammatory, analgesic, antibacterial, virucidal, anti-parasitic and anti-cancer properties [4]. Leaves were examined for their antimicrobial potential in broth extracts in different concentrations and it showed antibacterial activity against Escherichia coli [5]. The chemical constituents of L. inermis includes naphthalene derivatives, quinoids, β-sitosterol glycoside, xanthones, flavonoids, gallic acid, coumarins and lawsoniasides and lawsone, 2-hydroxy-1, 4-naphthoquinone 172 & The Institution of Engineering and Technology 2014
were responsible for Henna’s fungicidal activity [6]. Phytochemical investigations of L. inermis have shown predominantly the presence of phenolic compounds (namely, coumarins, flavonoids and naphthalene and gallic acid derivatives) which could be glycosylated [7]. Nanotechnology provides the cutting edge to engineer these properties of nanomaterials for need-based application in bioscience such as biomedicine, biosensor, etc. [8]. This plant is commonly known as Henna or Mehendi and abundantly available in tropical and subtropical areas. Ancient history of India describes its diverse uses and also plays appreciable role in Ayurvedic or natural herbal medicines [9]. Skin infections caused by fungi, such as Trichophyton and Candida species, have become more common in recent years [10]. In particular, fungal infections are more frequent in patients who are immunocompromised because of cancer chemotherapy or human immunodeficiency virus (HIV) infections [11]. Reports suggest that silver nanoparticles (Ag-NPs) can be effectively used against multi-drug-resistant bacteria [12] because of their small size and relatively large surface area in comparison with their volume makes them easy to interact with substances and increases their antibacterial efficacy. Ag-NPs can be the new generation of antimicrobials [13] and it can be used in many IET Nanobiotechnol., 2014, Vol. 8, Iss. 3, pp. 172–178 doi: 10.1049/iet-nbt.2013.0015
www.ietdl.org antimicrobial preparations. Gade et al. [14] also reported the antibacterial activity of Ag-NPs synthesised by fungi. Duran et al. [15] successfully developed Ag-NPs impregnated wound dressings and textile fabrics which can be used for burnt patients. Ag-NPs are also used for the preparation of surgical masks [16]. In the present study, we report L. inermis mediated synthesis of Ag-NPs and its efficacy against Candida albicans, Microsporum canis, Propionabacterium acnes and Trichophyton mentagrophytes. On the basis of the information obtained, a two-step mechanism was proposed for bioreduction and formation of an intermediate complex leading to the formation of capped nanoparticles and formulation of gel against M. canis and T. mentagrophytes.
2 2.1
Material and methods
2.2 Preparation of the extract of L. inermis and synthesis of Ag-NPs Extract of L. inermis (Henna) was prepared at room temperature by crushing the prewashed 20 gm of leaves in 100 ml of sterilised distilled water with the extraction of about 56 min. Later, mixture was filtered through Whatman filter paper no. 21. The filtered extract of L. inermis was challenged with silver nitrate solution (1 mM AgNO3).
3.1
Detection and characterisation of Ag-NPs Visual observation
The primary detection was carried out by visual observation. The change in colour of solution from light-yellow to dark brown indicated the formation of Ag-NPs and control (without treatment with 1 mM AgNO3) remained light-yellow after keeping in bright sunlight for 10 min. 3.2
Ultraviolet–visible (UV–vis) spectroscopy
Preliminarily, Ag-NPs were detected by UV–vis spectrophotometer (Shimadzu UV-1700, Japan) at the range of wavelength of 250–800 nm. 3.3
3.5
Zeta potential
Zeta potential of the synthesised Ag-NPs was analysed to determine the charges present on the particles and its stability at pH7. This analysis also provided an idea about the size of nanoparticles produced.
Plant
Lawsonia inermis (Henna) plant was collected from a local nursery, Amravati, Maharashtra, India.
3
laser-based light-scattering system, in which particles suspended in the liquid medium were injected into the LM viewing unit and viewed in the closed proximity to the optical element. Brownian motion of Ag-NPs present within the path of the laser beam were observed via a dedicated non-microscope optical instrument (LM-20, NanoSight Pvt. Ltd., UK) having charge-coupled device (CCD) camera. The motions of the particles in the field of view (approximately 100 × 100 μm) were recorded (at 30 fps) and the subsequent sample images and video were captured.
Fourier transform infrared spectroscopy (FTIR)
FTIR reveals the biomolecules responsible for the reduction of silver ions and stabilisation of Ag-NPs in the solution. The FTIR (Perkin–Elmer FTIR-1600, USA) analysis in the range of 500–4000 cm−1 was performed to determine the presence of capping agent and role of molecules involved in the synthesis of Ag-NPs. For sample preparation, 300 µl of colloidal Ag-NPs solution was mixed with 10 mg potassium bromide in clean crucible, until it becomes a fine powder. The sample was prepared and oven dried to remove the traces of moisture. 3.4 Nanoparticle tracking and analysis system (NTA) The Ag-NPs synthesised by the extract were characterised by LM-20 to find out the average size of the particles. LM 20 is a IET Nanobiotechnol., 2014, Vol. 8, Iss. 3, pp. 172–178 doi: 10.1049/iet-nbt.2013.0015
3.6
Transmission electron microscopy (TEM)
TEM analysis is useful to determine the size and topology of nanoparticles synthesised by extract of L. inermis. The Ag-NPs were characterised by TEM (Philips, CM 12), on conventional carbon-coated copper grids (400 meshes, Plano Gmbh, Germany); it was cleaned using plasma treatment under oxygen for 45 s. A 5 µl of sample was then placed on the grid and dried at room temperature for 1 h. The samples were inspected by operating at 120 kV. 3.7
X-ray diffraction (XRD)
XRD spectra was recorded on a PAN analytical X PRT PRO, D-8, Advanced Brucker instrument (Netherlands) and depicted number of Bragg reflections indexed on the basis of the face-centred cubic (FCC) structure of metallic silver. The average size of Ag-NPs was calculated by using Scherrer’s equation (D = 0.9λ/β cos θ), where, D is the average grain size of crystallite, λ is the incident wavelength, θ is the Bragg angle, β is the diffracted full width at half maximum (in radians), respectively [17].
4 Assessment of antimicrobial activity of Ag-NPs 4.1
Test organisms
The pure culture of a bacterium P. acnes (MTCC-1951) and two human pathogenic fungi, namely, T. mentagrophytes (MTCC-7687) and M. canis (MTCC-3270) were procured from culture collection centre, Institute of Microbial Technology (IMTECH), Chandigarh and yeast C. albicans (NCIM-3100) was procured from NCIM, Pune, India. 4.2
In vitro evaluation of antimicrobial activity
The antibacterial and antifungal activity of Ag-NPs was evaluated against P. acnes, T. mentagrophytes, M. canis and C. albicans, respectively. The standard antibiotic discs containing Levofloxacin and Fluconazole were purchased from Hi-Media Laboratories Pvt. Ltd., Mumbai, India. The overnight grown culture of P. acnes having 105–106 colony forming units per ml (CFU/ml) was used to assess the activity of Ag-NPs. The activities of Ag-NPs were evaluated singly and in combination with the antibiotics by impregnating 20 μl of Ag-NPs solution in the respective antibiotic discs. Similar experiments were carried out with 173
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www.ietdl.org Ag-NPs alone (disc containing only Ag-NPs) and both the positive (aqueous silver ions 1 mM) and negative (aqueous plant extract) control. A disc diffusion assay was performed on Muller–Hinton agar plates for P. acnes. A single colony of P. acnes was grown overnight in Muller–Hinton broth on a rotary shaker (100 rpm) at 37°C. The inocula (100 µl) were then spread onto the agar plates and thenstandard and prepared discs containing Ag-NPs were placed onto agar surface. After incubation at 37°C for 24 h, the zones of inhibition were measured. Similarly, potato dextrose agar and sabouraud dextrose agar plates were inoculated with the spore suspension (20 μl) of the test fungi (T. mentagrophytes, M. canis and C. albicans). The discs containing Ag-NPs were placed onto the agar surface and incubated at 25°C for 24 h followed by measurement of the zones of inhibition. The assays were performed in triplicate. 4.3 Determination of minimum inhibitory concentration (MIC) of silver nanoparticles MIC was determined by broth dilution method. For determination of MIC, the inoculum from the broth containing 1 × 108 CFU/ml was added to experimental tubes accordingly, so final cell number turn out to be 1 × 106. Now to these 10 tubes, Ag-NP’s concentration used were as follows 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 µg/ml. According to this concentration, the stock solution was added to the sets of test tubes containing test organisms, respectively. For the visual observation, the test tubes were incubated for 24 h at 37°C for bacteria and 25°C for fungus, respectively.
5
Formulation of the Ag-NP gel
Of about 80 ml Lukewarm distilled water was taken in a flask, then all the ingredients including AGNPs powder 20 mg was added and volume was makeup to 100 ml. To this mixture, 10 gm (10%) carbapol (gelling agent) was added. In order to homogenise the gel and Ag-NPs magnetic stirrer was used. This gel was tested for its antimicrobial activity against the test organism in vitro.
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Fig. 1 Synthesis of Ag-NPs a L. inermis leaves extract (control) b Extract after treatment of 1 mM AgNO3
Based on the study of the bioactive compounds secreted by L. inermis, it was reported that the plant secretes mainly lawsone, flavonoids, tannins, alkaloids, terpenoids, quinones, coumarins, xanthones and carbohydrates as the major components [21]. Lawsone is a major component of Heena, which is also known as hennotanic acid. Chemically it is 2-hydroxy-1,4 Napthoquinone. FTIR spectrum of plant extract shown in Fig. 3a (control) demonstrated prominent band at 3370, 2950, 2356 1625, 1529, 1262, 1111 and 869. The aromatic C = C stretching frequency appeared at 1625 cm−1−. The peak at 3370 could be assigned to phenolic –OH groups (lawsone) [22], which was not prominent in the experimental spectra. This suggests that the lawsone was detected in control spectrum (Fig. 3a), which was involved in the reduction of silver ions to Ag-NPs, Therefore the lawsone peak was not prominent in the experimental spectra (Fig. 3b). Lawsone may have ability to reduce silver ions possibly by the formation of an intermediate complex, where the electron density obtains shifted towards the silver
Results and discussion
The extract of the Henna was obtained by grinding method. Further, extract thus obtained was treated with AgNO3 (1 mM). It was observed that the colour of the solution changes light-yellow to dark-brown (Fig. 1) after 24 h of the reaction, indicating formation of Ag-NPs. This colour change provided an evidence of the reduction of silver ions into Ag-NPs. The change in colour was because of the surface plasma vibrations [18, 19]. The synthesised Ag-NPs were detected by using UV–vis spectrophotometer. Henna extract treated with AgNO3 (1 mM) showed the peak at about 445 nm (Fig. 2). FTIR measurements were carried out to identify the possible biomolecules responsible for the reduction of the Ag+ ions and capping of the bioreduced Ag-NPs. FTIR spectrum (Fig. 3b) revealed that Ag-NPs (experimental) from Henna leaves extract gave prominent bands at 2770, 2362, 1626, 1524, 1383 and 820 cm−1. The band at 1524 cm−1 may be for amide II and the band at 1383 cm−1 could be assigned to C–N stretching vibrations of aliphatic and aromatic amines [20]. 174 & The Institution of Engineering and Technology 2014
Fig. 2 UV–vis spectroscopy of the synthesised Ag-NP showing absorbance at 445 nm IET Nanobiotechnol., 2014, Vol. 8, Iss. 3, pp. 172–178 doi: 10.1049/iet-nbt.2013.0015
www.ietdl.org that polyphenols and flavonoids were responsible for synthesis of nanoparticles. Kora et al. [26] also reported, synthetic and natural polymers such as poly(ethylene glycol), poly-(N-vinyl-2-pyrrolidone), starch, heparin, poly-cationic chitosan (1–4-linked 2-amino-2-deoxyb-D-glucose), sodium alginate (a polysaccharide gum derived from the cell-walls of brown algae) and gum acacia as a reducing and stabilising agents for the biosynthesis of gold and Ag-NPs. Gade et al. [27] also reported the ability of quercetin a quinone compound in the reduction of silver ions to form Ag-NPs. Thus, it can be hypothesised that lawsone acts as a reducing and capping agent (along with glycosides) for the synthesis of Ag-NPs. On the basis of the information obtained in this work, two-step mechanism has been proposed: (i) bioreduction and (ii) formation of an intermediate resonance complex leading to the formation of capped nanoparticles (Fig. 4). Total concentration of Ag-NPs synthesised by L. inermis was found to be 7.6 particles/frame (i.e. 4.25 × 108 Fig. 3 FTIR spectroscopy of the a Control (plant extract) b Extract treated with 1 mM AgNO3 (Ag-NPs)
ions because of its hydroxyl groups (–OH). The leaf extract also showed the presence of alkaloids and glycosides. Therefore these heterocyclic compounds are assumed to act as capping agents for nanoparticles. In case of phytosynthesis, Iravani [23] has reviewed that phytochemicals like polyphenols, glutathiones, metallothioneins and ascorbates possibly were responsible for synthesis of nanoparticles in which metallothioneins have the capability to attach with xenobiotic and physiological heavy metals through the thiol group of their cysteine residues. Also Kasthuri et al. [24] reported the capacity of apiin compound having secondary hydroxyl and carbonyl groups derived from Henna leaves, to produce anisotropic gold and quasi-spherical Ag-NPs by bioreduction of metal salt. In other study, Begum et al. [25] reported production of stable gold and Ag-NPs by using black tea leaf extracts. Results of this study have confirmed
Fig. 5 Nanoparticle tracking analysis NTA (NanoSight-LM 20) histogram showing particle size distribution and the average size of Ag-NPs (30 nm)
Fig. 4 Mechanism for formation of Ag-NPs IET Nanobiotechnol., 2014, Vol. 8, Iss. 3, pp. 172–178 doi: 10.1049/iet-nbt.2013.0015
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Fig. 6 Zeta potential graph of the Ag-NPs showing zeta potential value − 32.8 mV at pH7
Fig. 7 XRD graph of Ag-NPs
particles/ml). The size of Ag-NPs analysed by an NTA was in the range of 6–72 nm. Distributions of particle size/ concentration of Ag-NPs have been shown in Fig. 5 and the particle populations of Ag-NPs using NanoSight LM-20 were studied. The average size of the Ag-NPs from the NanoSight (LM20) analysis was found to be 30 nm (Fig. 5). To see whether the synthesised Ag-NPs are stable or not the zeta potential of these Ag-NPs was measured. Fig. 6 revealed that zeta potential (mV) of the synthesised Ag-NPs was − 32.8 at pH7, which indicates that the nanoparticles were stable. The XRD patterns obtained for the Ag-NPs synthesised using Henna bark extract is shown in Fig. 7. The presence of intense peaks of Ag-NPs corresponding to the 111, 200, 220 and 311, which was indexed as crystalline silver FCC phase [28]. The direct electron microscopic visualisation allows measuring the size and shape of the Ag-NPs formed. Typical bright-field TEM image of the synthesised Ag-NPs was showed in Fig. 8. TEM micrograph showed the presence of polydispersed spherical nanoparticles having the size range of 5–45 nm. The in vitro antibacterial activity of Ag-NPs produced from L. inermis leaves extract and the combination of Ag-NPs and antibiotics were evaluated against bacterium P. acnes and three fungal strain M. canis, T. mentagrophytes and C. albicans. The antibiotic used for detection of antibacterial and antifungal activity was Levofloxacin and Fluconazole, respectively. Synthesised Ag-NPs showed significant activity against all the four test organisms (P. acnes, M. canis, T. mentagrophytes and C. albicans) individually and in combination with antibiotics showing synergistic effect, whereas AgNO3 and aqueous plant extract did not show significant activity against these test organisms (Fig. 9). L. inermis mediated synthesis of Ag-NPs showed maximum activity against P. acnes as compared with M. canis, T. mentagrophytes and C. albicans. The formulated nano gel was evaluated for its antimicrobial activity against all the four test organisms. T. mentagrophytes and M. canis demonstrated the maximum zone of inhibition than P. acnes and C. albicans
Fig. 8 TEM micrograph image showing spherical and polydispers Ag-NPs of synthesised Ag-NPs 176 & The Institution of Engineering and Technology 2014
IET Nanobiotechnol., 2014, Vol. 8, Iss. 3, pp. 172–178 doi: 10.1049/iet-nbt.2013.0015
www.ietdl.org creams in combination with the commercially available antibiotics.
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Fig. 9 Antibacterial and antifungal activity of Ag-NPs against P. acnes and three fungal strains M. canis, T. mentagrophytes and C. albicans
Rapid biosynthesis of Ag-NPs is possible by using L. inermis. Antimicrobial potential of Ag-NP in combination with antibiotics showed the synergistic effect against four different test organisms – P. acnes, C. albicans, T. mentagrophytes and M. canis. In addition, it was noted that the Ag-NP enhanced the activity of the antibiotics significantly. It can be concluded from the above discussion that biologically synthesised Ag-NPs significantly inhibited the growth of the test organisms. The recent approach for the effective, eco-friendly and economical management of microbial disease is, thus deserve urgent attention. The nanogel that has been formulated also showed the effective results against the test organism. Hence, it can be concluded that the skin infection causing microorganisms could be treated with the Ag-NP in the formulations like the gels and creams.
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Fig. 10 Antimicrobial activity of formulated nano gel against a T. mentagrophytes b M. canis[a – control (gel without Ag-NPs) and b – nano gel (gel with Ag-NPs) (zone of inhibition – 16 mm)]
when treated with formulated gel (Fig. 10). Similarly, biosynthesised Ag-NPs also screened against test organisms (P. acnes, M. canis, T. mentagrophytes and C. albicans) to evaluate MIC. P. acnes was found to be most sensitive to L. inermis leaves extract mediated synthesised Ag-NPs leading to its growth inhibition at concentration of 8 µg/per ml. In case of other microorganisms, Ag-NPs inhibits the growth of M. canis, T. mentagrophytes and C. albicans at concentration of 14, 18 and 12 µg per/ml, respectively.
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Future prospects
The fungal infections caused by various species of Trichophyton and Microsporum, are increasing with fast pace. The bacterium P. acnes causes the serious skin infection in teenagers. P. acnes is able to produce the enzyme that degrades the skin. C. albicans, a causal agent of opportunistic oral and genital infections in humans lives in 80% of the human population without causing harmful effects; although overgrowth of the fungus results in candidiasis. Candidiasis is often observed in immunocompromised individuals such as HIV-infected patients. These severe skin diseases can be treated with biosynthesised Ag-NPs, and its formulations as gel or IET Nanobiotechnol., 2014, Vol. 8, Iss. 3, pp. 172–178 doi: 10.1049/iet-nbt.2013.0015
Conclusion
References
1 Bandyopadhyay, U., Biswas, K., Chattopadhyay, I., Banerjee, R.K.: ‘Biological activities and medicinal properties of neem (Azadirachta indica)’, Curr. Sci., 2002, 82, (11), pp. 1336–1345 2 Cragg, G.M., Newman, D.J., Sander, K.M.: ‘Natural products in drug discovery and development’, J. Nat. Prod., 1997, 60, pp. 52–60 3 Chung, W.H., Chang, Y.C., Yang, L.J., Hung, S.I., Wong, W.R., Lin, J.Y.: ‘Clinicopathologic features of skin reactions to temporary tattoos and analysis of possible causes’, Arch. Dermatol., 2002, 138, pp. 88–92 4 Habbal, O.A., Al-Jabri, A.A., El-Hag, A.G.: ‘Antimicrobial properties of Lawsonia inermis (henna): a review’, Aust. J. Med. Herbalism, 2007, 19, pp. 114–125 5 Abulyazid, I., Elsayed, M.E., Mahdy, B., Ragaa, M., Ahmed, M.: ‘Biochemical study for the effect of henna (Lawsonia inermis) on Escherichia coli‘, Arab. J. Chem., 2013, 6, (3), pp. 265–273 6 Ahmadian, S., Fakhree, M.A.: ‘Henna (Lawsonia inermis) might be used to prevent mycotic infection’, Med. Hypotheses, 2009, 73, pp. 629–630 7 Siddiqui, B.S., Kardar, M.N., Ali, S.T., Khan, S.: ‘Two new and a known compound from Lawsonia inermis’, Helv. Chim. Acta, 2003, 86, (6), pp. 2164–2169 8 Thevenot, D.R., Toth, K., Durst, R.A., Wilson, G.S.: ‘Electrochemical biosensors: recommended definitions and classification’, Biosens. Bioelectron., 2001, 16, pp. 121–131 9 Lavhate, M.S., Mishra, S.H.: ‘A review: nutritional and therapeutic potential of Ailanthus excelsa‘, Pharmacognosy Rev., 2007, 1, (1), pp. 105–113 10 Woodfolk, J.A.: ‘Allergy and dermatophytes’, Clin. Microbiol. Rev., 2005, 18, pp. 30–43 11 Mirmirani, P., Hessol, N.A., Maurer, T.A., Berger, T.G., Nguyen, P., Khalsa, A.: ‘Prevalence and predictors of skin disease in the women’s interagency HIV study (WIHS)’, J. Am. Acad. Dermatol., 2001, 44, pp. 785–788 12 Ingle, A., Gade, A., Pierrat, S., Sonnichsen, C., Rai, M.: ‘Mycosynthesis of silver nanoparticles using the fungus Fusarium acuminatum and its activity against some human pathogenic bacteria’, Curr. Nanosci., 2008, 4, pp. 141–144 13 Rai, M., Yadav, A., Gade, A.: ‘Silver nanoparticles as a new generation of antimicrobials’, Biotechnol. Adv., 2009, 27, (1), pp. 76–83 14 Gade, A.K., Bonde, P., Ingle, A.P., Marcato, P.D., Duran, N., Rai, M.K.: ‘Exploitation of Aspergillus niger for synthesis of silver nanoparticles’, J. Biobased Mater. Bioenergy, 2008, 2, pp. 243–247 15 Duran, N., Marcarto, P.D., De Souza, G.I.H., Alves, O.L., Esposito, E.: ‘Antibacterial effect of silver nanoparticles produced by fungal process on textile fabrics and their effluent treatment’, J. Biomed. Nanotechnol., 2007, 3, pp. 203–208 16 Li, Y., Leung, P., Yao, L., Song, Q.W., Newton, E.: ‘Antimicrobial effects of surgical masks coated with nanoparticles’, J. Hosp. Infection, 2006, 62, pp. 58–63 177
& The Institution of Engineering and Technology 2014
www.ietdl.org 17 Montes-Burgos, D., Hole, W.P., Smith, J., Lynch, I., Dawson, K.: ‘Characterisation of nanoparticle size and state prior to nanotoxicological studies’, J. Nanoparticle Res., 2010, 12, pp. 47–53 18 Bonde, S.R.: ‘A biogenic approach for green synthesis of silver nanoparticles using extract of Foeniculum vulgare and its activity against Staphylococcus aureus and Escherichia coli’, Nusantara Biosci., 2011, 3, (2), pp. 59–63 19 Bonde, S.R., Rathod, D.P., Ingle, A.P., Ade, R.B., Gade, A.K., Rai, M. K.: ‘Murraya koenigii mediated synthesis of silver nanoparticles and its activity against three human pathogenic bacteria’, Nanoscience Meth., 2012, 1, pp. 25–36 20 Sun, L., Simmons, B.A., Singh, S.: ‘Understanding tissue specific compositions of bioenergy feedstocks through hyperspectral Raman imaging’, Biotechnol. Bioeng., 2011, 108, (2), pp. 286–95 21 Chaudhary, G., Golyal, S., Poonia, P.: ‘Lawsonia inermis Linnaeus: a phytopharmacological review’, Int. J. Pharm. Sci. Drug Res., 2010, 2, (2), pp. 91–98 22 Rajendran, R., Hemalatha, S., Akasakalai, K., MadhuKrishna, C.H., Sohil, B., Sundaram, M.R.: ‘Hepatoprotective activity of Mimosa pudica leaves against carbontetrachloride induced toxicity’, J. Nat. Prod., 2010, 2, pp. 116–122
178 & The Institution of Engineering and Technology 2014
23 Iravani, S.: ‘Green synthesis of metal nanoparticles using plants’, Green Chem., 2011, 13, pp. 2638–2650 24 Kasthuri, J., Veerapandian, S., Rajendiran, N.: ‘Biological synthesis of silver and gold nanoparticles using apiin as reducing agent’, Colloids Surf., B, 2009, 68, pp. 55–60 25 Begum, N.A., Mondal, S., Basu, S., Laskar, R.A., Mandal, D.: ‘Biogenic synthesis of Au and Ag nanoparticles using aqueous solutions of Black Tea leaf extracts’, Colloids Surf., B, 2009, 71, pp. 113–118 26 Kora, A.J., Sashidhar, R.B., Arunachalam, J.: ‘Gum kondagogu (Cochlospermum gossypium): a template for the green synthesis and stabilization of silver nanoparticles with antibacterial application’, Carbohydrate Polym., 2010, 82, (3), pp. 670–679 27 Gade, A.K., Gaikwad, S.C., Tiwari, V., Yadav, A., Ingle, A.P., Rai, M.K.: ‘Biofabrication of silver nanoparticles by Opuntia ficus-indica: in vitro antibacterial activity and study of the mechanism involved in the syntheses’, Curr. Nanosci., 2010, 6, pp. 370–375 28 Gade, A., Rai, M., Kulkarni, S.: ‘Phoma sorghina, a phytopathogen mediated synthesis of unique silver rod’, Int. J. Green Nanotechnol., 2011, 3, (3), pp. 153–159
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Lawsonia inermis-mediated synthesis of silver nanoparticles: activity against human pathogenic fungi and bacteria with special reference to formulation of an antimicrobial nanogel Arpita Gupta1, Shital R. Bonde1, Swapnil Gaikwad1, Avinash Ingle1, Aniket K. Gade2, Mahendra Rai1,3 1
Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati 444 602, Maharashtra, India Department of Biology, Utah State University, Logan, UT 84322-5305, USA 3 Laboratório de Química Biológica, Instituto de Química – Unicamp Cidade Universitária ‘Zefferino Vaz’, Barão Geraldo-CEP 13083-970, Caixa Postal 6150 Campinas, SP, Brazil E-mail: [email protected] 2
Abstract: Lawsonia inermis mediated synthesis of silver nanoparticles (Ag-NPs) and its efficacy against Candida albicans, Microsporum canis, Propioniabacterium acne and Trichophyton mentagrophytes is reported. A two-step mechanism has been proposed for bioreduction and formation of an intermediate complex leading to the synthesis of capped nanoparticles was developed. In addition, antimicrobial gel for M. canis and T. mentagrophytes was also formulated. Ag-NPs were synthesized by challenging the leaft extract of L. inermis with 1 mM AgNO3. The Ag-NPs were characterized by Ultraviolet-Visible (UV–Vis) spectrophotometer and Fourier transform infrared spectroscopy (FTIR). Transmission electron microscopy (TEM), nanoparticle tracking and analysis sytem (NTA) and zeta potential was measured to detect the size of Ag-NPs. The antimicrobial activity of Ag-NPs was evaluated by disc diffusion method against the test organisms. Thus these Ag-NPs may prove as a better candidate drug due to their biogenic nature. Moreover, Ag-NPs may be an answer to the drug-resistant microorganisms.
1
Introduction
Medicinal plants have been used by human beings to combat diseases, from the dawn of civilisation [1]. Herbal medicines are in great demand in the developed as well as developing countries for primary healthcare because of their wide biological activities, higher safety margins and lesser costs [2]. L. inermis (Henna) is a dwarf shrub and is well-known worldwide for its cosmetic use because of unique active principles that are present in the leaves. It has a diverse range of bioactive molecules, making them rich sources of different types of medicines. Henna is believed to give healthy and beautiful hair and also reduce body temperature in cases of high fever. L. inermis is grown in dry tropical and subtropical zones, including North Africa, India, Sri Lanka and the Middle East [3]. This plant showed strong fungicidal as well as anti-inflammatory, analgesic, antibacterial, virucidal, anti-parasitic and anti-cancer properties [4]. Leaves were examined for their antimicrobial potential in broth extracts in different concentrations and it showed antibacterial activity against Escherichia coli [5]. The chemical constituents of L. inermis includes naphthalene derivatives, quinoids, β-sitosterol glycoside, xanthones, flavonoids, gallic acid, coumarins and lawsoniasides and lawsone, 2-hydroxy-1, 4-naphthoquinone 172 & The Institution of Engineering and Technology 2014
were responsible for Henna’s fungicidal activity [6]. Phytochemical investigations of L. inermis have shown predominantly the presence of phenolic compounds (namely, coumarins, flavonoids and naphthalene and gallic acid derivatives) which could be glycosylated [7]. Nanotechnology provides the cutting edge to engineer these properties of nanomaterials for need-based application in bioscience such as biomedicine, biosensor, etc. [8]. This plant is commonly known as Henna or Mehendi and abundantly available in tropical and subtropical areas. Ancient history of India describes its diverse uses and also plays appreciable role in Ayurvedic or natural herbal medicines [9]. Skin infections caused by fungi, such as Trichophyton and Candida species, have become more common in recent years [10]. In particular, fungal infections are more frequent in patients who are immunocompromised because of cancer chemotherapy or human immunodeficiency virus (HIV) infections [11]. Reports suggest that silver nanoparticles (Ag-NPs) can be effectively used against multi-drug-resistant bacteria [12] because of their small size and relatively large surface area in comparison with their volume makes them easy to interact with substances and increases their antibacterial efficacy. Ag-NPs can be the new generation of antimicrobials [13] and it can be used in many IET Nanobiotechnol., 2014, Vol. 8, Iss. 3, pp. 172–178 doi: 10.1049/iet-nbt.2013.0015
www.ietdl.org antimicrobial preparations. Gade et al. [14] also reported the antibacterial activity of Ag-NPs synthesised by fungi. Duran et al. [15] successfully developed Ag-NPs impregnated wound dressings and textile fabrics which can be used for burnt patients. Ag-NPs are also used for the preparation of surgical masks [16]. In the present study, we report L. inermis mediated synthesis of Ag-NPs and its efficacy against Candida albicans, Microsporum canis, Propionabacterium acnes and Trichophyton mentagrophytes. On the basis of the information obtained, a two-step mechanism was proposed for bioreduction and formation of an intermediate complex leading to the formation of capped nanoparticles and formulation of gel against M. canis and T. mentagrophytes.
2 2.1
Material and methods
2.2 Preparation of the extract of L. inermis and synthesis of Ag-NPs Extract of L. inermis (Henna) was prepared at room temperature by crushing the prewashed 20 gm of leaves in 100 ml of sterilised distilled water with the extraction of about 56 min. Later, mixture was filtered through Whatman filter paper no. 21. The filtered extract of L. inermis was challenged with silver nitrate solution (1 mM AgNO3).
3.1
Detection and characterisation of Ag-NPs Visual observation
The primary detection was carried out by visual observation. The change in colour of solution from light-yellow to dark brown indicated the formation of Ag-NPs and control (without treatment with 1 mM AgNO3) remained light-yellow after keeping in bright sunlight for 10 min. 3.2
Ultraviolet–visible (UV–vis) spectroscopy
Preliminarily, Ag-NPs were detected by UV–vis spectrophotometer (Shimadzu UV-1700, Japan) at the range of wavelength of 250–800 nm. 3.3
3.5
Zeta potential
Zeta potential of the synthesised Ag-NPs was analysed to determine the charges present on the particles and its stability at pH7. This analysis also provided an idea about the size of nanoparticles produced.
Plant
Lawsonia inermis (Henna) plant was collected from a local nursery, Amravati, Maharashtra, India.
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laser-based light-scattering system, in which particles suspended in the liquid medium were injected into the LM viewing unit and viewed in the closed proximity to the optical element. Brownian motion of Ag-NPs present within the path of the laser beam were observed via a dedicated non-microscope optical instrument (LM-20, NanoSight Pvt. Ltd., UK) having charge-coupled device (CCD) camera. The motions of the particles in the field of view (approximately 100 × 100 μm) were recorded (at 30 fps) and the subsequent sample images and video were captured.
Fourier transform infrared spectroscopy (FTIR)
FTIR reveals the biomolecules responsible for the reduction of silver ions and stabilisation of Ag-NPs in the solution. The FTIR (Perkin–Elmer FTIR-1600, USA) analysis in the range of 500–4000 cm−1 was performed to determine the presence of capping agent and role of molecules involved in the synthesis of Ag-NPs. For sample preparation, 300 µl of colloidal Ag-NPs solution was mixed with 10 mg potassium bromide in clean crucible, until it becomes a fine powder. The sample was prepared and oven dried to remove the traces of moisture. 3.4 Nanoparticle tracking and analysis system (NTA) The Ag-NPs synthesised by the extract were characterised by LM-20 to find out the average size of the particles. LM 20 is a IET Nanobiotechnol., 2014, Vol. 8, Iss. 3, pp. 172–178 doi: 10.1049/iet-nbt.2013.0015
3.6
Transmission electron microscopy (TEM)
TEM analysis is useful to determine the size and topology of nanoparticles synthesised by extract of L. inermis. The Ag-NPs were characterised by TEM (Philips, CM 12), on conventional carbon-coated copper grids (400 meshes, Plano Gmbh, Germany); it was cleaned using plasma treatment under oxygen for 45 s. A 5 µl of sample was then placed on the grid and dried at room temperature for 1 h. The samples were inspected by operating at 120 kV. 3.7
X-ray diffraction (XRD)
XRD spectra was recorded on a PAN analytical X PRT PRO, D-8, Advanced Brucker instrument (Netherlands) and depicted number of Bragg reflections indexed on the basis of the face-centred cubic (FCC) structure of metallic silver. The average size of Ag-NPs was calculated by using Scherrer’s equation (D = 0.9λ/β cos θ), where, D is the average grain size of crystallite, λ is the incident wavelength, θ is the Bragg angle, β is the diffracted full width at half maximum (in radians), respectively [17].
4 Assessment of antimicrobial activity of Ag-NPs 4.1
Test organisms
The pure culture of a bacterium P. acnes (MTCC-1951) and two human pathogenic fungi, namely, T. mentagrophytes (MTCC-7687) and M. canis (MTCC-3270) were procured from culture collection centre, Institute of Microbial Technology (IMTECH), Chandigarh and yeast C. albicans (NCIM-3100) was procured from NCIM, Pune, India. 4.2
In vitro evaluation of antimicrobial activity
The antibacterial and antifungal activity of Ag-NPs was evaluated against P. acnes, T. mentagrophytes, M. canis and C. albicans, respectively. The standard antibiotic discs containing Levofloxacin and Fluconazole were purchased from Hi-Media Laboratories Pvt. Ltd., Mumbai, India. The overnight grown culture of P. acnes having 105–106 colony forming units per ml (CFU/ml) was used to assess the activity of Ag-NPs. The activities of Ag-NPs were evaluated singly and in combination with the antibiotics by impregnating 20 μl of Ag-NPs solution in the respective antibiotic discs. Similar experiments were carried out with 173
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www.ietdl.org Ag-NPs alone (disc containing only Ag-NPs) and both the positive (aqueous silver ions 1 mM) and negative (aqueous plant extract) control. A disc diffusion assay was performed on Muller–Hinton agar plates for P. acnes. A single colony of P. acnes was grown overnight in Muller–Hinton broth on a rotary shaker (100 rpm) at 37°C. The inocula (100 µl) were then spread onto the agar plates and thenstandard and prepared discs containing Ag-NPs were placed onto agar surface. After incubation at 37°C for 24 h, the zones of inhibition were measured. Similarly, potato dextrose agar and sabouraud dextrose agar plates were inoculated with the spore suspension (20 μl) of the test fungi (T. mentagrophytes, M. canis and C. albicans). The discs containing Ag-NPs were placed onto the agar surface and incubated at 25°C for 24 h followed by measurement of the zones of inhibition. The assays were performed in triplicate. 4.3 Determination of minimum inhibitory concentration (MIC) of silver nanoparticles MIC was determined by broth dilution method. For determination of MIC, the inoculum from the broth containing 1 × 108 CFU/ml was added to experimental tubes accordingly, so final cell number turn out to be 1 × 106. Now to these 10 tubes, Ag-NP’s concentration used were as follows 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 µg/ml. According to this concentration, the stock solution was added to the sets of test tubes containing test organisms, respectively. For the visual observation, the test tubes were incubated for 24 h at 37°C for bacteria and 25°C for fungus, respectively.
5
Formulation of the Ag-NP gel
Of about 80 ml Lukewarm distilled water was taken in a flask, then all the ingredients including AGNPs powder 20 mg was added and volume was makeup to 100 ml. To this mixture, 10 gm (10%) carbapol (gelling agent) was added. In order to homogenise the gel and Ag-NPs magnetic stirrer was used. This gel was tested for its antimicrobial activity against the test organism in vitro.
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Fig. 1 Synthesis of Ag-NPs a L. inermis leaves extract (control) b Extract after treatment of 1 mM AgNO3
Based on the study of the bioactive compounds secreted by L. inermis, it was reported that the plant secretes mainly lawsone, flavonoids, tannins, alkaloids, terpenoids, quinones, coumarins, xanthones and carbohydrates as the major components [21]. Lawsone is a major component of Heena, which is also known as hennotanic acid. Chemically it is 2-hydroxy-1,4 Napthoquinone. FTIR spectrum of plant extract shown in Fig. 3a (control) demonstrated prominent band at 3370, 2950, 2356 1625, 1529, 1262, 1111 and 869. The aromatic C = C stretching frequency appeared at 1625 cm−1−. The peak at 3370 could be assigned to phenolic –OH groups (lawsone) [22], which was not prominent in the experimental spectra. This suggests that the lawsone was detected in control spectrum (Fig. 3a), which was involved in the reduction of silver ions to Ag-NPs, Therefore the lawsone peak was not prominent in the experimental spectra (Fig. 3b). Lawsone may have ability to reduce silver ions possibly by the formation of an intermediate complex, where the electron density obtains shifted towards the silver
Results and discussion
The extract of the Henna was obtained by grinding method. Further, extract thus obtained was treated with AgNO3 (1 mM). It was observed that the colour of the solution changes light-yellow to dark-brown (Fig. 1) after 24 h of the reaction, indicating formation of Ag-NPs. This colour change provided an evidence of the reduction of silver ions into Ag-NPs. The change in colour was because of the surface plasma vibrations [18, 19]. The synthesised Ag-NPs were detected by using UV–vis spectrophotometer. Henna extract treated with AgNO3 (1 mM) showed the peak at about 445 nm (Fig. 2). FTIR measurements were carried out to identify the possible biomolecules responsible for the reduction of the Ag+ ions and capping of the bioreduced Ag-NPs. FTIR spectrum (Fig. 3b) revealed that Ag-NPs (experimental) from Henna leaves extract gave prominent bands at 2770, 2362, 1626, 1524, 1383 and 820 cm−1. The band at 1524 cm−1 may be for amide II and the band at 1383 cm−1 could be assigned to C–N stretching vibrations of aliphatic and aromatic amines [20]. 174 & The Institution of Engineering and Technology 2014
Fig. 2 UV–vis spectroscopy of the synthesised Ag-NP showing absorbance at 445 nm IET Nanobiotechnol., 2014, Vol. 8, Iss. 3, pp. 172–178 doi: 10.1049/iet-nbt.2013.0015
www.ietdl.org that polyphenols and flavonoids were responsible for synthesis of nanoparticles. Kora et al. [26] also reported, synthetic and natural polymers such as poly(ethylene glycol), poly-(N-vinyl-2-pyrrolidone), starch, heparin, poly-cationic chitosan (1–4-linked 2-amino-2-deoxyb-D-glucose), sodium alginate (a polysaccharide gum derived from the cell-walls of brown algae) and gum acacia as a reducing and stabilising agents for the biosynthesis of gold and Ag-NPs. Gade et al. [27] also reported the ability of quercetin a quinone compound in the reduction of silver ions to form Ag-NPs. Thus, it can be hypothesised that lawsone acts as a reducing and capping agent (along with glycosides) for the synthesis of Ag-NPs. On the basis of the information obtained in this work, two-step mechanism has been proposed: (i) bioreduction and (ii) formation of an intermediate resonance complex leading to the formation of capped nanoparticles (Fig. 4). Total concentration of Ag-NPs synthesised by L. inermis was found to be 7.6 particles/frame (i.e. 4.25 × 108 Fig. 3 FTIR spectroscopy of the a Control (plant extract) b Extract treated with 1 mM AgNO3 (Ag-NPs)
ions because of its hydroxyl groups (–OH). The leaf extract also showed the presence of alkaloids and glycosides. Therefore these heterocyclic compounds are assumed to act as capping agents for nanoparticles. In case of phytosynthesis, Iravani [23] has reviewed that phytochemicals like polyphenols, glutathiones, metallothioneins and ascorbates possibly were responsible for synthesis of nanoparticles in which metallothioneins have the capability to attach with xenobiotic and physiological heavy metals through the thiol group of their cysteine residues. Also Kasthuri et al. [24] reported the capacity of apiin compound having secondary hydroxyl and carbonyl groups derived from Henna leaves, to produce anisotropic gold and quasi-spherical Ag-NPs by bioreduction of metal salt. In other study, Begum et al. [25] reported production of stable gold and Ag-NPs by using black tea leaf extracts. Results of this study have confirmed
Fig. 5 Nanoparticle tracking analysis NTA (NanoSight-LM 20) histogram showing particle size distribution and the average size of Ag-NPs (30 nm)
Fig. 4 Mechanism for formation of Ag-NPs IET Nanobiotechnol., 2014, Vol. 8, Iss. 3, pp. 172–178 doi: 10.1049/iet-nbt.2013.0015
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Fig. 6 Zeta potential graph of the Ag-NPs showing zeta potential value − 32.8 mV at pH7
Fig. 7 XRD graph of Ag-NPs
particles/ml). The size of Ag-NPs analysed by an NTA was in the range of 6–72 nm. Distributions of particle size/ concentration of Ag-NPs have been shown in Fig. 5 and the particle populations of Ag-NPs using NanoSight LM-20 were studied. The average size of the Ag-NPs from the NanoSight (LM20) analysis was found to be 30 nm (Fig. 5). To see whether the synthesised Ag-NPs are stable or not the zeta potential of these Ag-NPs was measured. Fig. 6 revealed that zeta potential (mV) of the synthesised Ag-NPs was − 32.8 at pH7, which indicates that the nanoparticles were stable. The XRD patterns obtained for the Ag-NPs synthesised using Henna bark extract is shown in Fig. 7. The presence of intense peaks of Ag-NPs corresponding to the 111, 200, 220 and 311, which was indexed as crystalline silver FCC phase [28]. The direct electron microscopic visualisation allows measuring the size and shape of the Ag-NPs formed. Typical bright-field TEM image of the synthesised Ag-NPs was showed in Fig. 8. TEM micrograph showed the presence of polydispersed spherical nanoparticles having the size range of 5–45 nm. The in vitro antibacterial activity of Ag-NPs produced from L. inermis leaves extract and the combination of Ag-NPs and antibiotics were evaluated against bacterium P. acnes and three fungal strain M. canis, T. mentagrophytes and C. albicans. The antibiotic used for detection of antibacterial and antifungal activity was Levofloxacin and Fluconazole, respectively. Synthesised Ag-NPs showed significant activity against all the four test organisms (P. acnes, M. canis, T. mentagrophytes and C. albicans) individually and in combination with antibiotics showing synergistic effect, whereas AgNO3 and aqueous plant extract did not show significant activity against these test organisms (Fig. 9). L. inermis mediated synthesis of Ag-NPs showed maximum activity against P. acnes as compared with M. canis, T. mentagrophytes and C. albicans. The formulated nano gel was evaluated for its antimicrobial activity against all the four test organisms. T. mentagrophytes and M. canis demonstrated the maximum zone of inhibition than P. acnes and C. albicans
Fig. 8 TEM micrograph image showing spherical and polydispers Ag-NPs of synthesised Ag-NPs 176 & The Institution of Engineering and Technology 2014
IET Nanobiotechnol., 2014, Vol. 8, Iss. 3, pp. 172–178 doi: 10.1049/iet-nbt.2013.0015
www.ietdl.org creams in combination with the commercially available antibiotics.
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Fig. 9 Antibacterial and antifungal activity of Ag-NPs against P. acnes and three fungal strains M. canis, T. mentagrophytes and C. albicans
Rapid biosynthesis of Ag-NPs is possible by using L. inermis. Antimicrobial potential of Ag-NP in combination with antibiotics showed the synergistic effect against four different test organisms – P. acnes, C. albicans, T. mentagrophytes and M. canis. In addition, it was noted that the Ag-NP enhanced the activity of the antibiotics significantly. It can be concluded from the above discussion that biologically synthesised Ag-NPs significantly inhibited the growth of the test organisms. The recent approach for the effective, eco-friendly and economical management of microbial disease is, thus deserve urgent attention. The nanogel that has been formulated also showed the effective results against the test organism. Hence, it can be concluded that the skin infection causing microorganisms could be treated with the Ag-NP in the formulations like the gels and creams.
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Fig. 10 Antimicrobial activity of formulated nano gel against a T. mentagrophytes b M. canis[a – control (gel without Ag-NPs) and b – nano gel (gel with Ag-NPs) (zone of inhibition – 16 mm)]
when treated with formulated gel (Fig. 10). Similarly, biosynthesised Ag-NPs also screened against test organisms (P. acnes, M. canis, T. mentagrophytes and C. albicans) to evaluate MIC. P. acnes was found to be most sensitive to L. inermis leaves extract mediated synthesised Ag-NPs leading to its growth inhibition at concentration of 8 µg/per ml. In case of other microorganisms, Ag-NPs inhibits the growth of M. canis, T. mentagrophytes and C. albicans at concentration of 14, 18 and 12 µg per/ml, respectively.
7
Future prospects
The fungal infections caused by various species of Trichophyton and Microsporum, are increasing with fast pace. The bacterium P. acnes causes the serious skin infection in teenagers. P. acnes is able to produce the enzyme that degrades the skin. C. albicans, a causal agent of opportunistic oral and genital infections in humans lives in 80% of the human population without causing harmful effects; although overgrowth of the fungus results in candidiasis. Candidiasis is often observed in immunocompromised individuals such as HIV-infected patients. These severe skin diseases can be treated with biosynthesised Ag-NPs, and its formulations as gel or IET Nanobiotechnol., 2014, Vol. 8, Iss. 3, pp. 172–178 doi: 10.1049/iet-nbt.2013.0015
Conclusion
References
1 Bandyopadhyay, U., Biswas, K., Chattopadhyay, I., Banerjee, R.K.: ‘Biological activities and medicinal properties of neem (Azadirachta indica)’, Curr. Sci., 2002, 82, (11), pp. 1336–1345 2 Cragg, G.M., Newman, D.J., Sander, K.M.: ‘Natural products in drug discovery and development’, J. Nat. Prod., 1997, 60, pp. 52–60 3 Chung, W.H., Chang, Y.C., Yang, L.J., Hung, S.I., Wong, W.R., Lin, J.Y.: ‘Clinicopathologic features of skin reactions to temporary tattoos and analysis of possible causes’, Arch. Dermatol., 2002, 138, pp. 88–92 4 Habbal, O.A., Al-Jabri, A.A., El-Hag, A.G.: ‘Antimicrobial properties of Lawsonia inermis (henna): a review’, Aust. J. Med. Herbalism, 2007, 19, pp. 114–125 5 Abulyazid, I., Elsayed, M.E., Mahdy, B., Ragaa, M., Ahmed, M.: ‘Biochemical study for the effect of henna (Lawsonia inermis) on Escherichia coli‘, Arab. J. Chem., 2013, 6, (3), pp. 265–273 6 Ahmadian, S., Fakhree, M.A.: ‘Henna (Lawsonia inermis) might be used to prevent mycotic infection’, Med. Hypotheses, 2009, 73, pp. 629–630 7 Siddiqui, B.S., Kardar, M.N., Ali, S.T., Khan, S.: ‘Two new and a known compound from Lawsonia inermis’, Helv. Chim. Acta, 2003, 86, (6), pp. 2164–2169 8 Thevenot, D.R., Toth, K., Durst, R.A., Wilson, G.S.: ‘Electrochemical biosensors: recommended definitions and classification’, Biosens. Bioelectron., 2001, 16, pp. 121–131 9 Lavhate, M.S., Mishra, S.H.: ‘A review: nutritional and therapeutic potential of Ailanthus excelsa‘, Pharmacognosy Rev., 2007, 1, (1), pp. 105–113 10 Woodfolk, J.A.: ‘Allergy and dermatophytes’, Clin. Microbiol. Rev., 2005, 18, pp. 30–43 11 Mirmirani, P., Hessol, N.A., Maurer, T.A., Berger, T.G., Nguyen, P., Khalsa, A.: ‘Prevalence and predictors of skin disease in the women’s interagency HIV study (WIHS)’, J. Am. Acad. Dermatol., 2001, 44, pp. 785–788 12 Ingle, A., Gade, A., Pierrat, S., Sonnichsen, C., Rai, M.: ‘Mycosynthesis of silver nanoparticles using the fungus Fusarium acuminatum and its activity against some human pathogenic bacteria’, Curr. Nanosci., 2008, 4, pp. 141–144 13 Rai, M., Yadav, A., Gade, A.: ‘Silver nanoparticles as a new generation of antimicrobials’, Biotechnol. Adv., 2009, 27, (1), pp. 76–83 14 Gade, A.K., Bonde, P., Ingle, A.P., Marcato, P.D., Duran, N., Rai, M.K.: ‘Exploitation of Aspergillus niger for synthesis of silver nanoparticles’, J. Biobased Mater. Bioenergy, 2008, 2, pp. 243–247 15 Duran, N., Marcarto, P.D., De Souza, G.I.H., Alves, O.L., Esposito, E.: ‘Antibacterial effect of silver nanoparticles produced by fungal process on textile fabrics and their effluent treatment’, J. Biomed. Nanotechnol., 2007, 3, pp. 203–208 16 Li, Y., Leung, P., Yao, L., Song, Q.W., Newton, E.: ‘Antimicrobial effects of surgical masks coated with nanoparticles’, J. Hosp. Infection, 2006, 62, pp. 58–63 177
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www.ietdl.org 17 Montes-Burgos, D., Hole, W.P., Smith, J., Lynch, I., Dawson, K.: ‘Characterisation of nanoparticle size and state prior to nanotoxicological studies’, J. Nanoparticle Res., 2010, 12, pp. 47–53 18 Bonde, S.R.: ‘A biogenic approach for green synthesis of silver nanoparticles using extract of Foeniculum vulgare and its activity against Staphylococcus aureus and Escherichia coli’, Nusantara Biosci., 2011, 3, (2), pp. 59–63 19 Bonde, S.R., Rathod, D.P., Ingle, A.P., Ade, R.B., Gade, A.K., Rai, M. K.: ‘Murraya koenigii mediated synthesis of silver nanoparticles and its activity against three human pathogenic bacteria’, Nanoscience Meth., 2012, 1, pp. 25–36 20 Sun, L., Simmons, B.A., Singh, S.: ‘Understanding tissue specific compositions of bioenergy feedstocks through hyperspectral Raman imaging’, Biotechnol. Bioeng., 2011, 108, (2), pp. 286–95 21 Chaudhary, G., Golyal, S., Poonia, P.: ‘Lawsonia inermis Linnaeus: a phytopharmacological review’, Int. J. Pharm. Sci. Drug Res., 2010, 2, (2), pp. 91–98 22 Rajendran, R., Hemalatha, S., Akasakalai, K., MadhuKrishna, C.H., Sohil, B., Sundaram, M.R.: ‘Hepatoprotective activity of Mimosa pudica leaves against carbontetrachloride induced toxicity’, J. Nat. Prod., 2010, 2, pp. 116–122
178 & The Institution of Engineering and Technology 2014
23 Iravani, S.: ‘Green synthesis of metal nanoparticles using plants’, Green Chem., 2011, 13, pp. 2638–2650 24 Kasthuri, J., Veerapandian, S., Rajendiran, N.: ‘Biological synthesis of silver and gold nanoparticles using apiin as reducing agent’, Colloids Surf., B, 2009, 68, pp. 55–60 25 Begum, N.A., Mondal, S., Basu, S., Laskar, R.A., Mandal, D.: ‘Biogenic synthesis of Au and Ag nanoparticles using aqueous solutions of Black Tea leaf extracts’, Colloids Surf., B, 2009, 71, pp. 113–118 26 Kora, A.J., Sashidhar, R.B., Arunachalam, J.: ‘Gum kondagogu (Cochlospermum gossypium): a template for the green synthesis and stabilization of silver nanoparticles with antibacterial application’, Carbohydrate Polym., 2010, 82, (3), pp. 670–679 27 Gade, A.K., Gaikwad, S.C., Tiwari, V., Yadav, A., Ingle, A.P., Rai, M.K.: ‘Biofabrication of silver nanoparticles by Opuntia ficus-indica: in vitro antibacterial activity and study of the mechanism involved in the syntheses’, Curr. Nanosci., 2010, 6, pp. 370–375 28 Gade, A., Rai, M., Kulkarni, S.: ‘Phoma sorghina, a phytopathogen mediated synthesis of unique silver rod’, Int. J. Green Nanotechnol., 2011, 3, (3), pp. 153–159
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