Title of Invention	"SILVER NANOPARTICLES AGAINST MALASEZZIA FURFUR, A DANDRUFF CAUSING FUNGUS"
Abstract	This invention relates to A synthetic route of preparing silver nanoparticles against malassezzia furfur, in aqueous solution at a low temperature and coated with weak adsorbing agent like polyacrylic acid on the particle surface, having size less than 50 nm and the steps comprising, chemical reduction of silver salt solution using sodium borohydride in the presence of polyacrylic acid as a capping agent, wherein 800 µl of silver nitrate solution (5% w/v) and 800 µl of polyacrylic acid (50% w/v) are thoroughly mixed up in 40 ml double distilled water at 4°C with constant stirring, followed by further stirring for another hour, adding 100 ml of 0.01 M ice cooled NaBH4 solution when it becomes light green in colour, further stirring the resultant solution for 2 hours at 4 °C, dialyzing the solution through 24 k D dialysis bag for 3-4 hours against distilled water wherein the colour of the solution changes from light green to yellow, diluting the dialyzed solution containing silver particles of known concentration.

Title of Invention

"SILVER NANOPARTICLES AGAINST MALASEZZIA FURFUR, A DANDRUFF CAUSING FUNGUS"

Abstract

This invention relates to A synthetic route of preparing silver nanoparticles against malassezzia furfur, in aqueous solution at a low temperature and coated with weak adsorbing agent like polyacrylic acid on the particle surface, having size less than 50 nm and the steps comprising, chemical reduction of silver salt solution using sodium borohydride in the presence of polyacrylic acid as a capping agent, wherein 800 µl of silver nitrate solution (5% w/v) and 800 µl of polyacrylic acid (50% w/v) are thoroughly mixed up in 40 ml double distilled water at 4°C with constant stirring, followed by further stirring for another hour, adding 100 ml of 0.01 M ice cooled NaBH4 solution when it becomes light green in colour, further stirring the resultant solution for 2 hours at 4 °C, dialyzing the solution through 24 k D dialysis bag for 3-4 hours against distilled water wherein the colour of the solution changes from light green to yellow, diluting the dialyzed solution containing silver particles of known concentration.

Full Text	"Silver nanoparticles against Malasezzia furfur, a dandruff causing Fungus". FIELD OF INVENTION This invention relates to the synthesis, characterization and application of stable aqueous dispersion of silver nanoparticles as antifungal agent. BACKGROUND OF INVENTION Dandruff has been a major skin problem and is of great concern. Malasezzia fur fur (M.furfur), lipophilic dimorphic yeast like fungus produces white to silvery powder scales in the scalp region often with moderate to severe itching and hair fall. Silver and its compounds have been reported to have potent and broad-spectrum antimicrobial activity since ancient times. A very well known toxicity effect of silver has been reported to wide range of microorganism and silver nanoparticles have recently been shown to be a promising antimicrobial material. Various studies already have done with bactericidal effects of silver nanoparticles but less or very little focus was done with antifungal effect of silver nanoparticles. In present study, we have investigated the antifungal effect of silver nanoparticles on M.furfur, the causative organism of dandruff. We prepared very low polydispersity and stable aqueous dispersion of silver nanoparticles in aqueous medium by using biocompatible polymer as capping agent. We carried out the antifungal study of these nanoparticles against Malasezzia fur fur. We have also carried out the comparative hemolytic study of these silver nanoparticles, Ketoconazole, and itraconazole, which are usually available in the market as antidandruff shampoos. Considering the lower antifungal properties of ketoconazole and itraconazole and their harmful effects on the skin, hair and blood compared to same amount of silver nanoparticles it may be concluded that addition of silver nanoparticles in the commercial shampoos would be more effective than these chemicals. Moreover, silver nanoparticles is cost effective, non-toxic and remove the dandruff problem to large extent, which is a major health, concerns these days for dermatitis. To date no effective non-toxic treatment for treatment of dandruff is available. This invention relates to the development of new biocompatible & safe antidandruff agent. Luis- charles malassez in late 19th century identified this fungus which later in 1904 was found to be a dandruff causing organism by Raymon Saburaud. It is a lipophilic dimorphic and yeast like fungus occurring in human skin as an opportunistic pathogens causes diseases such as dandruff Pityriasis, versicolar, Sevorrheic, dermatisis etc. Dandruff is a condensation, which causes small white flakes of skin that separate and fall from scalp. People who suffer from dandruff have over active Sebaceous glands, which make this scalp oily. It has been investigated and reported that there was not complete cure for the disease. Earlier there was no reliable recovered method of M.furfur from human skin by using cultural media. Therefore this fungus was enumerated by direct examination of skin sample. M. furfur has been shown to reside at various body sites including scalp forehead, nose, shoulder, abdomen, lower axilla, groin, fore arm and palp due to inefficient recovery media, the quantification of M.furfur by culturally was not possible. The most reliable quantitative study of the carriage and distribution of M.furfur on normal adult skin utilized an efficient recovery media. A wide range of antifungal agents has been used to treat this fungus. To treat both pityrasis vesicular and dandruff, scientist used selenium sulphide and zinc pyrithione. However these tropical treatments lead to high lapse rate. Ketoconazole have been used systemically in patients with pity arises vesicular, Sebarrhoic dermatitis, and Folliculitis with high success rate; compared to older therapies the relapse rate was much lower with oral Itraconazole. The scientist also suggested the need of prophylaxis to prevent reoccurrence and long them therapy of a relapsing condensation. For Seborohoic dermatitis topical application of Ketoconazole has been found to be an effective therapy and is more suitable for long-term use because of lower toxicity. Itraconazole has been used on the treatment of pity arises versicolar and is better than Ketoconazole for long-term prophylactic use. Topical Greeseofulvin and Terbinafine have shown significant clinical improvement in Pityrasis vesicular and there is report of success with topical Lithium succinate in patients with serbohoic dermatitis. Several reports of the cases of systemic infection by M. furfur are available. Several common features has emerged from this report, the main concern is that although the antifungal were administered to some of patient in the majority of patient the therapy was unsuccessful even at the above MIC level of antifungal. The antifungal used most commonly for the treatment of M.furfur also forms part of the skin flora in the normal adult population is not associated with overt disease in the majority of individuals. Cutaneous disease associated M. furfur are superficial and not involve invasion of living hast tissue. Both normal healthy individuals and patients with M. furfur associated skin disease are immenologically sensitized to M. furfur antigens. No antigens of M.furfur have been well characterized. Since the relation between body's defence mechanism and M.furfur is fully understood the therapeutic approach of the dermal disease is not very successful. There is no other solution at present but to use mainly Ketoconazole and Itraconazole. The difficulties of using these compounds in shampoos have been described in the earlier section. The toxicity of ketokonazole and itraconazole to blood, skin and hair has been removed by using silver nanoparticles. OBJECTS OF INVENTION The main object of this invention is to develop and characterize stable aqueous dispersion of silver nanoparticles as antifungal agent. Other object is to develop an effective antidandruff agent avoiding the toxicity by ketokonazole and itraconazole to blood, skin and hair. Another object is to produce cheap and safe to use antidandruff agent. STATEMENT OF INVENTION This invention relates to a synthetic route of preparing silver nanoparticles against malassezzia furfur, in aqueous solution at a low temperature and coated with weak adsorbing agent like polyacrylic acid on the particle surface, having size less than 50 nm and the steps comprising, chemical reduction of silver salt solution using sodium borohydride in the presence of polyacrylic acid as a capping agent, wherein 800 µl of silver nitrate solution (5% w/v) and 800 µ1 of polyacrylic acid (50% w/v) are thoroughly mixed up in 40 ml double distilled water at 4°C with constant stirring, followed by further stirring for another hour, adding 100 ml of 0.01 M ice cooled NaBH4 solution when it becomes light green in colour, further stirring the resultant solution for 2 hours at 4 °C, dialyzing the solution through 24 k D dialysis bag for 3-4 hours against distilled water wherein the colour of the solution changes from light green to yellow, diluting the dialyzed solution containing silver particles of known concentration. BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS Fig 1. Shows the nanosized spherical Ag particles typically exhibit a surface plasmon Peak at around 423 nm (figl) This characteristic resonance corresponds to excitation of surface plasmon vibrations in the silver nanoparticles and is responsible for the striking yellow colour. Fig 2. Shows the XRD pattern recorded of PAA-capped silver nanoparticles A number of strong Bragg reflections can be seen which correspond to the (111), (200), (220), (311) reflections of fee silver. The XRD results thus show that the silver nanoparticles formed are crystalline in nature. The inset in figure2 represents the selected area electron diffraction (SAED) of these particles, which reveals only diffusive ring patterns, which are most probably attributed to the small particle size and polycrystalline nature of the particles core. Nevertheless the diffraction feature were consistent with the fee crystal structure of bulk metallic silver with two brightest rings corresponding to the diffraction planes of (111) and (220). Fig 3. Shows the FTIR spectra recorded in the region 800-1800 cm-1 of pure PAA (curve 1) and PAA-capped silver nanoparticles obtained (curve 2).Absorption bands are seen at 1122, 1190,1060,1540,and 1700 cm-1 in the case of pure PAA (curve 1). The band at 1122 cm-1 and 1190 cm-1 are assigned to the C-0 stretching vibration, peak at 1060 cm_l indicates the presence of a non-conjugated cyclic anhydride and peak at 1540cm-1 can be assigned to stretching of carboxylate anion where as peak in the region of 1395-1450 indicate COH in plane deformation in the PAA molecules and the bands in the 1639-1800 cm-1 is due to carbonyl stretching [COOH stretching and C-O-C anhydride stretching] vibrations with strong absorption at 1700cm-1 due to carboxylic group in the PAA molecules. The bands at 1122 and 1190 are shifted to 1160 ,1210 where as bands at 1060 and 1540 disappear in the PAA-capped silver nanoparticle (curve 2) indicate that capping agent is bound to the silver nanoparticle surface through the hydrophilic carboxylic acid groups. Fig 4. shows TEM micrographs of the obtained silver nanoparticles particles The well-defined nanoscale, highly monodisperse silver particles with spherical shape and low polydispersity were obtained.From the TEM picture it is apparent that the particle size is less than 50 nm diameter. Fig 5. Showing inhibitory effect of silver nanoparticles on Malasezzia furfur. It (Itraconozole) Kt (Ketoconazole). Negative control(sterile water). 1 (silver nanoparticles Conc.0.317mg/ml)2. silver nanoparticles conc.0.635mg/ml). Fig 6. showing inhibitory effects of silver on Malasezzia furfur l(0.631mg/ml) 2.(0.423mg/ml) 3.(0.317mg/ml) and minimum Inhibitory concentration to be 4,( 0.16mg/ml) of silver nanoparticles. Fig 7. shows already available antifungal drugs like ketoconazole showed significant extant of hemolysis at the same concentration(Figure-7). The toxicity of the most reported antibiotics to mammalian cells has become a major limitation in using them as leading molecules.for developing new antifungal drugs and using them for further formulations. Antifungal effect and much lower toxicity of silver Nanoparticles against M.furfur in current study thus emphasis on the usefulness of silver Nanoparticles in the treatment of dandruff. Fig 8 a. Shows Morphology of hair which kept in nano silver formulation for 30 days at different magnification a.(at 4X) b.(at 10X) c.(at 20X) d.( at 40X) Fig 8 b. Shows Morphology of normal hair at different magnification a. (at 4X) b.(at 10X) c.(at 20X) d.( at 40X) The invention relates to the synthesis, characterization and application of stable aqueous dispersion of silver nanoparticles as antifungal agent, M.furfur, a dendruff causing fungus. The average size of the nanoparticles measures less than 50nm in diameter and the particles have been tested on Malasezzia furfur fungus. The effects of these particles on the human body such as on skin, hair and blood cells have been tested and it was found that these silver nanoparticles are practically harmless. (i) Preparation of silver nanoparticles:- (ii) Growth of M.furfur in culture media (iii) Treatment of M.furfur with Silver nanoparticles (iv) Effect of silver nanoparticles on hair (v) Hemolytic study of silver nanoparticles Materials: Malasezzia furfur(MTCC 1374 ) was obtained from Institute of Microbial Technology, (IMTECH) Chandigarh. Sabouraud Dextrose Agar(SDA) and corn oil were used to grow and maintain the fungus cultures per the supplier's(Hi Media) protocol. Chemicals such as silver nitrate, polyacrylic acid and sodium borohydride were purchased from Spectrochem (India) Ltd. and these reagents used without further purification. Synthesis of Silver nanoparticles: Silver nanoparticles were prepared in aqueous solution by reducing Ag+ ions with sodium borohydride. The metallic silver produced is immediately capped by poly acrylic acid to prevent further growth. The detailed synthetic procedure of silver nanoparticles is as follows: The particles were prepared in aqueous phase by chemical reduction of silver salt solution using sodium borohydride in the presence of polyarylic acid as capping agent. 800µl of silver nitrate solution (5% w/v) and 800µl of polyacrylic acid (50%w/v) were thoroughly mixed up in 40 ml double distilled water at 4° C with constant stirring. After mixing the solutions the mixture was further stirred for another hour. 100 ml of 0.01M ice-cooled NaBH4 solution was then added to the stirred mixture when it became light green in colour. The resultant solution was further stirred for two hours at 4° C. After stirring was completed, the solution was dialyzed through 24 kD dialysis bag for 3-4 hours against distilled water when the solution was changed from light green to yellow.The dialyzed solution containing silver particles were diluted to get silver nanoparticles solution of known concentration to carry out the antifungal activity. Characterization of silver nanoparticles: UV-vis spectroscopic studies The absorption spectra of PAA capped silver nanoparticle solution were monitored on UV-1600 Shimadzu UV-VIS spectrophotometer.. X-ray diffraction measurements X-ray diffraction (XRD) analysis PAA-capped silver nanoparticles lyophilized powder was carried out on a Phillips PW1830 instrument operating at 40 kV and a current of 30 mA with CuKa radiation. Fourier transform infrared (FTIR) spectroscopy Measurements FTIR spectra of pure silver nanoparticles and PAA-capped silver nanoparticles were recorded on a Perkin Elmer Spectrum. The spectrum of pure PAA was also recorded for comparison. Transmission electron microscopy (TEM) Measurements TEM measurements were performed on a JEOL Model JEM 2000Ex200 electron microscope operated at an accelerating voltage of 120 kV. Samples were prepared by placing small drops of dispersed silver nanoparticles (lyophilized powder) in water on formvar coated copper grids and allowing the solvent to slowly evaporate at room temperature. Preparation of M.furfur culture: M.furfur (MTCC 1374) was sub cultured on SDA(sabouraud Dextrose Agar) supplemented with 0.2% (v/v) corn oil. Isolated colony was inoculated in SDA broth supplemented with corn oil and culture was grown for 5-6 days at 30°C. Antifungal activity of Nanoparticles: The antifungal activity of silver nanoparticles was analyzed by disc diffusion assay and the minimum inhibitory concentration (MIC) of the nanoparticles was determined from the concentration of silver particles giving the least inhibitory activity below which there was no further inhibition of growth of the fungal cells The experiment was performed as follows: Sabouraud Dextrose Agar, (SDA) as per suppliers instructions was dissolved in required amount of water and was autoclaved .Corn oil was added to final concentration of 0.2%. The prepared agar medium was poured in radiation-sterilized petriplates of 10cm diameter (Tarson) and solidified .SDA supplemented with corn oil plates were inoculated with 10 16 cells of M. furfur Sterile discs obtained from Hi-media were impregnated with l0µl solution of different concentration of nanoparticles (0.02mg/ml, mg/ml, 0.04mg/ml, 0.08mg/ml, 0.16mg/ml, 0.317mg/ml and 0.635mg/ml). The discs were placed on the surface of agar plates inoculated with the culture using sterile forceps. The plates were incubated at 30°C and examined after 4-5 days for zone of inhibition, if any, around the disc.Positive and negative controls were also put. The experiment was repeated thrice. In vitro toxicity test by hemolytic assay: The basic method of kotler brajtburg et al.[16]with slight modification was employed to determine the hemolytic effect of silver nanoparticles. Erythrocytes or red blood corpuscles (RBC) have been used as a convenient model for the measurement of cell death (hemolysis) caused by silver nanoparticles. Blood was collected from Wistar rats by cardiac puncture using syringe prewashed with heparin,5ml blood was centrifuged at 3000rpm for 10 mins and the supernatant was discarded. RBC were washed thrice with isotonic PBS. Cells were finally suspended in 0.1% isotonic PBS. To study hemolysis 2.4ml of the above suspension was mixed with 10\|il solution of different concentration of nanoparticles (0.16mg/ml, 0.317mg/ml 0.4235mg/ml and 0.635mg/ml) and in order to compare it with commercially available formulation same experiment is performed with ketoconazole. RBCs suspended in 2.4ml PBS was used as negative control to assess background lysis,if any,while 2.4ml of suspension pretreated with 10 µ1 of Triton X-100 for complete lysis of the erytrocytes.Each suspension was incubated at 37°C for 24 hours and then keeping them in ice for 5minutes to stop hemolysis. Non-lysed RBSs were removed by centrifugation (3000rpm) for 10 mins. The supernatant was collected and the hemoglobin content was estimated spectrophotometrically at λmax = 575nm. The percentage of hemolysis was determined by following equation %Hemolysis = [(Abss - abs0)/ (Absioo - Abs0) ] xl00 where Abs0 is the absorption of the blank sample, Abss is the absorption of the sample and Absioo is the absorption of the sample treated with tritonX-100. Colloidal dispersion of metal nanoparticles exhibit absorption bands or broad regions of absorption in the ultra violet-visible range. These are due to the excitation of plasmon resonance or interband transition and are a characteristic property of the metallic nature of the particle. The yellow colour observed in the resultant solution are symptomatic of the presence of silver nanoparticles in the solutions. Antifungal activity of silver nanoparticles was examined by disc diffusion assay and we found that under our experimental condition silver is quite effective in preventing the growth of malasezzia furfur(figure-5). The minimum inhibitory concentration of the silver nanoparticles against M.furfur was found to be 0.16mg/ml(figure-6).Table one indicates that the inhibitory effect of silver is directly proportional to the concentration of silver particles,as the concentration of silver nanoparticles increases the zone of inhibition also increases Table-1 showing zone of inhibition (Table Removed) Hemolysis experiment;- The results of the hemolytic experiments show that silver Nanoparticles were non-toxic upto 0.635mg/ml to rat erythrocytes which was significantly higher than minimum inhibitory concentration (0.16mg/ml).On the other hand already available antifungal drugs like ketoconazole showed significant extant of hemolysis at the same concentration(Figure-7). The toxicity of the most reported antibiotics to mammalian cells has become a major limitation in using them as leading molecules.for developing new antifungal drugs and using them for further formulations. Antifungal effect and much lower toxicity of silver Nanoparticles against M.furfur in current study thus emphasis on the usefulness of silver Nanoparticles in the treatment of dandruff. Effect of nano silver formulation on hair In order to study the effect of silver nanoparticles on hair we kept a hair in the nano silver formulation for 30 days and then obseseved its morphology under the microscope at different magnifications and compare it with normal hair. Results are shown in the figure 8a and 8b which clearly demonstrates that silver nano particles do not damage the hair. Here inventors have described a new synthetic route of preparing silver nanoparticles in aqueous solution at low temperature and coated with a weak adsorbing agent like polyaerylie acid on the particle surface which controls the particle size.The observations of the present study indicate that the silver nanoparticles exhibit strong antifungal effects on M.furfur cells with extremely low toxicity to Rat's RBCs, and no damaging effect on hair morphology such preparations may be important leading to development of new anti dandruff formulations and treating fungal infections. We Claim 1. A synthetic route of preparing silver nanoparticles against malassezzia furfur, in aqueous solution at a low temperature and coated with weak adsorbing agent like polyacrylic acid on the particle surface, having size less than 50 nm and the steps comprising i) chemical reduction of silver salt solution using sodium borohydride in the presence of polyacrylic acid as a capping agent, wherein 800 µl of silver nitrate solution (5% w/v) and 800 µl of polyacrylic acid (50% w/v) are thoroughly mixed up in 40 ml double distilled water at 4°C with constant stirring ii) followed by further stirring for another hour iii) adding 100 ml of 0.01 M ice cooled NaBH4 solution when it becomes light green in colour iv) further stirring the resultant solution for 2 hours at 4 °C v) dialyzing the solution through 24 k D dialysis bag for 3-4 hours against distilled water wherein the colour of the solution changes from light green to yellow vi) diluting the dialyzed solution containing silver particles of known concentration. 2. A synthetic route of preparing silver nanoparticles against malassezzia furfur as claimed in claim 1, wherein the coating with a weak adsorbing agent like polyacrylic acid on the particle surface controls the particle size. 3. A synthetic route of preparing silver nanoparticles against malassezzia furfur as described and illustrated herein.

Full Text

"Silver nanoparticles against Malasezzia furfur, a dandruff causing Fungus".
FIELD OF INVENTION
This invention relates to the synthesis, characterization and application of stable aqueous dispersion of silver nanoparticles as antifungal agent.
BACKGROUND OF INVENTION
Dandruff has been a major skin problem and is of great concern. Malasezzia fur fur (M.furfur), lipophilic dimorphic yeast like fungus produces white to silvery powder scales in the scalp region often with moderate to severe itching and hair fall. Silver and its compounds have been reported to have potent and broad-spectrum antimicrobial activity since ancient times. A very well known toxicity effect of silver has been reported to wide range of microorganism and silver nanoparticles have recently been shown to be a promising antimicrobial material. Various studies already have done with bactericidal effects of silver nanoparticles but less or very little focus was done with antifungal effect of silver nanoparticles. In present study, we have investigated the antifungal effect of silver nanoparticles on M.furfur, the causative organism of dandruff. We prepared very low polydispersity and stable aqueous dispersion of silver nanoparticles in aqueous medium by using biocompatible polymer as capping agent. We carried out the antifungal study of these nanoparticles against Malasezzia fur fur. We have also carried out the comparative hemolytic study of these silver nanoparticles,
Ketoconazole, and itraconazole, which are usually available in the market as antidandruff shampoos. Considering the lower antifungal properties of ketoconazole and itraconazole and their harmful effects on the skin, hair and blood compared to same amount of silver nanoparticles it may be concluded that addition of silver nanoparticles in the commercial shampoos would be more effective than these chemicals. Moreover, silver nanoparticles is cost effective, non-toxic and remove the dandruff problem to large extent, which is a major health, concerns these days for dermatitis.
To date no effective non-toxic treatment for treatment of dandruff is available. This invention relates to the development of new biocompatible & safe antidandruff agent.
Luis- charles malassez in late 19th century identified this fungus which later in 1904 was found to be a dandruff causing organism by Raymon Saburaud. It is a lipophilic dimorphic and yeast like fungus occurring in human skin as an opportunistic pathogens causes diseases such as dandruff Pityriasis, versicolar, Sevorrheic, dermatisis etc. Dandruff is a condensation, which causes small white flakes of skin that separate and fall from scalp. People who suffer from dandruff have over active Sebaceous glands, which make this scalp oily. It has been investigated and reported that there was not complete cure for the disease. Earlier there was no reliable recovered method of M.furfur from human skin by using cultural media. Therefore this fungus was enumerated by direct examination of skin sample. M. furfur has been shown to reside at various body sites including scalp forehead, nose, shoulder, abdomen,
lower axilla, groin, fore arm and palp due to inefficient recovery media, the quantification of M.furfur by culturally was not possible. The most reliable quantitative study of the carriage and distribution of M.furfur on normal adult skin utilized an efficient recovery media. A wide range of antifungal agents has been used to treat this fungus. To treat both pityrasis vesicular and dandruff, scientist used selenium sulphide and zinc pyrithione. However these tropical treatments lead to high lapse rate. Ketoconazole have been used systemically in patients with pity arises vesicular, Sebarrhoic dermatitis, and Folliculitis with high success rate; compared to older therapies the relapse rate was much lower with oral Itraconazole. The scientist also suggested the need of prophylaxis to prevent reoccurrence and long them therapy of a relapsing condensation. For Seborohoic dermatitis topical application of Ketoconazole has been found to be an effective therapy and is more suitable for long-term use because of lower toxicity. Itraconazole has been used on the treatment of pity arises versicolar and is better than Ketoconazole for long-term prophylactic use. Topical Greeseofulvin and Terbinafine have shown significant clinical improvement in Pityrasis vesicular and there is report of success with topical Lithium succinate in patients with serbohoic dermatitis. Several reports of the cases of systemic infection by M. furfur are available. Several common features has emerged from this report, the main concern is that although the antifungal were administered to some of patient in the majority of patient the therapy was unsuccessful even at the above MIC level of antifungal. The antifungal used most commonly for the treatment of M.furfur also forms part of the skin flora in the normal adult population is not associated with overt disease in the majority of individuals. Cutaneous disease associated M. furfur are
superficial and not involve invasion of living hast tissue. Both normal healthy individuals and patients with M. furfur associated skin disease are immenologically sensitized to M. furfur antigens. No antigens of M.furfur have been well characterized. Since the relation between body's defence mechanism and M.furfur is fully understood the therapeutic approach of the dermal disease is not very successful.
There is no other solution at present but to use mainly Ketoconazole and Itraconazole. The difficulties of using these compounds in shampoos have been described in the earlier section.
The toxicity of ketokonazole and itraconazole to blood, skin and hair has been removed by using silver nanoparticles.
OBJECTS OF INVENTION
The main object of this invention is to develop and characterize stable aqueous dispersion of silver nanoparticles as antifungal agent.
Other object is to develop an effective antidandruff agent avoiding the toxicity by ketokonazole and itraconazole to blood, skin and hair.
Another object is to produce cheap and safe to use antidandruff agent.
STATEMENT OF INVENTION
This invention relates to a synthetic route of preparing silver nanoparticles against malassezzia furfur, in aqueous solution at a low temperature and coated with weak adsorbing agent like polyacrylic acid
on the particle surface, having size less than 50 nm and the steps comprising, chemical reduction of silver salt solution using sodium borohydride in the presence of polyacrylic acid as a capping agent, wherein 800 µl of silver nitrate solution (5% w/v) and 800 µ1 of polyacrylic acid (50% w/v) are thoroughly mixed up in 40 ml double distilled water at 4°C with constant stirring, followed by further stirring for another hour, adding 100 ml of 0.01 M ice cooled NaBH4 solution when it becomes light green in colour, further stirring the resultant solution for 2 hours at 4 °C, dialyzing the solution through 24 k D dialysis bag for 3-4 hours against distilled water wherein the colour of the solution changes from light green to yellow, diluting the dialyzed solution containing silver particles of known concentration.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
Fig 1. Shows the nanosized spherical Ag particles typically exhibit a surface plasmon Peak at around 423 nm (figl) This characteristic resonance corresponds to excitation of surface plasmon vibrations in the silver nanoparticles and is responsible for the striking yellow colour.
Fig 2. Shows the XRD pattern recorded of PAA-capped silver nanoparticles A number of strong Bragg reflections can be seen which correspond to the (111), (200), (220), (311) reflections of fee silver. The XRD results thus show that the silver nanoparticles formed are crystalline in nature. The inset in figure2 represents the selected area electron diffraction (SAED) of these particles, which reveals only diffusive ring patterns, which are most probably attributed to the small particle
size and polycrystalline nature of the particles core. Nevertheless the diffraction feature were consistent with the fee crystal structure of bulk metallic silver with two brightest rings corresponding to the diffraction planes of (111) and (220).
Fig 3. Shows the FTIR spectra recorded in the region 800-1800 cm-1 of pure PAA (curve 1) and PAA-capped silver nanoparticles obtained (curve
2).Absorption bands are seen at 1122, 1190,1060,1540,and 1700 cm-1 in the case of pure PAA (curve 1). The band at 1122 cm-1 and 1190 cm-1 are assigned to the C-0 stretching vibration, peak at 1060 cm_l indicates the presence of a non-conjugated cyclic anhydride and peak at 1540cm-1 can be assigned to stretching of carboxylate anion where as peak in the region of 1395-1450 indicate COH in plane deformation in the PAA molecules and the bands in the 1639-1800 cm-1 is due to carbonyl stretching [COOH stretching and C-O-C anhydride stretching] vibrations with strong absorption at 1700cm-1 due to carboxylic group in the PAA molecules. The bands at 1122 and 1190 are shifted to 1160 ,1210 where as bands at 1060 and 1540 disappear in the PAA-capped silver nanoparticle (curve 2) indicate that capping agent is bound to the silver nanoparticle surface through the hydrophilic carboxylic acid groups.
Fig 4. shows TEM micrographs of the obtained silver nanoparticles particles The well-defined nanoscale, highly monodisperse silver particles with spherical shape and low polydispersity were obtained.From the TEM picture it is apparent that the particle size is less than 50 nm diameter.
Fig 5. Showing inhibitory effect of silver nanoparticles on Malasezzia furfur. It (Itraconozole) Kt (Ketoconazole). Negative control(sterile water). 1 (silver nanoparticles Conc.0.317mg/ml)2. silver nanoparticles conc.0.635mg/ml).
Fig 6. showing inhibitory effects of silver on Malasezzia furfur l(0.631mg/ml) 2.(0.423mg/ml) 3.(0.317mg/ml) and minimum Inhibitory concentration to be 4,( 0.16mg/ml) of silver nanoparticles.
Fig 7. shows already available antifungal drugs like ketoconazole showed significant extant of hemolysis at the same concentration(Figure-7). The toxicity of the most reported antibiotics to mammalian cells has become a major limitation in using them as leading molecules.for developing new antifungal drugs and using them for further formulations. Antifungal effect and much lower toxicity of silver Nanoparticles against M.furfur in current study thus emphasis on the usefulness of silver Nanoparticles in the treatment of dandruff.
Fig 8 a. Shows Morphology of hair which kept in nano silver formulation for 30 days at different magnification a.(at 4X) b.(at 10X) c.(at 20X) d.( at 40X)
Fig 8 b. Shows Morphology of normal hair at different magnification a. (at 4X) b.(at 10X) c.(at 20X) d.( at 40X)
The invention relates to the synthesis, characterization and application of stable aqueous dispersion of silver nanoparticles as antifungal agent, M.furfur, a dendruff causing fungus. The average size of the
nanoparticles measures less than 50nm in diameter and the particles have been tested on Malasezzia furfur fungus. The effects of these particles on the human body such as on skin, hair and blood cells have been tested and it was found that these silver nanoparticles are practically harmless.
(i) Preparation of silver nanoparticles:-
(ii) Growth of M.furfur in culture media
(iii) Treatment of M.furfur with Silver nanoparticles
(iv) Effect of silver nanoparticles on hair
(v) Hemolytic study of silver nanoparticles
Materials: Malasezzia furfur(MTCC 1374 ) was obtained from Institute of Microbial Technology, (IMTECH) Chandigarh. Sabouraud Dextrose Agar(SDA) and corn oil were used to grow and maintain the fungus cultures per the supplier's(Hi Media) protocol. Chemicals such as silver nitrate, polyacrylic acid and sodium borohydride were purchased from Spectrochem (India) Ltd. and these reagents used without further purification.
Synthesis of Silver nanoparticles: Silver nanoparticles were prepared in aqueous solution by reducing Ag+ ions with sodium borohydride. The metallic silver produced is immediately capped by poly acrylic acid to prevent further growth. The detailed synthetic procedure of silver nanoparticles is as follows: The particles were prepared in aqueous phase by chemical reduction of silver salt solution using sodium borohydride in the presence of polyarylic acid as capping agent. 800µl of silver nitrate
solution (5% w/v) and 800µl of polyacrylic acid (50%w/v) were thoroughly mixed up in 40 ml double distilled water at 4° C with constant stirring. After mixing the solutions the mixture was further stirred for another hour. 100 ml of 0.01M ice-cooled NaBH4 solution was then added to the stirred mixture when it became light green in colour. The resultant solution was further stirred for two hours at 4° C. After stirring was completed, the solution was dialyzed through 24 kD dialysis bag for 3-4 hours against distilled water when the solution was changed from light green to yellow.The dialyzed solution containing silver particles were diluted to get silver nanoparticles solution of known concentration to carry out the antifungal activity.
Characterization of silver nanoparticles:
UV-vis spectroscopic studies
The absorption spectra of PAA capped silver nanoparticle solution were monitored on UV-1600 Shimadzu UV-VIS spectrophotometer..
X-ray diffraction measurements
X-ray diffraction (XRD) analysis PAA-capped silver nanoparticles lyophilized powder was carried out on a Phillips PW1830 instrument operating at 40 kV and a current of 30 mA with CuKa radiation.
Fourier transform infrared (FTIR) spectroscopy Measurements
FTIR spectra of pure silver nanoparticles and PAA-capped silver nanoparticles were recorded on a Perkin Elmer Spectrum. The spectrum of pure PAA was also recorded for comparison.
Transmission electron microscopy (TEM) Measurements
TEM measurements were performed on a JEOL Model JEM 2000Ex200 electron microscope operated at an accelerating voltage of 120 kV. Samples were prepared by placing small drops of dispersed silver nanoparticles (lyophilized powder) in water on formvar coated copper grids and allowing the solvent to slowly evaporate at room temperature.
Preparation of M.furfur culture: M.furfur (MTCC 1374) was sub cultured on SDA(sabouraud Dextrose Agar) supplemented with 0.2% (v/v) corn oil. Isolated colony was inoculated in SDA broth supplemented with corn oil and culture was grown for 5-6 days at 30°C.
Antifungal activity of Nanoparticles: The antifungal activity of silver nanoparticles was analyzed by disc diffusion assay and the minimum inhibitory concentration (MIC) of the nanoparticles was determined from the concentration of silver particles giving the least inhibitory activity below which there was no further inhibition of growth of the fungal cells The experiment was performed as follows: Sabouraud Dextrose Agar, (SDA) as per suppliers instructions was dissolved in required amount of water and was autoclaved .Corn oil was added to final concentration of 0.2%. The prepared agar medium was poured in radiation-sterilized petriplates of 10cm diameter (Tarson) and solidified .SDA supplemented with corn oil plates were inoculated with 10 16 cells of M. furfur Sterile discs obtained from Hi-media were impregnated with l0µl solution of different concentration of nanoparticles (0.02mg/ml, mg/ml, 0.04mg/ml, 0.08mg/ml, 0.16mg/ml, 0.317mg/ml and 0.635mg/ml). The discs were placed on the surface of agar plates inoculated with the culture using
sterile forceps. The plates were incubated at 30°C and examined after 4-5 days for zone of inhibition, if any, around the disc.Positive and negative controls were also put. The experiment was repeated thrice.
In vitro toxicity test by hemolytic assay: The basic method of kotler brajtburg et al.[16]with slight modification was employed to determine the hemolytic effect of silver nanoparticles. Erythrocytes or red blood corpuscles (RBC) have been used as a convenient model for the measurement of cell death (hemolysis) caused by silver nanoparticles. Blood was collected from Wistar rats by cardiac puncture using syringe prewashed with heparin,5ml blood was centrifuged at 3000rpm for 10 mins and the supernatant was discarded. RBC were washed thrice with isotonic PBS. Cells were finally suspended in 0.1% isotonic PBS. To study hemolysis 2.4ml of the above suspension was mixed with 10|il solution of different concentration of nanoparticles (0.16mg/ml, 0.317mg/ml 0.4235mg/ml and 0.635mg/ml) and in order to compare it with commercially available formulation same experiment is performed with ketoconazole. RBCs suspended in 2.4ml PBS was used as negative control to assess background lysis,if any,while 2.4ml of suspension pretreated with 10 µ1 of Triton X-100 for complete lysis of the erytrocytes.Each suspension was incubated at 37°C for 24 hours and then keeping them in ice for 5minutes to stop hemolysis. Non-lysed RBSs were removed by centrifugation (3000rpm) for 10 mins. The supernatant was collected and the hemoglobin content was estimated spectrophotometrically at λmax = 575nm. The percentage of hemolysis was determined by following equation
%Hemolysis = [(Abss - abs0)/ (Absioo - Abs0) ] xl00
where Abs0 is the absorption of the blank sample, Abss is the absorption of the sample and Absioo is the absorption of the sample treated with tritonX-100.
Colloidal dispersion of metal nanoparticles exhibit absorption bands or broad regions of absorption in the ultra violet-visible range. These are due to the excitation of plasmon resonance or interband transition and are a characteristic property of the metallic nature of the particle. The yellow colour observed in the resultant solution are symptomatic of the presence of silver nanoparticles in the solutions.
Antifungal activity of silver nanoparticles was examined by disc diffusion assay and we found that under our experimental condition silver is quite effective in preventing the growth of malasezzia furfur(figure-5). The minimum inhibitory concentration of the silver nanoparticles against M.furfur was found to be 0.16mg/ml(figure-6).Table one indicates that the inhibitory effect of silver is directly proportional to the concentration of silver particles,as the concentration of silver nanoparticles increases the zone of inhibition also increases
Table-1 showing zone of inhibition
(Table Removed)
Hemolysis experiment;- The results of the hemolytic experiments show that silver Nanoparticles were non-toxic upto 0.635mg/ml to rat erythrocytes which was significantly higher than minimum inhibitory concentration (0.16mg/ml).On the other hand already available antifungal drugs like ketoconazole showed significant extant of hemolysis at the same concentration(Figure-7). The toxicity of the most reported antibiotics to mammalian cells has become a major limitation in using them as leading molecules.for developing new antifungal drugs and using them for further formulations. Antifungal effect and much lower toxicity of silver Nanoparticles against M.furfur in current study thus emphasis on the usefulness of silver Nanoparticles in the treatment of dandruff.
Effect of nano silver formulation on hair
In order to study the effect of silver nanoparticles on hair we kept a hair in the nano silver formulation for 30 days and then obseseved its morphology under the microscope at different magnifications and compare it with normal hair. Results are shown in the figure 8a and 8b which clearly demonstrates that silver nano particles do not damage the hair.
Here inventors have described a new synthetic route of preparing silver nanoparticles in aqueous solution at low temperature and coated with a weak adsorbing agent like polyaerylie acid on the particle surface which controls the particle size.The observations of the present study indicate that the silver nanoparticles exhibit strong antifungal effects on M.furfur cells with extremely low toxicity to Rat's RBCs, and no damaging effect on hair morphology such preparations may be important leading to development of new anti dandruff formulations and treating fungal infections.

We Claim
1. A synthetic route of preparing silver nanoparticles against malassezzia furfur, in aqueous solution at a low temperature and coated with weak adsorbing agent like polyacrylic acid on the particle surface, having size less than 50 nm and the steps comprising
i) chemical reduction of silver salt solution using sodium borohydride in the presence of polyacrylic acid as a capping agent, wherein 800 µl of silver nitrate solution (5% w/v) and 800 µl of polyacrylic acid (50% w/v) are thoroughly mixed up in 40 ml double distilled water at 4°C with constant stirring
ii) followed by further stirring for another hour
iii) adding 100 ml of 0.01 M ice cooled NaBH4 solution when it becomes light green in colour
iv) further stirring the resultant solution for 2 hours at 4 °C
v) dialyzing the solution through 24 k D dialysis bag for 3-4 hours against distilled water wherein the colour of the solution changes from light green to yellow
vi) diluting the dialyzed solution containing silver particles of known concentration.
2. A synthetic route of preparing silver nanoparticles against malassezzia furfur as claimed in claim 1, wherein the coating with a weak adsorbing agent like polyacrylic acid on the particle surface controls the particle size.
3. A synthetic route of preparing silver nanoparticles against malassezzia furfur as described and illustrated herein.

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Patent Number

277227

Indian Patent Application Number

1779/DEL/2010

PG Journal Number

48/2016

Publication Date

18-Nov-2016

Grant Date

16-Nov-2016

Date of Filing

30-Jul-2010

Name of Patentee

JAMIA HAMDARD (HUMDARD UNIVERSITY)

Applicant Address

JAMIA HAMDARD (HUMDARD UNIVERSITY) HAMDARD NAGAR, NEW DELHI 110062.

Inventors:

#	Inventor's Name	Inventor's Address
1	MOHM. SAMIM	DEPARTMENT OF CHEMISTRY, JAMIA HAMDARD, NEW DELHI 110062. INDIA.
2	AMARNATH MAITRA	DEPARTMENT OF CHEMISTRY, JAMIA HAMDARD, NEW DELHI 110062. INDIA.
3	REENA ARORA	DEPARTMENT OF CHEMISTRY, JAMIA HAMDARD, NEW DELHI 110062. INDIA.
4	SOUVIK BISWAS	DEPARTMENT OF CHEMISTRY, JAMIA HAMDARD, NEW DELHI 110062. INDIA.

PCT International Classification Number

A01H

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA