AU2021103328A4 - Synthesis and pharmacological evaluation of ibuprofen entrapped silver nanoparticles using herbals - Google Patents

Abstract

The present invention relates to synthesis of ibuprofen loaded silver nanoparticles by using herbal extract. The method used for the synthesis of silver Nano particles by green synthesis and to formulate a cost-effective antibacterial dosage form. The prepared ibuprofen loaded silver nanoparticles by using fruit extracts were effective against the gram negative bacteria E.coli.

Description

SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF IBUPROFEN ENTRAPPED SILVER NANOPARTICLES USING HERBALS FIELD

[0001] The present invention relates to a Potentiation of Antibacterial Efficacy of Ibuprofen Entrapped Silver Nanoparticles by Green Synthesis.

BACKGROUND

[0002] Antibiotic resistant problem is a global concern because of the ability of bacteria to cause community acquired infection. Many scientific approaches have been developed to deal with antibiotic resistance and to make antibiotic more therapeutic, safe and effective. In this development silver was found as antimicrobial agent with distinctive properties of conductivity, stability, and activity. Previous data show that nanoparticles of silver are effective against a wide spectrum of bacteria, fungi, viruses, some infectious diseases and bum wounds. With the rise in microbial resistance of to various antibiotics, researchers are urged to grow silver nanoparticles free of resistance and cost by various green sources. The antibacterial efficacy of silver nanoparticle was found more when they are loaded with antibacterial agent by green synthesis.

[0003] Silver (Ag) is a soft, white, lustrous transition metal having 47 atomic number. Silver has the highest thermal and electrical conductivity and one of the highest optical reflectivity of any metal. The unique properties of silver nanoparticles like, optical, electrical, and magnetic etc makes them suitable to be used in antimicrobial applications, biosensors, cosmetics etc. Specific physical and chemical approaches have been used in Ag nanoparticles synthesis and stabilization. Chemical reduction is the most popular chemical approach which includes an array of organic and inorganic reducion agents.

[0004] In recent days, synthesis of Ag nanoparticle is the most interesting research field in science, and nanoparticles are being developed with growing focus using environmentally friendly methods (green chemistry). Silver and its nanoparticles have a ample application in medicinal products such as skin ointments and silver creams that prevent burns, open wounds and microbial attack.

[0005] In last few years, silver nanoparticles have been reported for their synthesis from the sources that occur naturally and their products or extracts. The silver nanoparticles be placed on the microbial cell wall, thereby disrupt the cell wall permeability and cell respiration. The nanoparticles may damage cell by penetrating deep into the cell wall and interacting with cell organelles like DNA and protein. Silver nanoparticles release Ag ions which confer the bactericidal activity.

[0006] Antibacterial effect mechanism of silver nanoparticle

[0007] The actual antimicrobial mechanism of silver nanoparticles is not well understood and is a topic of discussion. Various hypotheses are there about the activity of Ag nanoparticles against microbes to induce antimicrobial effect. One mechanism suggests that Ag nanoparticles cause cell death by structural change of bacterial cell wall after binding on it. Nanoparticles accumulate on the cell surface by forming pits or holes on surface. Other mechanism suggests that Ag nanoparticles form the free radicals by resonance of moving electrons and these free radicals cause cell death.

[0008] The free radicals of Ag nanoparticle makes pores on the bacterial cell membrane by damaging it, this can eventually cause cell death. It was suggested that nanoparticles may release silver ions, and these Ag+ may inhibit various enzymes by interacting with their thiol group. Silver ions entrapped in bacterial cells and inhibit the functioning of cell. Then, respiratory enzymes produce reactive oxygen species (ROS), which aggresses the cell itself. Silver is categorized as weak acid which has a tendency to react with a weak base. In case of cells, they are formed by weak base like sulfur and phosphorus. This acid base reaction of silver nanoparticle and cell is also responsible for cell death. The DNA of cells is also made up of sulfur and phosphorus so that Ag nanoparticles can also act on these bases and prevents DNA replication which may cause cell death.

[0009] Phosphorylation of protein substrates in bacteria affects the transduction of bacterial signalsDephosphorylation is reported only in the gram -ive bacterial tyrosine residues. Nanoparticles change the phosphotyrosine profile of bacterial peptides, which inhibits the signal transduction and bacterial growth. Nevertheless, it is necessary to understand that further work on the subject is needed in order to establish the statements.

[00010] The silver nanoparticles were synthesized using a local plant (lemon fruit), Ibuprofen because lemon has a high content of ascorbic acid and Ibuprofen is a local non steroidal drug. Both are also easier to obtain and more cost effective. Furthermore, the antimicrobial properties of the silver nanoparticles serve well as an alternate antiseptic against bacteria and by loading Ibuprofen in silver nanoparticles, a new alternative of antibacterial agent was found.

[00011] The research objective was achieved by focusing following studies. Firstly, lemon fruit extract was used in order to synthesize the silver nanoparticles. Secondly, the Ibuprofen was loaded into the silver nanoparticles to find an alternative use of non steroidal drug as antimicrobial agent. Thirdly, the characteristics of silver nanoparticles were studied through spectroscopy, UV, FTIR, TEM and XRD. Fourthly, antimicrobial properties of Ibuprofen loaded silver nanoparticles and plane silver nanoparticles were studied on gram-negative bacteria Escherichia coli.

[00012] The following prior art is being reported:

[00013] US10828328: The method of preparing biogenic silver nanoparticles includes preparing an aqueous plant extract by boiling cut leaves of Alternanthera bettzickiana (Regel) G. Nicholson in distilled water, retaining the aqueous extract. The aqueous plant extracts were mixed with aqueous solutions of silver ions derived from different silver salt precursors (e.g., silver nitrate, silver sulfate, etc.). The resulting biogenic silver nanoparticles exhibit antimicrobial activity against various strains of gram-positive and gram-negative organisms, including some strains of drug-resistant microorganisms. The biogenic silver nanoparticles also exhibit anticancer activity against certain human cancer cell lines. Surprisingly, biogenic silver nanoparticles prepared from nitrate precursor exhibited greater anticancer activity than nanoparticles from sulfate precursor, while biogenic silver nanoparticles prepared from sulfate precursor exhibited greater antibacterial activity than nanoparticles from nitrate precursor.

SUMMARY

[00014] The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the present invention. It is not intended to identify the key/critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concept of the invention in a simplified form as a prelude to a more detailed description of the invention presented later.

[00015] The present invention is generally directed to formulation ofIbuprofen loaded silver nanoparticles with the help of green synthesis

[00016] An embodiment of the present invention is to evaluate the antibacterial properties of nanoparticles against bacteria.

[00017] Another embodiment of the invention is the formed nanoparticles were crystal, cube shaped with size approx. 40 nm.

[00018] Yet another embodiment of the invention isIbuprofen loaded nanoparticles were able to damage the outer membrane of bacteria.

[00019] Yet another embodiment of the invention structural disintegration in the bacterial membrane by AgNPs was increased when they were loaded with the increasing concentrations of the Ibuprofen

BRIEF DESCRIPTION OF DRAWING

[00020] Figure.1. a) Drug loaded and plane silver nanoparticles, b) after centrifugation

[00021] Figure.2. UV-Vis spectrometry results for silver nanoparticles (AgNPs) with 1mM(-) and 2mM(-) concentration

[00022] Figure.3. XRD pattern ofIbuprofen loaded AgNPs

[00023] Figure.4. TEM micrographs of prepared AgNPs, a) at 1mM conc, b) at 2mMconcentration.

[00024] Figure.5. Drug release graph of 1mM and 2mM formulation

DESCRIPTION OF EMBODIMENTS

[00025] The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way.

Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.

[00026] Preparation of Fruit Extract

[00027] Approx 25 g of Citrus limon was weighed and washed with distilled water and dried. It was converted into small segments and smashed into 100 ml sterile distilled water and filtered through Whatman filter paper. For further tests the extract was processed at 40 C.

[00028] Synthesis of Ibuprofen loaded Ag nanoparticles by fruit extract

[00029] Nanoparticles were formulated by following procedure. Firstly 1 mM, 2mM solution of silver nitrate was prepared. Ibuprofen was separately dissolved in methanol/water to form 0.5mM solution. In a conical flask, silver nitrate solution was boiled at 100°C and then drug solution was added and mixed. Then fruit extract was added in 4:1 ratio of silver nitrate and fruit extract. Solution was heated for 10-15 minutes. Silver nanoparticle formation was confirmed by color change of solution from colorless to yellowish and then brown. The solution was then cooled at room temperature and allowed to centrifuge at 12000 rpm for 15 minutes, and then it was washed with distilled water. Final product was dried under vacuum and used without further processing.

[00030] Preparation of AgNO 3 , drug solutions

S. NO. FOR QTY DISTILLED WATER REQUIRED

1. 1mM AgNO3 16.99 mg 100 ml AgNO3

2. 2mM AgNO3 33.98 mg 100ml

AgNO3

3. 0.5mM ibuprofen 25.7 mg 250 ml solution Ibuprofen

[00031] Synthesis of silver:

[00032] The color of silver nitrate solution was changed when we added the fruit extract as reducing agent. The color of silver nanoparticle solution was brown or reddish. The color of solution was getting darker with the increased heating time. Color was yellowish red in beginning and brown at last. After cooling at room temperature the color was changed to dark brown. (Fig.1) This color change was due to the quantum confinement which means that size of nanoparticle increases with darkness in color.

[00033] EXAMPLE 1:

[00034] UV Spectroscopy Analysis:

[00035] Synthesis of Ag nanoparticles by Citrus limon fruit extract as reducing agent may be easily absorbed by UV-visible spectroscopy. The absorption spectra of fruit extract quantities and silver concentration was measured using 200-800range. Ag nanoparticle uses UV light in visible ranges and absorbs it directly. Molecules undergo electronic transitions in this region of the electromagnetic spectrum.

[00036] Silver nanoparticles give the UV absorption peak at between 400 nm - 450 nm, Fig.2. shows the UV absorption peaks silver nanoparticles. It was approx 421.00nm, this indicates the formation of Ag nanoparticle in lemon fruit extract. The peak at 421 nm was due to the surface plasmon resonance phenomenon, which occurs because silver nanoparticles surface gets excited by applied electromagnetic field.

[00037] Fig.2. also shows an absorbance peak at 221 nm, this peak confirms the presence of Ibuprofen in silver nanoparticles.

[00038] EXAMPLE 2:

[00039] XRD Analysis

[00040] Synthesized AgNP was analyzed for XRD measurements, XRD pattern of Ibuprofen loaded silver nanoparticles by fruit extract were listed on X-ray diffractometer. The machine was operated at 30 kV voltage and 40 mA current with Cu K (a) (X = 1.5406 A), radiation. Material was identified and determination for the crystalline phase. The samples of silver nanoparticles was taken in lid and shifted in equipment for analysis. Diffractogram was compared with reference to the JCPDS data file No. 04-0783.

[00041] Fig.3. shows the XRD patterns of drug loaded silver nanoparticles. With reference to the JCPDS data file No. 04-0783 it was concluded that the nanoparticles were crystal, cube shaped with size approx 40 nm and with no contaminations. XRD analysis has shown that AgNPs with well-defined dimensions could be produced by reduction of Ag ions due to fruit extract of Citrus limon.

[00042] EXAMPLE 3:

[00043] Transmission Electron Microscopy (TEM)

[00044] The size and morphics of silver nanoparticles was studied via TEM (Jeol, Japan). Dispersion of silver nanoparticle was sonicated for 20 min with the help of UV sonicator. One drop of sample solution was diluted to a concentration of 75 mcg/ml. Then sample was placed on carbon grid and dried overnight. The microscope was adjusted at an accelerating voltage of 80 kV and sample was observed.

[00045] TEM micrographs (Fig.4.) show that both AgNPs with molar variations, 1mM and 2mM, were approximately spherical in shape. Silver nanoparticles with 1mM concentration were observed with smaller size diameter in range of 50-60 nm and most of the particles were in range of 40 nm. Silver nanoparticles with 2mM concentration were observed approx 80-90 nm in diameter.

[00046] EXAMPLE 4:

[00047] Ibuprofen loading efficiency

[00048] 10mg, 20mg, 30mg, 40mg of Ibuprofen was weighed and dispersed separately into Silver nanoparticles solution. The mixture solution was incubated for 24 hours with stirring. Then the resulting nanoparticle solution was centrifuged at 12,000 rpm for 30 minutes. The pellets was separated from supernatant solution and washed with distilled water for further characterization. Drug loading efficiency of nanoparticles was quantified by measuring the amount of drug left in supernatant with a UV-spectrophotometer at 221 nm. The standard curve for Ibuprofen was constructed by measuring the absorbance of standard Ibuprofen and diluted concentrations of the Ibuprofen.

[00049] Ibuprofen loading efficiency could also be confirmed by FTIR analysis. The formula used for calculating loading efficiency was

% loadingloadngeficincytotal in suparnatant * 100 efficiency = total drug-drugdrug Equation 1

[00050] Surface adsorption is the basic principle of drug loading on Silver Nanoparticles. We have added increasing quantity of drug to silver nanoparticles and observed their efficacy

[00051] Table: % Ibuprofen loading in silver nanoparticles

S. Formulation Drug Amount AgNP conc. % Drug loading No.

1. Fl 10 mg 1mM 77.4%

2. F2 20 mg 1mM 80.1%

3. F3 30 mg 1mM 55.6%

4. F4 40 mg 1mM 46.7%

[00052] EXAMPLE 5:

[00053] Ibuprofen release capacity

[00054] Ibuprofen release capacity of AgNPs was analyzed by using phosphate buffer with 7.2 pH. 400-500 mcg/ml ibuprofen loaded nanoparticles were placed in buffer solution up to 30 hours at 37°C. The amount of drug release from AgNPs was calculated by measuring the absorbance of the supernatant at specific time gaps at 221nm by a UV-spectrophotometer. A calibration curve of ibuprofen was made with known drug concentration for its quantification.

[00055] Drug release of silver nanoparticle was carried out in phosphate buffer pH 7.2. Correlation coefficient was found to be fit in first order equation with regression value R2 = 0.990. Diffusion was found as the main mode of drug release from nanoparticles. The percentage Ibuprofen release is shown in table 2. and Fig.5.

[00056] Table 2: In Vitro drug release of1mM and 2mM formulations

S. No. Time Cumulative Percentage Cumulative Percentage (hours) drug release (1mM drug release (2mM solution) solution)

1. 0 0 0

2. 2 9.2 11.2

3. 4 14.9 13.4

4. 6 15.3 16.6

5. 8 16.4 17.4

6. 10 17.3 18.5

7. 12 19.1 21.6

[00057] EXAMPLE 6:

[00058] Anti microbial activity

[00059] Disc-diffusion method was employed to test the antimicrobial sensitivity of drug loaded nanoparticles. Agar plates were developed for inoculation of bacteria. 10 L of the drug loaded nanoparticles were impregnated on 6 mm diameter sterilized paper and introduced to agar plates streaked with clinical isolates ofE.coli. This procedure was repeated using plane silver nanoparticles. Ciprofloxacin (10 mcg/disc) was used as the standard antibiotics in the study and as reference standards to determine the sensitivity of one strain per isolate for microbial specie tested. The agar streaked plates were incubated in an oven for 24 hours at 37°C. The antimicrobial activity was studied by measuring the zone of inhibition for the test microbe.

[00060] Zone of inhibition ofE.coli on agar disc plates has shown in Fig. The fruit extract AgNPs and drug loaded AgNPs were used to inhibit the growth of gram -ive E.coli which was clinically isolated for the study. Multiple studies have evaluated the interaction of the AgNPs with bacteria but it was observed that the AgNPs were present on the membranes of bacterial cells. Ibuprofen loaded nanoparticles were able to damage the outer membrane of bacteria. Structural disintegration in the bacterial membrane by AgNPs was increased when they were loaded withIbuprofen.

[00061] While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.

Claims (1)

1. A method of preparing ibuprofen loaded silver Nano particles comprising the steps of:

a. preparation of 1 mM, 2mM, silver nitrate solution;

b. Ibuprofen was separately dissolved in methanol/water to form 0.5mM solution;

c. silver nitrate solution was boiled at 100°C then drug solution was added;

d. adding the solution of fruit extract and silver nitrate in the ratio of in 4:1;

e. heat the solution for 10-15 minutes;

f. formation of Silver nanoparticle was confirmed by color change of solution from colorless to yellowish and then brown;

g. cooling of solution at room temperature followed by centrifuge at 12000 rpm for 15 minutes:

h. finally, it was washed with distilled water and;

i. final product was dried under vacuum and used without further processing.