Study of Antimicrobial activity of Star Anise loaded poly (DL-lactide-co-glycolide) nanoparticles

 

Vinod Kumari*, Aditi Sangal

Department of Chemistry, Amity Institute of Applied Sciences, Amity University, Sector 125, Noida

Uttar Pradesh-201313 (India)

 

ABSTRACT:

Nanoencapsulation using polymeric material can be done for Star Anise as it increases its efficacy. Earlier studies reveal that nanoencapsulation increases the antimicrobial activity, bio availability of the plant extract. For preparing the Star Anise loaded poly (DL-lactide-co-glycolide) PLGA nanoparticles ‘solvent evaporation method’ was used. Solvent evaporation method is used for producing nanoparticles of small size (10 nm for Star Anise), high entrapment efficiency (88.53% for Star Anise) and also for the improvement of inhibition of microbial growth. Further morphology, drug loading, entrapment efficiency, release profile and antimicrobial activities of nanoparticles are characterized. Usually studies related to drug release are conducted in vitro at 37°C as the Star Anise shows the initial burst of 36% and it is followed by quite slow rate. During microbial analysis, the minimum inhibitory concentration(MIC) of Star Anise loaded nanoparticles against the Staphylococcus aureus and Bacillus pumilus shows the inhibition zone of 9.84 mm and 10.20mm respectively at 6000 ppm whereas in case of Pseudomonas aeruginosa and Escherichia coli the nanopaticles shows inhibition zone of 8.21mm and 7.21 mm respectively at 4000ppm.Nanoparticles prepared here in shows suitable sizes as well as morphology. Antimicrobial studies show that nanoparticles prepared are beneficial for food and biomedical applications. The main objective behind the work in hand is the preparation of Star Anise loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles for increasing the efficacy of antimicrobial activity related to Star Anise. 

 

 

INTRODUCTION:

Nanotechnology is the nano-formulation of substances at nanoscale (1 to 100 nm) to produce substances which possess superiority than others, in one form or another. The conceptualization of nanotechnology was done by Richard Feynman in 1959.Now a days, Nanotechnology is considered as quite significant in the field of research and technology. So, Nanoencapsulation of hydrophobic biologically active compounds was done for enhancing the effectiveness and efficiency of antimiocrobial compound present in the traditional herbal plant in the drug delivery system.

 

Nanoparticles have numerous advantages due to its small size, as they can easily penetrate in the areas both (intracellular and extracellular areas) that could be inaccessible for other possible drug delivery systems. Nanoparticles can protect any drug against its degradation and it can also reduce the side effects^[1]. Among all the biodegradable polymers which are used in the preparation of nanoparticles, the PLGA shows lot of potentiality of a drug delivery vehicle, the most accepted among all available.  PLGA are biodegradable polymers due to its long clinical experience and also its degradation characteristics. Consequently they are favorable and they have potentiality of sustained drug delivery^[2]. The improvement of PLGA nanoparticles have been very encouraging, whereas a study has revealed that the PLGA nanoparticles of anethole show different degrees of growth in inhibition^[3]. Keeping in view here we synthesize Star Anise loaded PLGA nanoparticles and as per the studies reported earlier the principle components comprising  star anise is Anethole^[3]. Anethole (E)-1-methoxy-4-(1-propenyl) benzene), a phenylpropene, is a clear, transparent colorless to pale-yellow liquid having the  freezing point 20°C and the  boiling points is 234°C. Anethole is hydrophobic compound, and has very low solubility especially in water. While synthesizing the Star Anise loaded PLGA nanoparticles, solvent evaporation method was used with altogether different formulations so as to improve the antibacterial and encapsulation efficiency of Star Anise in uniform and also to attain the small size of PLGA nanoparticles. The Nanoparticles can be characterized and even compared as per their sizes, size distribution, morphology, drug loading, entrapment efficiency and drug release profile. Kinetic drug release study and in-vitro discharge profiles of methanolic extract as well as Star anise loaded PLGA nanoparticles were done to ascertain its oral release^[4-6].  Finally the antimicrobial effects of these compounds were tested keeping in view the earlier antimicrobial results reported by various researchers.^[4-19].

 

MATERIALS AND METHODS:

1 Materials:

Star Anise was purchased from local market. The polymer poly (D,L-lactide-co-glycolide) (PLGA)having a copolymer ratio of 50:50 (M_w = 24000 to 38000) was obtained from Sigma-Aldrich). The surfactants Pluronic F-68 and Polyvinyl alcohol (PVA)(Mw 30,000-70,000 Da) was procured from Sigma-Aldrich (St. Louis, MO, USA). The Organic reagents and solvents used Acetonitrile, Acetone and methanol is considered of analytical grade. Sodium dihydrogen phosphate, disodium hydrogen phosphate and trisodium phosphate used are of Analytical grade. Dialysis bag (Spectra/Por, Mw 12,000 Da) is used for drug release test.

 

2 Preparation of Star Anise loaded PLGA Nanoparticles by Solvent Evaporation Method:

Initially methanolic extract of Star Anise was prepared. For this purpose 8gm of powdered Star Anise was dissolved in 100 ml of Methanol (AR grade) and kept for 3 days, after which extract was filtered using whatmann filter paper, and the extract was then sun dried. The dark brown resinous crude methanolic extract of Star Anise was obtained. Solvent evaporation method was used for preparing Star Anise loaded PLGA nanoparticles.10 mg methanolic extract of Star Anise as drug sample along with 50 mg of PLGA (1:5) was used to prepare Organic phase by dissolving in 10 ml acteonitrile. The Organic phase so prepared was then added to 25ml of 1% Pluronic F-68 aqueous phase by using micro-syringe gradually drop by drop. The mixture was carefully sonicated with the help of microtip probe sonicator (set at 50 W volts). Thus the nanoparticles were formed and the organic phase present was evaporated by stirring the solution using magnetic stirrer continuously for 4 hrs at the speed of 400 rpm. After evaporation of organic phase, the centrifugation (Remi Cooling Centrifuge, C-24 BL) at the rate of 4000 rpm for about 5 minutes was carried out, to separate the nanoparticles. As after that 1st pellet could be separated out and the supernatant after washing with distilled water once again centrifuged at the rpm of 15000 for about 20minutes. The Nanoparticles obtained in this way were later dried and stored at 4⁰C for further use.

 

3 Particle Size and Morphology Characterization:

Particle Size and morphology of Star Anise loaded PLGA nanoparticles were confirmed by using TEM (Transmission Electron Microscopy, at IARI, New Delhi). Nanoparticles morphology was examined by Transmission Electron Microscopy using FEI Morgagni TEM (FEI Co., Hillsboro, OR, USA). Particles were suspended in water (1 mg/mL) and placed on 0.037 mm copper grids and stained with a 2 g/100 mL uranyl acetate aqueous stain to provide contrast under magnification. Excess liquid on the Mesh was removed with filter paper and the grid was allowed to air dry before viewing under 50,000 to 120,000 times magnification. Observations were performed at 80 kV^[20].

 

4 XRD and FTIR Study:

The XRD patterns of methanolic extract, PLGA and nanoparticle of drug loaded into PLGA were examined by utilizing X-beam diffractrometer. The estimations were taken at a voltage of 45 kV & an anodic current of 40 mA. XRD patterns were acquired at diffraction edges (2Ɵ) extending from 0º to 90º at an examining speed of 2º every moment.XRD patterns were done to find out the nature of compound formed whether is crystalline or amorphous.Further, FTIR studies were also done on Star Anise loaded PLGA nanoparticles to determine whether the polymer interacted with drug during the nanoencapsulation.

 

5 Determination Of Entrapment Efficiency And Percentage Yeild:

The UV analysis (Shimadzu, UV-1800)at 264 nm determined the amount of Star Anise entrapment. Supernatant obtained after centrifugation and removal of 1^st pellet was UV monitored at 264 nm wavelength and corresponding absorption maxima was recorded. A standard calibration curve was drawn using different concentration of methanolic extract (1-10 mg into 10ml water) versus maximum absorbance of the supernatant and as stated UV monitoring was used for examining the samples in direct determination of encapsulation efficiency and percentage yield. The following equations were used for calculating the amount of encapsulation efficiency and percentage yield.

% EE = Drug added - Free "unentrapped drug"   *100

                                 Drug added

 

Percentage Yield =    Weight of Nanoparticles      *100

         Weight of PLGA+ Weight of methanolic Extract

 

6 In Vitro Drug Release:

Star Anise released out from the nanoparticle drug could be studied by using the dialysis technique. 5mg of nanoparticles were measured and placed on the dialysis membrane, which was then suspended in 20 mL of phosphate buffer saline solution (pH7.4) and under magnetic stirring condition for 3 days; also the temperature was maintained at 37 C and solution was given 100 rpm speed. The amount of drug released is quantified by UV.

 

7 Kinetic Analysis of Drug release Profiles:

Mathematical models are generally used for computing the data released by drug. The Zero order kinetics, first order kinetics, Hixon Crowell model and the resultant data were fitted to the Higuchi release equation to initiate the mechanism involved in the release of the drug. Using these models, percentage of drug released could be calculated and data was feed in various equations.

 

8 Antimicrobial activity of Star Anise loaded PLGA nanoparticles:

The activities and properties of Star Anise-loaded nanoparticle when examined against Enterococcus coli, Psedomonas aeruginosa, B.pumilus and Staphylococcus aureus. The MIC of these test compounds were determined using cup plate method. In cup plate method, the antimicrobial substance diffuses from the cup through a solidified agar layer in a  petri-dish to  some  extent so that the  growth of added micro - organism is inhibited entirely in a circular area or zone around  the cavity containing the solution of a known quantity of antimicrobial substance. The antimicrobial activity is expressed as the zone of inhibition in millimeters, which is measured with a zone reader. (The amount of nanoparticles which were in suspension form was used to study the antimicrobial activity). Methanolic extract of pure drug along with that contain drug loaded PLGA nanoparticles were used for comparative study. After a period of 24 hours of incubation at the temperature of 37 C, the plates were then examined to determine the MIC of the entire test compounds were determined as lowest concentration which could inhibit the visible growth of bacteria.

 

RESULTS AND DISCUSSION:

1. Formulation, Optimization and Characterization Of Nanoparticles:

The methanolic extract of medicinal plant obtained as sticky and dark brown in colour. The nanoparticles were formulated using acetone and acetonitrile solvents with Pluronic F68 and Poly (vinyl alcohol) as surfactants were optimized for further studies based on the particles size and encapsulation efficiency. The percentage yield and encapsulations efficiency obtained was discussed in the Table (1):

 

From the table 1, it was concluded that the percentage yield of formulations F1 and F4 was come out to be moderate but in the formulation F1, the drug encapsulation efficiency was high. So, different parameters such as Morphological analysis, size, and in vitro drug release and antimicrobial study was carried out using F-1.

 

2. The Particle size and Morphological analysis:

PLGA encapsulated Star Anise nanoparticles were successfully prepared during the study using Solvent Evaporation Method. With the objective to study the size of particles in the aqueous solution where nanoparticles suspension have been analyzed after removing the  organic solvent by using magnetic stirring continuously  for at least 4 hr. Hence the results obtained through TEM images of the formulation F1 showed that the spherical nanoparticles (prepared by using acetonitrile and Pluronic F-68 ranging from 8nm to 20 nm. The image of the drug loaded PLGA nanoparticles is shown in Fig.(1 and 2).

 

Table 1: Characteristics of drug loaded PLGA nanoparticles: 

 

 

Fig 1: Spherical shaped Star Anise loaded PLGA nanoparticles diameter in between 8.71 nm to 16.63 nm

 

 

Fig 2: Spherical shaped Star Anise loaded PLGA nanoparticles

 

3. XRD And FTIR Studies:

The structural characteristics of the nanoparticles of the drug loaded PLGA were studied by the FTIR spectroscopy technique. The FTIR spectra of the nanoparticles of the drug loaded into PLGA are presented in the Fig (3). The PLGA showed bands at 2998.46 and 2953.79 cm^-1 corresponds to –CH, –CH2, –CH3 stretching vibrations, C=O stretching of carboxyl group appeared at 1753.45 cm^-1 and C—O stretching at range 1088 to 1270 cm^-1 as shown in Fig (3-a). The methanolic extract showed peaks at 3477.34 and 3376.56 cm^-1 due to O—H stretching, C—C stretching at 1506 cm^-1, bands at 2943 and 2857.41 cm^-1 corresponds to C—H stretching and other peaks at range 1450 to 850 cm^-1 due to C—H bends. In the encapsulated nanoparticles, some peaks were slightly moved to lower wavelength in Fig 3(c, d) of PLGA and methanolic extra confirmed that there was no chemical interaction between the drug and the polymer PLGA.

 

The XRD spectra for the drug loaded polymeric nanoparticles were obtained Fig. 3(e) and indicated that there is no characteristic peaks were observed for the drug as well as the drug entrapped into PLGA, possibly due to the amorphous nature of the drug.

 

 

Wave number (cm^-1)

Fig 3a: FTIR spectra of PLGA poly (DL-lactide-co-glycolide)

 

 

 

ave number (cm^-1)

Fig 3b: FTIR spectra of Methanolic extract of Star Anise

 

 

Wave number (cm^-1)

Fig 3c: FTIR spectra of Formulation 2 ( Star Anise loaded PLGA nanoparticles having acetonitrile as Organic Phase and PVA as surfactant)

 

 

Fig 3d: FTIR spectra of Formulation 1 ( Star Anise loaded PLGA nanoparticles having acetonitrile as Organic Phase and Pluronic F-68 as surfactant)

 

 

Fig3e: XRD of drug loaded PLGA nanoparticle showing amorphous nature

 

Fig.4 (a) – In vitro release kinetics of the nanoparticles of drug loaded into PLGA for the   period of 1 day.

 

Fig.4(b) – In vitro release kinetics of the nanoparticles of drug loaded into PLGA for the period of 2 days.

 

4. Entrapment Efficiency:

Entrapment efficiency related to Star Anise loaded PLGA nanoparticle for active compound Star Anise was found to be 88.5%. The selection of the method used in the production of nanoparticles strongly depends on the drugs that were about to be encapsulated.

 

5. The release of the methanolic extract:

The in vitro discharge profile of the methanolic concentrate or PLGA stacked medication nanoparticles were examimed by UV-spectroscopy as appeared in Fig(4a,4b). Nanoparticles were studied over a period of 2 day. It was observed from the graph (fig-4a) that there was a biphasic release which depicts outburst of drug in initial 1 hour period of methanolic extract, thereafter slow and steady release of the drug. However in the case of the drug loaded PLGA nanoparticles there was slow and steady release from the initial stage due to encapsulation of drug by PLGA which confirmed that the when drug would be taken orally, it will also release slowly in the body and hence giving out the impact for a longer duration.

 

The ether group present in active compound anethole of Star Anise adds the properties of lipophil, causing lot of encapsulation of the Star Anise, thus takes long enough time to diffuse it from the nanoparticles to buffer phosphate medium. This initial burst could be due to antimicrobial agent which were distributed evenly exactly beneath the nanoparticles. Later on the constant release was because of the drug diffusion as well as matrix erosion mechanism. Then after this the release was usually due to diffusion of drug through the polymeric matrix of nanoparticles.

 

6. Antimicrobial activities study:

The MIC of Star Anise-loaded nanoparticles against B.pumilus and Staphylococcus aureus the gram-positive bacteria show higher MIC than Enterococcus coli, Pseudomonas aeruginosa gram-negative bacteria, (6000 ppm and 4000 ppm, respectively). Gram-negative bacteria are known for resistance to different types of antimicrobial agents in comparison to gram-positive bacteria.  The efficiency related to nanoparticle during inhibiting growth in bacteria was because of penetration of  nanoparticles in bacterial cells and comparatively better delivery of the Star Anise at  the site^[3]. Somehow the use of the nanoparticles to entrap antimicrobial hydrophobic compounds could improve the activity because of 3 factors i.e., sustained release, improved hydrophilicity and better penetration due to small size. The MIC with different gram positive and gram negative bacteria and the zone of inhibition obtained is listed in Table 2, and the zones obtained are depicted in Figure5 (a,b,c.d)

 

Table 2: Various zone of inhibition obtained against different concentration for different bacterial stains.

S. No	Bacteria	MIC	Zone of inhibition (NP)
1	E.Coli	4000 ppm	7.16mm
2	Pseudomonas aeruginosa	4000 ppm	8.21 mm
3	Staphylococcus aureus	6000 ppm	9.84mm
4	B.pumilus	6000 ppm	10.20 mm

 

 

Antimicrobial Study

Fig 5(a)- Zone of inhibition against Enterococcus coli

 

 

Antimicrobial Study

Fig 5(b)- Zone of inhibition against Pseudomonas aeruginosa

 

 

Antimicrobial Study

Fig 5(c) - Zone of inhibition against Bacillus pumilus

 

 

Antimicrobial Study

Fig 5(d) - Zone of inhibition against Staphylococcus aureus

 

Fig 6 (a)- Zero order Kinetics

X  axis : Time in mins           Y axis: Cumulative % of drug release

Formula Used : Q_t= Q_o - K_ot

 

 

Fig 6 (b) - First Order Kinetics Study

X  axis : Time in mins                          Y axis: log % of drug release

Formula Used : log Q_t= log Q_o-K.t/2.303

 

 

Fig 6 (c) - Hixson Crowell Release model

X  axis : Time in mins

Y axis:

Formula Used : log Q_t= log Q_o-K.t/2.303

 

Fig 6 (d) - Higuchi Release Equation

X  axis : Square root of Time in hrs

Y axis: Cumulative % of drug release

Formula Used: Q = K_h. √t

 

7. Kinetic Analysis of Drug release Profiles:

Model dependent methods were used to determine kinetic Analysis of Star Anise loaded PLGA nanoparticles release profiles. For evaluating the release profile, suitable functions have to be selected. Linear regression model was followed to calculate the order of the Reaction.

 

Table 3: Amount of drug dissolved in solution at various intervals of time.

S. No	Time (in mins)	Q0	Qt
1	0	0	0
2	30	0	0.055556
3	60	0	0.074879
4	90	0	0.166667
5	120	0	0.215217
6	150	0	0.285266
7	180	0	0.287198
8	210	0	0.328261
9	240	0	0.357971
10	270	0	0.397585

Where Q_o = Initial amount of drug in the solution.( most times Q_o =0)

Qt = Amount of drug dissolved in time t.

 

CONCLUSION:

Solvent evaporation method shows less extensive, an easier, less energy consuming and it is a commonly used method especially without an additives specially for producing spherical nanoparticles. A variety of formulations with different surfactant combinations, drugs, polymer and volumes are prepared by using solvent evaporation method. Our result demonstrates the use of solvent evaporation allows significant improvement in encapsulation efficiencyi.e.88.5%, particle size is lesser than 50nm. The in- vitro release indicates that after an initial burst  of release a controlled release of the Star Anise continues for 2 days and even more than that. The controlled release profile of PLGA encapsulated Star Anise nanoparticles shows that the biodegradable nanoparticles have got great potential, so it should be given a special consideration in the delivery system of antimicrobial. The antimicrobial activities of the nanoparticles are evaluated against the gram positive and the negative bacteria along with MICs with the range from 1000 ppm to 15,000 ppm. Antimicrobial results show that such type of nanoparticles which are surely more effective in the growth of inhibition of gram-positive and gram negative bacteria. Kinetic analysis of drug release profile studies show that it follows Zero Order Kinetics and Higuchi Model.\

 

ACKNOWLEDGEMENTS:

The authors thank Amity University, Uttar Pradesh, Noida for providing the required chemicals and infrastructure for carrying out the research work. They also thank IARI, New Delhi and Jamia Milia University, New Delhi for providing the sample analysis assistance.

 

 

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Accepted on 18.11.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(2):499-507.