INTRODUCTION:

The prominence of multidrug-resistant bacteria is a huge challenge for global health. Killing of antibiotic-resistant bacteria requires manifold pricey drugs that may have side effects. This will lead to expensive treatments for a long period of time. It is the need of the hour to develop herbal products that can substitute existing products in antimicrobial therapy. The antimicrobial property of compounds extracted from medicinal plants is of great significance for medical and food application¹. These compounds are synthesized in the secondary metabolism of the plant such as flavonoids, tannin and essential oil.

Nanoparticles can offer a new strategy to tackle multidrug-resistant bacteria², therefore it is wisely proposed to combine herbal medicine with nanotechnology, because nanostructured systems might be able to potentiate the action of plant extracts, reducing the required dose and side effects, delivering it to the desired site of action and thus improving activity³.

Polymeric nanoparticles are made from biodegradable and biocompatible polymers, represent an option for controlled drug delivery and are promising formulation used for drug delivery systems, because they can be targeted where as hydrogels are polymeric networks with three-dimensional configuration capable of imbibing high amounts of water or biological fluids^4,5.

Mimosa pudica L., also called sensitive plant or touch-me-not, belongs to the genus Mimosa (Family: Mimosaceae). M.pudica has been used as a traditional medicine for the treatment of some diseases and conditions including diarrhoea, insomnia, tumour, headache, skin conditions, fever and blood pressure⁶. Some studies showed the presence of various bioactive compounds in this plant like tannins, steroids, flavonoids, glycosides, non-protein amino acid leucenine (mimosine), alkaloids⁷.

In view of its medicinal importance of M.pudica, attempts has made to formulate a nanoparticle hydrogel for topical application and to validate its antimicrobial properties and checking the effect of the polymer PLGA on its antimicrobial properties.

MATERIALS AND METHODS:

Materials:

Poly Lactic Glycolic Acid (PLGA 50:50) was procured from Sigma Aldrich, Bangalore and H.P.M.C K4M was obtained from Yarrow Chem products, Mumbai.

Preparation of plant extract:

Leaves of Mimosa pudica were collected from local area of Bantwal Taluk and authenticated. Leaves were dried, powdered and then extracted by cold maceration method using 95% ethanol as solvent for 7 days. After the extraction solution was filtered and filtrate was evaporated to dryness and percentage yield was calculated. Prepared extract was subjected to different chemical tests according to standard procedure in order to determine the presence of various phytoconstituents⁸.

Formulation of Polymeric Nanoparticles:

Nanoparticles of plant extract were prepared by emulsion solvent evaporation method⁹ employing PLGA as polymer. The organic phase was made by various concentrations of PLGA and plant extract in DMSO. The organic phase was then added dropwise at the rate of 1ml/min into an aqueous phase containing surfactant (PVA -0.5%) dissolved in water as aqueous solvent. The nanoparticles suspension was stirred constantly at 300 rpm for 3 h at 30°C to allow the complete evaporation of DMSO, leaving behind the colloidal suspension of PLGA nanoparticles holding plant extract in aqueous phase. The colloidal nanosuspension was centrifuged at 12,000 rpm (Remi, Mumbai, India) for 30min at 4°C to get the final nanoparticulate containing pellet as encapsulated plant extract. The pellet was washed with deionized water twice to remove unentrapped drug from the surface of nanoparticles. Nanoparticulate pellets were redispersed in water.

Table 1: Composition of pyrazoline loaded polymer nanoparticle

Formulation	Drug (mg)	Polymer (mg)	Surfactant (%)
MP1	25	50	0.5
MP2	25	100	0.5
MP3	25	200	0.5

Characterization of Polymeric Nanoparticles:

Measurement of Particle size, PDI and Zeta Potential of the nanoparticles:

Average particle size (z-average), polydispersity index (PDI) and zeta potential of the nanoparticles were found by laser dynamic light scattering using Malvern Zetasizer (Nano ZS, Malvern Instruments, UK)¹⁰.

Entrapment efficiency of the polymeric herbal nanoparticle:

The nanoparticle suspension formulated with the extract and polymer was ultra-centrifuged at 18,000 rpm for 30 minutes in a cooling centrifuge apparatus and then the supernatant solution was diluted suitably to measure the absorbance, from which the concentration of drug in supernatant was calculated using the standard calibration data¹¹. The entrapment efficiency of the extract in the polymeric nanoparticles was calculated using the formula,

Total amount of Durg added - amount of Drug is supernatant

Entrapment ----------------------------------------------------------------x100

Effciency (%) Total amount of amount added

Based on the outcomes of particle size analysis, PDI and entrapment efficiency, optimised formulation of nanoparticles was chosen and scanning electron microscopy (SEM) and transmission electron microscopy (TEM) was carried out.

Formulation of Nanoparticulate Hydrogel:

The optimized pyrozoline loaded polymer nanoparticle (MP2) was incorporated in hydrogel matrix. In a separate containers, the hydrogel forming polymers were dissolved in small amount of double distilled water in various proportions as shown in table 2 and then remaining ingredients i.e. glycerine and sodium benzoate were added. The Prepared nanoparticle formulation was added to it and the weight up to 50 g. Then, probe sonicated at amplitude of 25, 30 sec. The above formulation was allowed to stand for 24 hrs at room temperature. The pH of this gel preparation was maintained 6.5 ± 05 by using Triethanolamine and stored in well closed¹².

Table 2: Composition of various Hydrogel formulations

Formulation	HGN1	HGN2	HGN3
MP2	10 mg	10 mg	10 mg
Carbopol 934	500 mg	500 mg	250 mg
HPMC 15 LV	250 mg	500 mg	500 mg
Glycerine	2 ml	2 ml	2 ml
Sodium benzoate	100 mg	100 mg	100 mg
Triethanolamine	q.s.	q.s.	q.s.
Distilled water	q.s 50 g	q.s 50 g	q.s 50 g

Antimicrobial screening of extracts:

Ethanolic extract of Mimosa pudica were evaluated for their antimicrobial activities against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonous aeruginosa. The antimicrobial activity was determined by the agar well diffusion method using Muller Hinton agar media¹³. Each Petri dish containing Muller-Hinton agar medium was inoculated with one bacterial culture by spreading the suspension of the organism with a sterile glass rod with a bended tip. Wells were made on the agar surface with 6 mm cork borer. The first cup was filled with Ciprofloxacin, second was filled with DMF and other is filled with extracts (50mg/ml) using sterile syringe. The plates were incubated at 37±2oC for 24 hours. The plates were observed for the zone formation around the wells. The zone of inhibition was calculated by measuring the diameter of the inhibition zone around the well (in mm) including the well diameter. The readings were taken in three different fixed directions in all 3 replicates and the average values were tabulated.

In Vitro Drug Release Study of Hydrogel:

Prepared nanoparticulate hydrogel formulations (HGN1, HGN2, HGN3) was evaluated for the percentage of release of the extract constituents, for 24 h. In vitro release of drug across the dialysis bag (12 Kda, Hi Media) soaked in deionized water for 12 h before use was performed by using diffusion cell (containing 1 ml of sample) and 80 ml of phosphate buffer pH 6.8 as the dissolution medium (n=6). The dissolution medium was maintained at 37 ± 0.5ºC and the medium was stirred at 100 rpm with the help of small teflon coated magnetic bead. Aliquots of the medium were withdrawn at suitable time interval and were replaced with the same volume of fresh medium to maintain the sink condition. These samples were filtered through 0.45 µm membrane filter and the collected samples were analyzed using UV-visible spectrophotometer at the lmax of 203 nm¹⁴.

RESULTS AND DISCUSSION:

Ethanolic extract of Mimosa pudica were greenish in colour and percentage yield was found to be 14% respectively. The preliminary phytochemical analysis showed the presence of flavonoids, tannins, steroids, carbohydrates, proteins in the extract.

Preparation and Characterisation of Polymeric Nanoparticles:

Polymeric nanoparticles were prepared by solvent evaporation method with different ratios of Mimosa pudica extract and polymer PLGA. All the formulations had shown particle in nanosize range 95.7-455.2 nm. As shown in table 3., as the concentration of polymer was increased, particle size was also increased, may be at the higher concentration of polymer in the sample may have led to an increased frequency of collisions, resulting in fusion of semiformed particles and producing finally an overall increase in the size of nanoparticles. Polydispersibility index (PDI) of formulations was found to be in a range of 0.236- 0.298. The zeta potential of the synthesized herbal nanoparticles was found to be -2.07 mV to -3.5 mV. The different nanoparticle formulation had entrapment efficiency in the range of 42.5– 69.3 %. Results revealed that as polymer concentration increases till 100 mg of PLGA, EE increases and further it was decreased. At high polymer concentration (200 mg), the viscosity was so high that the efficiency of emulsion was reduced and allowed the production of large particles (455.2 nm in mean diameter) with reduced entrapment efficiency (63.9%).

Table 3: Characterization of nanoparticles loaded with extract of Mimosa pudica

Formulation code	Particle Size (nm)	PDI	Zeta Potential (mV)	Entrapment Efficiency (%)
MN1	95.7	0.256	-2.07	42.5
MN2	180.3	0.226	-3.14	69.3
MN3	455.2	0.298	-3.50	63.9

Based on results of particle size and entrapment efficiency, nanoparticle formulation MP2 containing Mimosa pudica ethanolic extract with 100 mg of PLGA polymer was selected as optimised formulation as it has nanosize range (<200 nm) particles with low PDI and sufficient entrapment efficiency.

Scanning electron microscopy (SEM) and TEM:

The SEM study reveals that polymeric nanoparticles were spherical in shape with an average particle size around 97.3 nm as shown in Fig.1. The transmission electron microscope revealed a positive image in which nanoparticles appeared dark with bright surroundings as shown in Fig.2. The average droplet size of sample was less than 1000 nm. These results confirmed that the droplets were in nanosize range. Moreover, z-average gives the hydrodynamic size when the particles are suspended in aqueous media. TEM images would give a better understanding of the real geometric size of the particles.

Fig. 1

Fig. 1: SEM image of optimized nanoformulation loaded with extract

Fig. 2

Fig. 2: TEM image of optimized nanoformulation loaded with extract

Formulation and characterisation of nanoparticulate Hydrogel formulation:

Optimzed polymeric nanoparticle (MP2) incorporating hydrogel formulations HGN1, HGN2, HGN3 were prepared by varying ratios of HPMC 15 LV:Carbopol 934 hydrogel forming matrix 1:2, 1:1 and 2:1 respectively. Prepared hydrogel were subjected for determination of pH, spreadability, viscosity and drug content and results of which is shown in table 4. The drug content of nanoparticulate hydrogel formulation was in the range of 90.6 ± 0.38% to 95.4±0.21%. The results showed that the drug was uniformly distributed throughout the formulation and drug loss was minimum while formulating nanoparticulate hydrogel. The pH values of different nanoparticulate hydrogel formulations were found to be in a range of 6.4–6.8 (nearly neutral), permitting the use of the formulation on the skin. The spreadability of the all formulations exhibited slip and drag phenomenon with higher diameters. Viscosity of hydrogel formulation decrease as the ratio of carbopl : HPMC decreases. In HGN1 and HGN2 conatin same amount of cabopole but different HGN1 (250 mg) and HGN2 (500 mg) concentration of HPMC.

Table 4: Characterization of Hydrogel

Formulation code	Drug content	pH	Spreadability g.cm/sec	Viscosity (m.PaS)
HGN1	94.8±0.23	6.4±0.2	5.0±0.32	4231±0.21
HGN2	95.4±0.21	6.5±0.3	6.5±0.22	4995±0.18
HGN3	90.6±0.38	6.8±0.1	5.3±0.42	3311±0.16

All the values are expressed as mean ± SD (n= 3).

Antibacterial activity:

Among the four bacterial strains tested for antibacterial activity, S.aureus was most susceptible with inhibition zones ranging from 13-27 mm (Table 5). Antibacterial activity may be attributed to the presence of phytoconstituents namely flavonoids, tannins and saponins in the extracts. Zone of inhibition of Mimosa pudica loaded PLGA nanoparticles and hydrogel was almost similar to that of isolated plant extract activity. This shows that incorporation of extracts into polymeric hydrogel does not decrease its activity.

Release studies from nanoparticulate hydrogel formulation:

In-vitro release studies were carried out by using modified Franz diffusion cell. In order to obtain the rate of release, the release study showed that the nanoparticle loaded m.pudica was released in a sustained manner from hydrogel, due to the presence of the sustained release polymer carbopol and HPMC ( Fig.3). The drug release of formulation HGN1 was found to be 44.67 % due to the presence of large concentration of Carbopol 934, on the other hand formulation HGN2 and HGN3 shown increased release of 75.42% and 89.01% respectively, due to increase in HPMC concentration in the formulation which is more hydrophilic in nature. On the basis of drug release study, formulation HGN3 was found to be optimized prepared.

Fig 3: In-vitro drug release profile of nanoparticulate hydrogel loaded with extract of Mimosa pudica

CONCLUSION:

The novel nanoparticulate hydrogel formulation loaded with herbal extract of appropriate viscosity was effectively formulated for transdermal application. PLGA nanoparticles with Mimosa pudica incorporated

into the hydrogels showed controlled release of active constituents which might be a likely carrier for transdermal delivery of active constituents in the extract

and will be good at exerting antibacterial action.

ACKNOWLEDGMENT:

Thanks to authorities of Nitte University and NGSM Institute of Pharmaceutical Sciences for providing the financial support and facilities for performing this project. I am also grateful to Dr. Nagalaxmi, Botany Department, Aloysius College, Mangalore for assisting in the identification of plant material.

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Received on 15.02.2018 Modified on 27.03.2018

Research J. Pharm. and Tech 2018; 11(7): 2876-2880.

DOI: 10.5958/0974-360X.2018.00530.9

Plant extracts	Zone of inhibition (mm)
Plant extracts	*Staphylococcus aureus*	*Bacillus subtilis*	*Escherichia coli*	*Pseudomonous aeruginosa*
Mimosa pudica extract	26	12	13	16
Mimosa pudica loaded PLGA nanoparticles	25	12	12	16
Mimosa pudica loaded hydrogel	25	11	12	16
Ciprofloxacin (Standard )	29	22	17	19