An antibacterial delivery system based on Eucalyptus camaldulensis loaded in starch microspheres

Antibacterial Delivery System Based on Eucalyptus Camaldulensis Loaded in Starch Microspheres

Leila Sadeghzadeh ¹, Mohammad Rafienia ^2,*, Fatemeh Sefidkon ³, Parviz Owlia ⁴, Babak Negahdari ⁵,  Shahin Bonakdar ^6,*

¹ Nanotechnology Researchers Company, Tehran, Iran.

² Biosensor Research Center, Isfahan University of Medical Sciences, Isfahan, Iran.

³ Research Institute of Forests and Rangelands, Tehran, Iran.

⁴ Molecular Microbiology Research Center, Shahed University, Tehran, Iran.

⁵ School of Advanced Technologies in Medicine, Tehran University of Medical Science. Iran.

⁶ National Cell bank. Pasteur Institute, Tehran, Iran.

Correspondence to: Bonakdar S. (E-mail: Shahinbonakdar@yahoo.com)

Keywords: Antibacterial, Drug delivery, Eucalyptus oil, Microspheres, Starch

Received: 28 December 2018, Accepted: 14 February 2019

DOI: 10.22034/jtm.2019.93448

Abbreviations
Eucalyptus camaldulensis oil (EO)

water-in-oil (w/o)

water-in-water (w/w)

1. Introduction

Starch as the most abundant storage reserve carbohydrate in plants is found in many different organs, including seeds, fruits, tubers and roots. It is a natural useful polymer not only as an available low-cost material but also due to its diversity in versatile applications. Because of its attractive characteristics (non-toxicity, biodegradability, edibility and good film forming ability), starch has been widely studied as raw material for preparing microparticles for food industries [1-5] and pharmaceutics [1, 3, 6-8]. Moreover, the physic-chemical properties of this bio-polymer can be easily modified through chemical or enzymatic alteration and/or physical treatment. Some of its main limitations are listed as low shear resistance, low thermal resistance, low thermal decomposition and retrogradation tendency [9, 10]. Self-associating (induced by changes in pH, ionic strength or physical and thermal means), mixing with salts and covalent cross-linking are some of the widely adopted strategies to overcome inadequacies of the starch [11].

Biodegradable starch microspheres have been used for controlled release of different drugs such as ceftriaxone [12], ampicillin [13], calcein and timolol [14, 15], theophylline [16], protein or peptide like insulin [17, 18], and rotavirus vaccine [19]. Different approaches have been investigated for preparation of starch microspheres including spray drying, precipitation, solvent evaporation and emulsion-cross-linking. Among these, water-in-oil (w/o) and water-in-water (w/w) emulsion-cross-linking techniques have been extensively used and rapidly developed [20-23].

Eucalyptus extracts are well characterized for different biological activities such as antibacterial, anti-fungal or anti-oxidant [24-27]. They have been suggested to be utilized in pharmaceutical applications including anti-inflammatory, analgesic, antipyretic remedies, asthma, pulmonary tuberculosis, osteoarthritis, joint pain, wound healing and diabetics [28-32].

 In previous study, the starch microspheres containing ceftriaxone were prepared with the emulsion cross-linking technique, using glutaraldehyde as cross-linker agent [12]. The results suggested feasibility of ceftriaxone encapsulation within starch microspheres which could deliver bioactive agents to the physiological milieu. In the present study, herbal drug was extracted from Eucalyptus leaves and loaded into the starch microspheres. The sustain release and anti-bacterial activity of this system were evaluated by alteration in drug/polymer ratio and cross-linking time.

1. Materials and Methods

2.1.   Materials

Soluble starch (C₆H₁₀O₅) with 70-80% amylopectin and 20-30% amylase, cyclohexane, chloroform and acetone, glutaraldehyde were purchased from Merck (Germany) and used as received.

2.2.   Extracting natural drug

Air-dried leaves of Eucalyptus camaldulensis (Collected from Khuzistan province, Research Station of Kushkak, Shushtar, cultivated plant, Origin: Australia) were subjected to hydro-distillation for 2.5 h using a clevenger-type apparatus. The oils was separated from water, dried over anhydrous sodium sulfate and stored in sealed vials at low temperature before analysis.

2.3.   Gas Chromatography (GC) and GC/Mass Spectrometry analysis

GC analysis was performed using a Shimadzu GC-9A gas chromatograph equipped with a DB-5 fused silica column (30 m x 0.25 mm i.d., film thickness 0.25 mm). Oven temperature was held at 40 °C for 5 min and then programmed to 280 °C at a rate of 4 °C/min. In addition, injector and detector (FID) temperature were set at 290 °C and helium used as carrier gas with a linear velocity of 32 cm/s.

GC-MS analysis was carried out on a Varian 3400 GC-MS system equipped with a DB-5 fused silica column (30 m x 0.25 mm i.d.); Oven temperature was set at 40 C to 250 C at a rate of 4 C/min, transfer line temperature 260 C, carrier gas helium with a linear velocity of 31.5 cm/s, split ratio 1/60, ionization energy 70 eV; scan time 1 s and mass range of 40-300 amu. The percentages of compounds were calculated by the area normalization method, without considering response factors. The components of the oil were identified by comparison of their mass spectra with those of a computer library or with authentic compounds and confirmed by comparison of their retention indices either with those of authentic compounds or with data published in the literature [33, 34]. The retention indices were calculated for all volatile constituents using a homologous series of n-alkanes.

2.4.   Preparation of starch microspheres

This experiment was designed based on the Taguchi methods to optimize the sample preparation (using MINITAB software, Release 14.20). Four factors including 3 levels of variation were selected. These factors were drug loading percentage, volume of cross-linking agent, rate of homogenizing and duration of cross-linking; three different values being chosen for each. Therefore with 9 experiments, 81 variations were possible. The starch microspheres were prepared by an emulsification technique. This process, described in detail in previous work [12], can be summarized as follows: 0.1, 0.15 and 0.2 mg of Eucalyptus oil were diluted into 2 ml ethanol solution (70 % (v/v)) and 500 mg starch dissolved in 30 ml distilled water separately. These two solutions were mixed and added to a 20 ml stirred solution of cyclohexane-chloroform (60:40 v/v) as oil phase. The aqueous phase was emulsified in this solution using a homogenizer under 8500, 13500 and 20000 rpm for 10 min at 25 ºC. Afterwards, a cross-linking solution consisting different ratios of glutaraldehyde-acetone was added, and the mixture held for 4 and 6 h at 25 ºC to complete the cross-linking process (Table 1). Finally, the Eucalyptus oil loaded starch microspheres were isolated by centrifugation, washed twice with distilled water and freeze-dried for 12 h.

2.5.   Evaluation of antimicrobial effect

Antimicrobial effects of microspheres were evaluated against Micrococcus luteus PTCC 1110 and Escherichia coli PTCC 1330 using disk diffusion method (Anhalt and Washington, 1985). Micrococcus luteus is a Gram positive saprotrophiccoccus that belongs to the family of Micrococcaceae. An obligate aerobe, M. luteus is found in soil, dust, water, air and some part of the normal flora of the mammalian skin. This bacterium also colonizes the human mouth, mucosae, oropharynx and upper respiratory tract [35]. Escherichia coli (commonly E. coli), is a gram negative bacterium that is commonly found in the lower intestine of warm-blooded animals. Most E. coli strains are harmless, but some, such as sero type O157:H7, can cause serious food poisoning in humans, and are occasionally responsible for costly product recalls [36, 37]. Plates of Muller Hinton agar were inoculated using sterile swabs with the bacterial suspension at 0.5 Mac Farland’s scale. Blank disks with 6 mm diameter were loaded with 20 µl of sample with concentration of 30 µg/ml (30 mg microspheres in 1 ml PBS) and placed on the surface of the inoculated plate. After incubation at 37 ºC for 24 h, inhibition zone was measured by vernier caliper. Each study was performed in triplicate, i.e. three samples taken from one formulation and repeated three times. As a control, some plates were loaded with EO and suspension of starch microspheres without EO.

2.6.   Fourier Transform Infrared Spectroscopy

   FTIR spectra were recorded on an Equinox 55IR Spectrometer (Bruker, USA). Scans for samples were recorded at a resolution of 2 cm^-1 over the wave number region 400–4000 cm^-1. Samples were mixed with KBr spectroscopy grade and pressed into a disc by compaction. IR spectra were recorded of drug before and after sterilization. The weight ratio of KBr:powder was considered 100:1.

2.7.   Morphology and particle size analysis

Morphology of microspheres was investigated with a scanning electron microscope (SEM) model XL30 (Germany), operating at 10 KV. They were sputter coated with gold using a Polaron sputter coater before mounting on stabs. Dynamic laser light diffraction technique (Euromex, Netherlands) was employed to measure mean diameter of microparticles.

2.8.   In vitro release study

The release profiles were obtained by dispersing 10 mg of each freeze-dried sample in 20 ml phosphate buffer saline (pH 7.4), as a release media. The release data were collected for more than 500 h under the sink condition. An ultraviolet-visible spectrometer (Milton Roy Spectronic 601, Canada) set at 546 nm was used to measure the release rate. A calibration curve was prepared at different Eucalyptus oil concentrations (0.1–1 mg/ml) in the phosphate buffer. At predetermined intervals, 3 ml of release media was removed to assay drug concentration, and 3 mL of fresh buffer added. In order to determine total amount of loaded Eucalyptus oil in each formulation, 10 mg of each sample was dispersed in 50 ml of PBS for 24 hours at 37 °C. This was followed by centrifugation (35000 rpm) to completely separate the precipitated polymer. The efficiency of extraction and recovery of Eucalyptus oil from starch microspheres were measured independently in triplicate. The loading efficiency (LE) of EO and loading capacity (LC) were calculated according to the following equations:

(1)

(2)

RI = Retention indices in elution order from DB-5 column    MS = Mass Spectroscopy      

CoI = Co-Injection             

1. Results and Discussion

3.1.   FTIR study

Figure 1 shows the FTIR spectroscopy of all the samples. The bands at 3450 cm^-1 and 2950 cm^-1 are related to the O-H stretching, and 1230 cm^-1 assigned to the O-H bending vibration. In addition C-H stretching vibrations can be observed at 1454 cm^-1 [13].

3.2.   Loading efficiency and loading capacity of microparticles

The loading efficiency and loading capacity of starch microspheres were determined by UV-visible spectrophotometry. The initial concentration of Eucalyptus oil (EO) plays an important role in the LE and LC of starch microparticles (Table 1). These results indicate that the LE of EO increases with an increase in EO percentage. Moreover, un-crosslinked samples show the minimum LEs (Samples N1, N6 and N8). It was found that increasing the cross-linking time and cross-linking amount enhances the LE and LC of drug.

3.3.   In vitro release study

The release profiles of two formulations are illustrated in Figure 2. In all specimens, the release rates were extended up to 100 h after the starch microspheres immersed into release medium.  Initially, 67% of loaded drug was immediately released from the superficial part of the dispersed microspheres. Subsequently, sustain release of EO continued based on swelling- controlled mechanism to release more than 80 % of the drug (38).

Figure 2. Cumulative release of Eucalyptus oil from two samples in PBS (pH 7.4)

By the immersion of microspheres in the dissolution medium, due to the water absorption of the matrix, a gel diffusion layer is formed. The presence of water at the outer layer, delayed the outward transport of the drug [39].

Drug in the microspheres might also act as inert filler and occupy free volume spaces inside the swollen hydrogel. This would create tortuous paths for water molecules to transport freely, but the degree of tortuosity depends upon the volume fraction of the filler [40]. On the other hand, by increasing in swelling, greater number of EO molecules diffuse out of the starch microspheres and pass into the release medium through numerous pores and channels in the microspheres [38]. Release profiles of drugs were greatly influenced by percent loading of the drug. Therefore, with an increase in drug loading, the release rate of drug was intensified. The amount and time of cross-linking influence on particle degradation and prolong drug release [13, 38]. In Figure 3(a) it can be seen that increasing the initial drug loading amount, enhances the drug release rate. On the other hand, increase in rate of homogenizing, amount of cross-linking agent and duration of cross-linking slightly decrease release rate.

3.4.   Oil Analysis

The oil isolated by hydro-distillation from the leaves of Eucalyptus was found to be colorless to pale yellow. This oil was analyzed by capillary gas chromatography using flame ionization and mass spectrometric detection. The oil yield was calculated 1.78% (w/w) based on dried weight. In addition, twenty- six components were identified in the oil of Eucalyptus and the major components were 1, 8-cineole (50.9%), p-cymene (3.6%), a-pinene (11.3%) and globulol (5.2%). The chemical composition of the oil can be seen in Table 2. The components are listed in order of their elution on the DB-5 column.

3.5.   Antibacterial activity study

The antibacterial activity of the starch microspheres was determined by measuring the inhibition zone. All samples showed antibacterial effects, which means that all matrices released a significant amount of EO and inhibited the growth of both bacteria. Formation of an inhibition zone shows that EO can easily diffuse

Figure 3. Effect of different factors on drug release (a) and average diameter of inhibition zone for Micrococcus luteus (b).

into the agar media from loaded microspheres and its antibacterial activity remains unaffected after loading into the starch microspheres. Table 3 demonstrates results of measuring inhibition zone in all samples and controls. The average diameters of inhibition zone were calculated in the range of 14 to 32 mm.

Figures 4(a) and 4(b) show the effect of different amounts of drug loading, homogenizing rate, cross-linking agent and duration of cross-linking on the production of inhibition zone for Micrococcus luteus and Escherichia coli. The effects of these parameters on the inhibition zone diameter illustrated the same trends for both bacteria. Regarding these data, the inhibition zone diameter for cross-linked samples was larger than uncross-linked ones. Moreover, the diameter of inhibition zone was increased by

Figure 4. Effect of different factors on average diameter of inhibition zone for Escherichia coli (a) and average diameter of starch microspheres (b).

increasing the cross-linking time, as shown in Table 3 and Figures 3(b) and 4(a). The sample N4 cross-linked for 6 h and displayed the largest inhibition zone at the vicinity of Escherichia coli with maximum diameter of 31.3±3.8 mm. It can be suggested that increasing in the cross-linking time decreases the rate of drug release. Larger diameter of inhibition zones were observed for the samples in the vicinity of Escherichia coli in comparison with Micrococcus luteus. These higher values were related to the higher sensitivity of Escherichia coli to EO.

3.6.   Particle size analyzing

Particle size and size distribution were analyzed by Dynamic laser light diffraction technique. Mean diameter of the microspheres as well as their different characteristics were summarized in Table 3. Particle size analysis of starch microspheres containing Eucalyptus camaldulensis oil indicates that the mean diameter of microsphere is affected by homogenizer stirring speed, time of cross-linking and amount of cross-linking agent in all formulations. The homogenizer speed plays significant role in the emulsification step. Because, it supplies the energy to

Figure 5.  Scanning electron micrograph of starch microspheres with different magnifications.

disperse starch solution in the oil phase. A decline in microsphere size was observed with increase in homogenizer speed up to 13500 (rpm). On the other hand, mean diameter of starch microspheres was increased in the speed of 20000 (rpm).

The results revealed that mean particle size of microspheres was inversely related to stirring speed. Consequently, increase in stirring speed (until 13500) decreased the size of microspheres. It may be proposed that the second emulsion was broken up into smaller droplets at a higher input power in agreement