Generation of Porous Structure from Basil Seed Mucilage via Supercritical Fluid Assisted Process for Biomedical Applications

Mucilage’s are plant derived natural polymer which are valuable due to their nontoxicity, low cost and nonirritating nature, with wide range of applications. In this work, extracted mucilage from basil seeds (BSM) was dried using three various drying methods including (1) laboratory oven drying (2) water substitution with organic solvent and laboratory oven drying and (3) water substitution with organic solvent and supercritical carbon dioxide (SC-CO2) gel drying process. The obtained products were characterized by SEM, BET and FTIR and were compared both qualitatively and quantitatively. The results of this study show that, using SC-CO2 assisted process, the 3-D BSM nanostructured networks were obtained with the pores size diameter about 40 nm, without any agglomeration. Furthermore, specifi c area of the fi nal products was increased from 69 to 92 m2/g by SC-CO2 gel drying in compression with air gel drying. Our observations show that, the amount of solvent residual in the SC-CO2 dried product was affected by the weight of sample, CO2 addition rate and drying time. The residual amount of organic solvent (ethanol) for CO2 fl ow rate of 2 mL/min was found to be 8 ppm after 90 min drying time. The FTIR analyses indicated that the nature of fi nal product did not change during supercritical drying procedure. Overall, the ability to form 3-D structures, and bio adhesive property make BSM as a suitable low cost polysaccharide for biomedical applications such as drug delivery medium, wound dressing and also tissue engineering. Research Article Generation of Porous Structure from Basil Seed Mucilage via Supercritical Fluid Assisted Process for Biomedical Applications Iman Akbari and Seyyed Mohammad Ghoreishi* Department of Chemical Engineering, Isfahan University of Technology, 84156-83111, Isfahan, Iran Dates: Received: 24 May, 2017; Accepted: 14 June, 2017; Published: 15 June, 2017 *Corresponding author: Seyyed Mohammad Ghoreishi, Department of Chemical Engineering, Isfahan University of Technology, 84156-83111, Isfahan, Iran, Tel: +98 31 33915604, Fax: +98 31 33912675, E-mail:


Introduction
In recent years, natural polymers have been investigated and employed for technological and industrial-related applications. Nowadays, a lot of the research effort in the biomedical fi eld is based on natural polymers [1][2][3]. These natural materials have advantages over synthetic ones since they are chemically inert, nontoxic, less expensive, biodegradable, and widely available [4]. Furthermore, owing to their similarity with the extracellular matrix (ECM), natural polymers may also avoid the stimulation of chronic infl ammation or immunological reactions and toxicity, often detected with synthetic polymers [5]. Mucilage's are plant derived natural polymers which are well known since ancient times for their medicinal use. Acacia, tragacanth, gum ghatti, gum karaya, guar gum, and ocimum bascilium are popular examples of plant mucilage's with a wide range of pharmaceutical applications [4,6,7]. Nowadays, mucilages are widely used in the pharmaceutical industries as thickeners, suspending agents, binders, etc. [8].
Ocimum basilicum L. also known as basil is a common herb plant found in many parts of the world especially in the tropical regions of Asia, Africa and central and South America well known since ancient times for their medicinal use [9]. Basil seeds have been used in traditional medicine for a long time to treat colic ulcer, dyspepsia, diarrhea and infl ammations, among others ailments [10]. Basil seeds are black in color and oval in shape and due to the presence of a polysaccharide layer, when they are soaked in water, the outer pericarp swells into mucilage which could be extracted from seeds and dried or concentrated for further applications.
Pharmaceutical applications of various natural mucilage's and their modifi ed forms were reviewed by Prajapati et al. [8]. In the Nerkar and Gattani study, buckle mucoadhesive gel using cress seed mucilage was prepared and the diffusion of venlafaxine from obtained gel was evaluated [11]. Almeida et al., studied the rheological and thermal properties of hydrogel mixtures containing xanthan gum, konjac gum, iota-carrageenan, and kappa-carrageenan for skin scaffold applications [12]. Oliviera et al., were analyzed gellan gum hydrogels for cartilage tissue engineering applications [13].
In our recently published work, extracted mucilage from basil seeds was dried using supercritical CO2 (SC-CO2) to form a 2-D Applications. Int J Pharm Sci Dev Res 3(1): 030-035.
nonporous structure for pharmaceutical applications [14]. In the mentioned work, drying procedure was performed using a modifi ed method based on Temtem et al. [15] study in which methanol was used as co-solvent to improve water solubility in SC-CO2. In the other previous work, ocimum basilicum mucilage was employed as a cost effective polymeric medium for precipitation of paclitaxel for controlled drug delivery [14].
In this work, extracted mucilage from basil seeds was dried using supercritical CO2 (SC-CO2) to form a 3-D nonporous structure 64 for pharmaceutical applications such as tissue replacing applications and drug delivery matrices. A wide variety of technologies are now available to fabricate 3-D scaffolds. Most of previous techniques proposed for gel drying present some limitations such as time-consuming, presence of organic solvents, that are diffi cult to be eliminated and that can remain entrapped inside the polymeric network, limitation in the obtainment and preservation of various levels of porosity and the three-dimensional structure. To overcome these limitations, SC-CO2 assisted processes have been recently implemented. SC-CO2 has been used as an alternative nonsolvent in phase inversion processes to generate polymeric and bio polymeric porous structure [16][17][18]. Although, the obtained structures have a good interconnectivity and high porosity, but it is very diffi cult to obtain complex 3-D structures (fl at membranes are usually generated. However, SC-CO2 shows a very limited compatibility with polar components, like water, at the ordinary temperatures and pressures used in SC-CO2 processing; for example, at 40 °C and 100 bar, water solubility is around 0.5% [19]. Thus, the common supercritical gel drying process is not directly applicable to polymeric hydrogels and a water/solvent substitution method is required. Therefore a new SC-CO2 assisted process for the production of poly (L-lactic acid) (PLLA) scaffolds has been proposed by Reverchon et al. [20]). In this work, the drying procedure was performed using mentioned method in which a water/solvent substitution step is added to the process [20,21]. The obtained nonporous product was also characterized from a macroscopic and microscopic point of view using SEM imaging and also surface area measuring. The Fourier transforms infrared spectroscopy (FTIR) analyses were performed to determine the suitability of SC-CO2 process to produce unchanged structure. Furthermore, the effect of drying time and CO2 fl ow rate on the solvent residue was determined.

Materials
Basil seeds used in this study were purchased from a local market in Isfahan, Iran. Ethanol and acetone (99.99% purity) was obtained from Merck and industrial grade carbon dioxide (≥ 99.9%) was purchased from Zamzam Co. (Isfahan, Iran). All materials were used as received.

Mucilage extraction procedure
The procedure of mucilage extraction from basil seeds was described in details in our previous work [15]. As stated in the mentioned study, the basil seeds were completely swelled in distilled water. The swelled seeds were washed with ethanol/ water solution. Seeds solution was passed through an extractor (Pars Khazar 700P, Rasht, Iran) to scrape the mucilage layer off the seed surface. The separated mucilage was passed through a vacuum fi lter to remove all likely seed residuals. In order to remove protein and ash content, after fi ltration of the crude extract, pure ethanol was added to the extracted mucilage in

SC-CO2 drying experimental set-up
The employed experimental apparatus in this work is similar to our previous works [14,22,23].  temperature were obtained (220-240 bar and 35-45 °C), drying was performed with a SC-CO2 fl ow rate of 1, 2 and 3 mL/min. The drying time lasted in the range of 40-90 min. The vessel depressurized for about 10 min to bring back the system to atmospheric pressure.

Scanning electron microscopy characterizations
To investigate microscopic structure of products, scanning electron microscopy (SEM) was utilized. The samples were sputter coated with gold (SC 7620, Quorum Technologies, UK) at 30 mA for 180 s and analyzed by a scanning electron microscope (DSM 960A, Carl Zeiss AG, Germany) to evidence the micro-and nanostructure and to measure the diameter of the pores and fi bers forming the structure.

Surface area characterization
Surface areas were determined from the isotherm of adsorption of liquid nitrogen using the Brunauer-Emmett-Teller (BET) method (measured on a Quantachrome ChemBET 3000, Florida, and USA, equipped with a thermal conductivity detector) at degasing temperature of 60 °C.

FTIR characterization
The FTIR spectra are considered as useful information for partial characterization of seaweeds [24]. In this work, FTIR (Tensor 27; Bruker) analysis was used to characterize the chemical structure of BSM. Spectra were taken in the region between 4000 and 500 cm−1. Furthermore the suitability of SC-CO2 process to produce unchanged structure was determines using FTIR spectra.

Solvent residue analysis
After carrying out each SC-CO2 drying procedure at specifi ed time, the dried sample was removed from the vessel and the amount of organic solvent (ethanol) residue was measured using an Agilent HP-6890N gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) with HP-5 5% phenyl methyl siloxane capillary column (30m~320μm~0.25μm, nominal) equipped with a G1540N-210 FID detector.

Results and Discussion
In this work, supercritical gel drying was utilized to generate nonporous structure from mucilaginous seeds of ocimum basilicum for tissue engineering applications. Our attention was focused on and the difference between obtained structure in three various drying methods including (1) no water substitution and laboratory oven drying (2) water substitution and laboratory oven drying (3) water substitution and supercritical gel drying. Furthermore, FTIR analyses were performed to determine the chemical structures of the BSM and also identify the effect of drying procedure on the fi nal products structure.

Laboratory oven drying of virgin sample
For the reference sample, hydrogel (with water to dried mucilage ratio of 200) with no further treatment was put on the stainless steel plate and dried in laboratory oven at 50 °C.
As expected, the fi nal product demonstrated a 2-dimensional membrane ( Figure 2) which its SEM image (Figure 3) shows non-porous structure. The solid sphere formed on plate surface is because of gel coagulation and impurities.

Water substitution and laboratory oven drying
The next sample was prepared by water substitution and drying in laboratory oven. To this purpose extra water content of gel was substituted by ethanol via putting in bath for 24 h at -20 °C and dried in laboratory oven at 50 °C. The substitution of water with an organic solvent is one of the most common procedures used for the drying of hydrogels [25]. Ethanol and acetone were tested as water substituting agent. Since during ethanol substitution a complete preservation of gel shape and size was observed in contrast to acetone substitution, thus, ethanol was selected as water substituent. In water substitution step, gel precipitated in ethanol bath as a 3-D structure, thus, the fi nal product from sample No. 2 was not obtained as fl at structure like the fi rst sample. Nevertheless, in this case, the initial 3-D shape was partly shrined due to drying and subsequently a size reduction of fi nal structure was observed

Water substitution followed by supercritical gel drying
In order to overcome collapsing of precipitated fi laments, and to obtain nonporous structure with high porosity, after substitution of water with organic solvent, drying procedure was carried out using SC-CO2, in this part of work. The water substitution with an organic solvent readily soluble in SC-CO2 eliminated the solubility problem of water in CO2. The same results were reported in processing of chitosan [21] and PLLA [20].
The obtained hydrogel was precipitated in pure ethanol and centrifuged (5000 rpm, 10 min at 4 °C) to remove extra water/ ethanol content. Resulted thick gel in a bath of ethanol was dried using SC-CO2 as mentioned in 2.2 (Mucilage extraction procedure). Processing temperature was set in regard to the CO2 critical temperature (T=304.2 K) and the possible gum thermal degradation. Processing pressure was selected to assure a suffi cient SC-CO2 solvent power for ethanol/water mixture. The drying temperature and pressure were kept constant during the experimentation. The required fl ushing time to ensure that the obtained fi nal products were free of solvent, water and ethanol, was different for each experiment. Initial investigations indicated that, for all cases, fl ushing time of 2 h was a reasonable and suffi cient time to obtain free solvent (ethanol/water) products. Thus, fl ushing time of 2 hours was selected as the optimum for all experiments. After fl ushing time, the dried BSM was taken from the vessel and was analyzed by SEM, BET and FTIR. In this case, the 3-D shape and the sample size not only was successfully preserved, but also increased in size due to diffusion of SC-CO2 in gel structure ( Figure 6). The reported SEM images in fi gure 7 show the presence of micro porous structure with a uniform sub nonmetric network. The mean diameter of the nano metric fi bers was measured to be about 40 nm. Specifi c surface areas of obtained samples were measured using BET method. The results showed a value of 92 m2/g which was increased more than 30 percent and indicate high porosity of the obtained sample.
In the SC-CO2 process, volume expansion of solution occurs when the CO2 is dissolved in the solution as modelled in our previous work thermodynamically [23]. The volume expansion of solution, water and ethanol in this case, leads to expansion of polymer structure and thus high level of porosity was obtained using SC-CO2 gel drying method in contrast to oven drying method. Furthermore, during the expansion, because of high diffusivity, supercritical mixture diffuse to the polymer structure and thus nonmetric fi bers and pores were obtained. The biocompatible scaffold obtained by this method demonstrated a high level of porosity, with a good interconnectivity among the pore network system. The obtained structure has a high level of porosity, with a good interconnectivity among the pore network system. Thus, the obtained structures present morphology very similar to an extracellular matrix (ECM), i.e. a fi nely interconnected nonmetric substructure, suitable for scaffolding applications in tissue engineering. Furthermore the obtained scaffold provides support for cell attachment continued by their proliferation and differentiation and also allowing controlled release of biomaterial exposed to cells.

Solvent residual
The results of this study indicated that the amount of solvent residual in the SC-CO2 dried product was affected by the weight of sample, CO2 addition rate and drying time.   diffusion to the polymeric network becomes the controlling step in the drying process and its effect governs the solvent residual amount. Thus, the solvent residual reduction at higher drying time (75-90 min) reaches an almost steady state trend despite of higher CO2 fl ow rate. The residual amount of ethanol for CO2 fl ow rate of 2 mL/min was found to be 8 ppm after 90 min drying.

FTIR characterization of the obtained products
The FTIR spectra are considered as useful information for partial characterization of seaweeds [24]. In our previous work FTIR spectra of BSM was discussed in details. As shown in fi gure 9, FTIR spectra of three fi nal products show same peaks which indicate that the structure of the BSM was not affected by the drying procedures. Furthermore, as mentioned in the previous work and the obtained peaks of fi gure 9 indicated that, BSM has good bio adhesive property and thus this porous polysaccharide can be employed formation for tissue engineering and wound dressing applications. Many actives can be released through such bio adhesives, as steroids, anti-infl ammatory agents, pH sensitive peptides and small proteins such as insulin, and local treatments to alleviate pain in the buckle cavity.

Conclusions
The generation of polymeric nonmetric structured from mucilaginous seeds of ocimum basilicum was successfully carried out for scaffolding applications using SC-CO2 based methods for hydrogels drying. Various drying methods were used and fi nally an effi cient low temperature water-solvent substitution step followed by SC-CO2 drying was developed in order to obtain a nonporous structure. It was shown that a collapsed structure with minor porosity was obtained by using water/solvent substitution and oven drying, while a nonmetric fi brous network was achieved by the SC-CO2 assisted process. The results of IR data show that BSM has good bio adhesive property and many actives can be released through such bio adhesives, as steroids, anti-infl ammatory agents, pH sensitive peptides and small proteins such as insulin, and local treatments to alleviate pain in the buckle cavity.
Furthermore, the amount of organic solvent residual in the fi nal product could be controlled by varying CO2 fl ow rate and