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Evaluation of jelly fig polysaccharide as a shell composite ingredient of colon-specific drug delivery
Journal of Drug Delivery Science and Technology xxx (xxxx) xxxx
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Evaluation of jelly fig polysaccharide as a shell composite ingredient of colon-specific drug delivery Thangavel Ponrasua, Jhao-Syuan Gua, Jia-Jiuan Wub, Yu-Shen Chenga,∗ a b
Department of Chemical and Materials Engineering, National Yunlin University of Science and Technology, Douliu, Yunlin, 64002, Taiwan Department of Nutrition, China Medical University, No. 91, Hsueh-Shih Road, Taichung, 404, Taiwan
ARTICLE INFO
ABSTRACT
Keywords: Ficus pumila var. awkeotsang Pectin Polysaccharide Hard capsule Colon-targeted delivery
The polysaccharide extracted from the achenes of jelly fig (Ficus pumila var. awkeotsang) was characterized as low methoxyl pectin and was blended with commercial high methoxyl pectin in film casting to evaluate the suitability of the combinations for developing a composite encapsulant for colon target drug delivery. The physiochemical properties of the casted films were characterized by Fourier Transform Infrared (FT-IR) spectroscopy, color measurement, thickness test, water content analysis, and mechanical strength measurement. Among all tested blending ratios, the film prepared from pure jelly fig polysaccharide has higher tensile strength and better performance of anti-erosion in simulated gastrointestinal fluids. Hence, for evaluation of applicability, the composite hard capsules were prepared by using jelly fig polysaccharide as the main component blending with xanthan gum and resistant starch. The results of the in vitro release study showed that jelly fig polysaccharide composite capsules were stable without significant deformation and releasing of encapsulated substance in the simulated gastrointestinal fluids during the test period. Then, the capsules were degraded in simulated colonic fluid in 2 h and the release rate was increased from 15% to 90%. Therefore, pectinous polysaccharide extracted from jelly fig could be a useful material for the preparation of a colon-targeted capsule.
1. Introduction Colon-specific drug delivery systems (CDDS) play a vital role in increasing the bioavailability of drugs in the colon through oral administration [1,2]. CDDS has a great impact to improve the treatment of local diseases affecting the colon and minimizing systemic side effects [3]. The delivery of drugs specifically to the colon without being absorbed first in the upper gastrointestinal tract [4] allowing high concentrations of drugs to reach colon with minimal body resorption. This system has been applied to reduce the delay of delivering the drugs to achieve high concentration, dosing frequency, and achieve sustained release during the treatment of inflammatory disease of the gut [5]. CDDS through oral admission can be classified into different types according to the functional mechanisms including time-dependent, pHresponsive, osmotically controlled, and bacterial triggered [6,7]. Nevertheless, time-dependent, pH-responsive, and osmotically controlled release systems are not always reliable among different cases. Since the colon contains more than 400 species of microorganisms that can produce a variety of enzymes capable of effectively degrade undigested carbohydrates reaching the large intestine [8], the bacterial triggered mechanism for drug release has become a popular research
∗
topic in CDDS. Successful delivery of a drug to the colon requires suitable protection for the drug from degradation or release in the stomach and sustained release in the colon [9]. Therefore, proper care must be taken while formulating drugs and dosage for CDDS to overcome the obstacles caused by the gastrointestinal tract during the drug delivery. The desired properties of CDDS can be achieved by using some polymers either alone or in a combination [10]. Some nature polymers like pectin [11], chitosan [6], alginates [12], and gum arabica [13] are inexpensive and available in various structures. Jelly fig (Ficus pumila var. awkeotsang) is a native plant of Taiwan and its achenes are a rich source of pectinous polysaccharide which can be easily extracted by warm water with great yield [14]. The pectinous polysaccharide extracted from the achenes of jelly fig is mainly composed of low methyl pectin (LMP) because of the release of pectinesterase (PE) during water extraction [15]. Pectin is a type of polysaccharide found in the primary cell wall and intercellular tissues of the plant, especially in the fruits [16]. Pectin is a natural heteropolysaccharide composed of galacturonic acid with a varying degree of methyl esterification (DM) and other neutral sugars. The DM varies depending on the plant type, tissue part, and maturity [17]. When DM
Corresponding author. E-mail address: [email protected] (Y.-S. Cheng).
https://doi.org/10.1016/j.jddst.2020.101679 Received 17 January 2020; Received in revised form 16 March 2020; Accepted 17 March 2020 1773-2247/ © 2020 Elsevier B.V. All rights reserved.
Please cite this article as: Thangavel Ponrasu, et al., Journal of Drug Delivery Science and Technology, https://doi.org/10.1016/j.jddst.2020.101679
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is higher than 50%, it is known as high methoxyl pectin (HMP) or DM lower than 50%, it is called as low methoxyl pectin (LMP) [18]. HMP can form a gel under acidic conditions in the presence of high sugar concentrations [19]. On the other hand, LMP forms gels by interaction with divalent cations, according to the “egg-box” model [20]. LMP has been suggested as potential material for CDDS due to its partial degradation by side-chain hydrolysis when exposure to the acidic conditions of the stomach (around pH 2) and by beta elimination or de-esterification of polygalacuronan at conditions of the small intestine (around pH 6). However, pectin is a water-soluble natural polysaccharide and tends to swell and erosion in aqueous solution. The swollen and eroded pectin based matrix is prone to release the drug prematurely in the upper digestive tract and thus limiting the capability of pectin to serve as a carrier of CDDS. Therefore, many different formulations have been developed to prevent pectin-based matrix from releasing the drug in the upper gastrointestinal tract, including crosslinking with multi-valent cations, mixing with viscous polymer, coacervation with a positively charged polyelectrolyte, or coating with pH resistant or low aqueous soluble polymer. Blending pectins with other polysaccharides showed various synergic effects that could be useful for the preparation of microcapsules [21,22]. For example, pectin/alginate composites were first prepared and exhibited an excellent gelling characteristic in drug delivery through the synergistic interaction between galacturonic acid chains of pectin and the glucuronic acid moiety of alginate. The intermolecular binding of pectin and alginate improved molecular interaction [23]. Chitosan and pectin molecules can form polyelectrolyte complexes through electrostatic interaction between amino groups of chitosan and the carboxyl groups of pectin. These polyelectrolyte complexes were widely used in food, pharmaceutical industry and in CDDS due to the possess the ability to protect the drug in the upper intestinal tract [24]. The mixture of LMP and HMP was also found to have strong synergistic effects that occurred in the gelation behavior [25]; however, there is no previous study described the combination of jelly fig polysaccharide and commercial HMP as an encapsulant for the CDDS. Therefore, in this present work, the jelly fig polysaccharide (JFP) was mixed with different ratios of commercial HMP to prepare the encapsulant for the CDDS. In order to evaluate the colonic delivery performance, hard capsules were prepared and their physicochemical properties such as thickness, color, moisture content, water vapor permeability, mechanical strength, morphology, in vitro degradation and release behavior were thoroughly investigated.
Table 1 Film formulation based on the desinged ratio between JFP and HMP. Film #
1 2 3 4 5
Ratio of the total polysaccharides
Final concentration of polysaccharide in the mixture (% w/v)
JFP
HMP
JFP
HMP
100% 87.5% 75% 62.5% 50%
0 12.5% 25% 37.5% 50%
0.6 0.525 0.45 0.375 0.3
0 0.075 0.15 0.225 0.3
knife mill and stored in a moisture-proof cabinet until further use. 2.3. Preparation of JFP and JFP/HMP films Five mixtures with a concentration of total polysaccharides at 0.6% (w/v) were prepared by solubilizing jelly fig polysaccharide (JFP) and high-methoxyl pectin (HMP) at different ratios in deionized water and 0.3% (w/v) of glycerol was added as the plasticizer (Table 1). Then, the mixture was homogenized at 300 rpm for 45 min at 60 °C, air bubbles were removed by ultrasonic shock. The films were cast by adding calcium ions to the mixture for the crosslinked mixture and transferred to Teflon dishes. Then, the dishes were kept for drying at 40 °C for 24 h in a convection oven. 2.4. Fourier transform infrared (FT-IR) spectrometer analysis FT-IR spectra of prepared films were obtained with a spectrometer (Spectrum One and Autoimagic system, Perkin Elmer). The spectra were scanned over the wavenumber range of 4000 to 450 cm−1 at ambient temperature. The most important two characteristic peaks were found to be 1615 cm−1 and 1744 cm−1 which corresponding respectively to the carboxylic group and methyl esters of carboxylic acids. These peak areas were used to determine the degree of methoxylation (DM) using the following equation [26]. (1)
DM = A1744 /(A1615 + A1744) × 100% 2.5. Thickness measurement
Thickness was measured using the Film Thickness Gauge (MET-CM, SEAT) at the four corners and the middle portion of each specimen. The mean thickness of each type of film was determined from an average measurement of five films at five different positions of each film sample [27].
2. Materials and methods 2.1. Materials Jelly fig achenes were purchased from the local market (Nantou, Taiwan). All chemicals used in this study were analytical grade obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Pepsin from porcine gastric, pancreatin from porcine pancreas, and pectinase from Aspergillus niger were purchased from Sigma-Aldrich, Inc. (Saint Louis, MO, USA). Resistant starch, xanthan gum, high methoxy pectin used were food-grade purchased from Emperor Chemical Co., Ltd. (Taipei, Taiwan).
2.6. Color measurment The color of the prepared films was determined with a color reader (KONICA MINOLTA, CR-20) which was calibrated and operated according to the official manual. The CIELAB color parameters (lightness and darkness, L∗; red and green, a∗; yellow and blue, b∗) were employed and the for the films was calculated to present differences of color parameters of sample films. The numerical value represents the average of five measurements (one at the center and four around the perimeter) on each film. The total difference in color (ΔE∗) was calculated according to the following equations [28]:
2.2. Extraction and precipitation the polysaccharides from jelly fig Jelly fig achenes were extracted with deionized water at a ratio of 1:40 (w/v) at 50 °C for 40 min. After the extraction was completed, the jelly fig achenes were removed by filtration through a cheesecloth. Four volumes of ethanol (95%) was added to the filtrate to precipitate the polysaccharides. The mixture was kept at room temperature over the time to completely precipitated the polysaccharides. Then, the precipitate was collected in a glass plate and dried at 80 °C in a convection oven for approximately 24 h. The dried samples were powdered using a
E=
(LR
Ls )2 + (aR
as )2 + (bR
bs )2
(2)
2.7. Moisture content analysis The prepared film samples of 3 cm average diameter were dried at 105 °C in a convection oven for 24 h and then the moisture content was 2
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determined. The initial weight (W0) and dry weight (Wd) of each film samples were accurately weighed and the water content (WC) was calculated by the following equation:
WC = (W 0
Wd )/ W 0 × 100%
2.12. Preparation of hard capsule The formulation of the hard capsule was achieved by optimizing the concentration of jelly fig polysaccharide, thickener, and stabilizer. For each hard capsule, JPF (1.2%, w/v), resistant starch (0.8%, w/v), xanthan gum (0.2%, w/v) and glycerol were solubilized in 50 mL of deionized water. Then this dispersion was homogenized for 45 min at 60 °C, 300 rpm and air bubbles formed in the mixture were removed by vacuum ultrasonic approach. The capsules were prepared by dipping a stainless steel rod into the mixture solution and dried at 40 °C for 12 h in a convection oven. Finally, the capsules were removed from the rod carefully, cut, capped and sealed for the further study.
(3)
2.8. Water vapor permeability (WVP) analysis The water vapor permeability (WVP) was determined using the cup method standardized by ASTM E96-80 (ASTM, 1987). The film samples were mounted over the cups, in order to maintain a 75% relative humidity (RH) gradient across the film, anhydrous CaCl2 (0% RH) was placed inside the cup and a saturated NaCl solution (75% RH) was added to the glass chamber. Under steady-state conditions, the osmotic membrane is weighed at the same time and regular intervals for 24 h. The water vapor permeability of the samples was determined in triplicate with the following equation:
WVP = W × L /(A × t × p)
2.13. In vitro release profile of hard capsule To determine the release behavior of the capsule, methylene blue was used as a model drug. After filling with methylene blue, the capsule was sealed and immersed simultaneously in SGF for 2 h, SIF for 4 h and finally in SCF for 5 h. The capsules were thoroughly washed in every step before transferring into subsequential simulation fluid. The whole experiment was performed at 37 °C in a shaker incubator at 50 rpm (LM-570R). The aliquots (2 mL) were withdrawn and replaced by the same simulation fluid periodically at a regular time interval to measure the release content. The release of methylene blue was observed for 9 h at a predetermined time interval of 30 min. The drug release was measured at 660 nm using a UV/Vis spectrophotometer (LKU-5200, LINKO).
(4)
where w is the weight of the water permeated through the film (g), L is the thickness of the film (mm), A is the permeation area (m2), t is the time of permeation (h), and Δp is the water vapor pressure difference between the two sides of the film (kPa). 2.9. Mechanical strength measurement The mechanical strength of the films was tested in terms of tensile strength and elongation. The tensile strength (TS) and elongation (E) at the break of each film was measured with Tinius Olsen Benchtop Tester (H5KT, Atec, Inc., USA) based on the ASTM Standard Method D882-12 (ASTM, 2012). Tensile strength (TS) is the maximum stress that the material can withstand while being stretched or pulled before failing or breaking. The elongation (E) at the breaking point is the change percentage of the original length of a material. The samples were cut into the size of 8 cm × 2.5 cm and incubated for 48 h at 25 °C with 50% relative humidity before the measurement. The initial grip separation and cross-head speed were set at 4 cm and 20 mm/min, respectively. Three replications of the measurements were performed for the statistical analysis.
2.14. Statistics All the experiments were performed in triplicate and the data are expressed as means ± standard deviation (SD). Data were subjected to analysis of students t-test using GraphPad Prism version 5 (GraphPad Software Inc. San Diego CA, California, USA). The values of ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 were considered as statistically significant. 3. Results and discussion 3.1. Film castsing and physiochemical characterization
2.10. Scanning electron microscopy (SEM) analysis
The jelly fig polysaccharide (JFP) was extracted from jelly fig achenes and ground into fine powder after drying. The yield of jelly fig polysaccharide was 82 ± 5 mg/g. The prepared polysaccharide was employed to cast the films. Preliminary experiments were conducted to determine the concentration of JFP in each film-forming solution. The study revealed that film-forming can be achieved using 0.6% w/v of JFP. However, JFP films were found to be brittle in nature and did not have flexibility. Thus, 50% (w/w, based on total polysaccharide weight) glycerol was added to the film-forming solutions to improve the flexibility of the films. In addition, HMP has been commonly used as a low-cost gelling and thickening agent and the mixture of LMP and HMP was reported to have strong synergistic effects on the gelation behavior [25], therefore, HMP was introduced into the JFP film preparation at the different ratios to test the effect of adding high-methoxyl pectin on the film formation and resistance in simulated gastrointestinal. The jelly fig polysaccharide and JFP/HMP films were analyzed by FTIR spectroscopy (Fig. 1 a) and their spectra were compared against two commercial pectin standards. The fingerprint region of FTIR (between the wavenumbers of 950 cm−1 and 1200 cm−1) is considered as specific for carbohydrates. The position and intensity of the bands are also specific for every polysaccharide. This region allows the identification of major chemical groups in polysaccharides. The characteristic peak observed at 2936 cm−1 is attributes C–H bond of the methylene
The surface morphology of the prepared films was examined by SEM (S3000H, HITACHI, Japan). The films were fixed on the specimen stage by using a double-sided adhesive tape and then sprayed with a layer of platinum for the clear surface visualization. The accelerating voltage used in this process was 10 kV. The photomicrographs were magnified (100× and 1000 x) and recorded. 2.11. In vitro degradation/erosion study The in vitro degradation test was performed to determine the suitability of the jelly fig polysaccharide complex for the colon-specific drug delivery system. The degradation study was performed in simulation solutions such as Simulated Gastric Fluid (SGF; pH 1.2), Simulated Intestinal Fluid (SIF; pH 6.0) and Simulated Colonic Fluid (SCF; pH 6.8) [29,30]. The film samples were cut into 30 mm × 30 mm size, weighed (W0), and then were degraded in vitro by simulated fluids. First, the samples were incubated in SGF for 2 h and washed with distilled water. Second, the samples were incubated in SIF for 4 h and washed with distilled water. Finally, the samples were incubated in SCF for 5 h. The experiment was conducted at 37 °C at a constant rotation of 50 rpm in an orbital shaking incubator (LM-570R, YIHDER TECHNOLOGY CO., LTD., Taiwan).
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Fig. 1. (a) FTIR spectra and (b) thickness of the JFP/HMP films. Data are represented here is the mean ± SD (n = 3) and the level of significance is denoted as ∗p < 0.05.
group (CH2) and the O–H bond is observed as a wideband at 3443 cm−1. However, the two most important characteristic peaks responsible for the degree of methylation were found at 1615 cm−1 and 1744 cm−1 respectively. The peak at 1615 cm−1 was produced by carboxylate (COO−), whereas the peak at 1744 cm−1 was methyl
esterified carboxyl. The structure of (COOMe) and the characteristic peak area of carboxylate and methyl esterified carboxyl groups are commonly used to determine the degree of methylation of the polysaccharide [31]. The degree of methylation for jelly fig polysaccharide was found to be 24.8%. As its degree of methylation values is less than
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significantly lower than the previously reported films like galactomannan films (0.235 ?? × ????⁄ ??2 × ℎ × ??????) [40], and polyester film (0.0091 ?? × ????⁄ ??2 × ℎ × ??????) [41]. This is probably due to the presence of calcium ions in the JFP/HMP film. The presence of calcium ions can crosslink the pectin molecule and form a network structure which lowers the WVP. Furthermore, the increased HMP ratio would also increase the density of film due to the decreased thickness. The films with higher bulk density will provide increased resistance to mass transfer and lower the WVP.
Table 2 Color measurements and appreance of JFP and JFP/HMP films. Film #
L∗
1 2 3 4 5
87.14 86.52 86.35 85.88 85.75
± ± ± ± ±
0.02 0.04 0.03 0.03 0.02
a∗
b∗
0.31 ± 0.01 0.22 ± 0.01 0.03 ± 0.01 - 0.13 ± 0.01 - 0.09 ± 0.01
5.11 4.23 4.19 3.38 2.99
ΔE∗ ± ± ± ± ±
0.02 0.03 0.03 0.02 0.01
11.18 11.56 11.72 12.04 12.13
± ± ± ± ±
0.01 0.04 0.03 0.03 0.03
3.4. Mechanical strength and film morphology
50%, the presence of pectin molecule in jelly fig polysaccharide is low methyl pectin.
Mechanical strength was characterized by assessing the tensile strength (TS) and elongation (E) of film materials. The mechanical properties of JFP and JFP/HMP films are presented in Table 3. The results revealed that the TS and E values obtained in our study were not significantly different from other biopolymer films such as pectinchitosan/carrageenan/starch mixture films (18,41 MPa/58.75%) [42] and gelatin films (19.5 MPa/122.0%) [43]. The addition of high methyl pectin in films caused significant differences in TS and E. Increasing the concentration of high methyl pectin in JFP films from 0% to 50% (w/ w), decreased the TS of these films from 22.94 MPa to 13.86 MPa. However, it increased the E from 67.43% to 120.43%. This could be attributed to the reducing intermolecular forces formed between the LMP molecules and therefore increase the mobility of polymer chains [44,45]. Scanning electron microscopy (SEM) was employed to investigate the microstructures of the surfaces of the JFP films and JFP/ HMP films. The SEM micrographs were given in Fig. 3. The microscopic view of the prepared films showed a relatively smooth and uniform surface morphology without cracks or breaks on the surfaces of the JFP films. However, JFP/HMP films showed slightly rough surface morphology compared to JFP film.
3.2. Thickness and color measurements of casted films The results showed that the JFP and JFP/HMP films had thicknesses within a range from around 57.8 to 93.3 μm at the casting condition applied in this study (Fig. 1b). The film thickness was reduced when the ratio of HMP in the film was increased. A similar result had also been reported by Christine Byun [32]. In the reference, the addition of calcium ions resulted in LMP films which were significantly thicker than HMP films. This could be attributed to the formation of intermolecular junction zones between LMP molecules by calcium ions, which makes the structure of LMP film is stiffer and less compressible than HMP film [33,34]. The color measure provided a convenient and non-destructive way to track the effects of variations on the process parameters on the resulting material. In addition, the color presentation is important because film opacity is a critical property governing the applicability of conditioned films, particularly if the film is to be used in industrial applications [35]. Color measurement by observation of CIELAB color space parameters for the films based on different blending ratios of JFP and HMP are presented in Table 2. The results showed that JFP films and JFP/HMP films were transparently opaque and slightly yellowish. With increasing JFP content, the value of a∗ was slightly decreased while the values of L∗ and b∗ increased. The positive value of b∗ indicated a yellow coloration associated with the natural color of JFP. The color properties of the JFP and JFP/HMP-based films were similar to the polysaccharide-based composite films reported by other studies [36–38], but the ΔE values of all prepared films were slightly higher than the films prepared from gelatin. These results agreed with visual observation. It was also observed that the opacity and transparency of JFP/HMP films depended on not only the concentration of polysaccharides but also the film thickness.
3.5. In vitro erosion behavior The erosion studies were carried out for prepared JFP film and JFP/ HMP films and the results were given in Fig. 4a. First, the film was incubated in SGF for 2 h, washed and transferred to SIF for 4 h. The results showed that the film made by pure JFP was around 50% eroded but the film was observable after the incubation in SGF and SIF. However, the films prepared from JFP/HMP mixtures were almost completely eroded and dissolved. This characteristic feature indicates that pure JFP can be used as a new material for a colon-specific drug delivery system. However, the JFP cannot be used exclusively to cast hard capsules because of its low viscoelasticity [46]. Therefore, composite capsules were formulated using JFP as the main ingredient blended with resistant starch and xanthan gum for the applicability evaluation of JFP in hard capsule preparation.
3.3. Moisture content and water vapor permeability Moisture content and water vapor permeability are frequently used as important measures in film preparation because moisture can pass through the film and subsequently change the properties of the film as well as the substance being packaged or encapsulated [39]. The moisture content of the JFP and JFP/HMP films are depicted in Fig. 2a and the results indicated that the addition of high-methyl pectin concentration from 0% to 50% (w/w) in JFP/HMP significantly increased the moisture content from 25.68% to 45.18%. Addition of high methyl pectin produced roughness and nonuniform surfaces in the JFP/HMP films. The rough surfaces of the films are capable of high interaction with water and hence increased moisture content was observed in JFP/ HMP films. WVP of the JFP and JFP/HMP films are provided in (Fig. 2b). The effect of high-methyl pectin in JFP/HMP films showed decreased WVP from 5.55 × 10−3 to 2.7 × 10−3 (?? × ????⁄ ??2 × ℎ × ??????). The WVP of the films developed in this study was
3.6. Hard capsule preparation and in vitro release profile Methylene blue was used as a model encapsulated substance to examine the release profile of the JFP composite hard capsule. The results (Fig. 4b) indicated that methylene blue was protected by the composite capsules in SGF during the test period with a minimal release (less than 2%) and the outer layer of the capsule remained fairly intact (Fig. 4c). Then, the capsule was removed from SGF and put into the SIF, the composite capsule remained integral over the test period without significant degradation but the release amount of methylene blue was slightly increased from ∼2% to ∼15%. The release of methylene blue in SIF could be attributed to the unsealed connection between the capsule cap and body (as indicated by the red arrow in the middle picture of Fig. 4c) that allows water molecules to penetrate the capsules
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Fig. 2. (a). Moisture content of JFP/HMP films. (b). Water vapor permeability of JFP/HMP films. Each value represented here is the mean ± SD (n = 3) and the significance difference is denoted as ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.01.
and causes the leak of the methylene blue. The resistance of simulated gastrointestinal fluids of JFP composite was in agreement with observation obtained in the film erosion test and the results reported in other LMP derived drug delivery systems [22,47,48]. Finally, the JFP composite capsule was removed from SIF and placed into the SGF. The rate of methylene blue release from the capsule was increased significantly from 15% to 90% in 2 h because of the substantial degradation of the composite capsule (as indicated by the red arrow in the picture on the right side of Fig. 4c). Based on the results of the in vitro release study, JFP can be a potential material for the preparation of colon-targeted hard capsules.
Table 3 Mechanical properties of JFP and JFP/HMP films. Film #
TS (MPa)
1 2 3 4 5
22.94 18.82 16.70 14.57 13.86
± ± ± ± ±
E (%) 2.58 4.82 3.71 3.49 1.07∗
67.43 ± 14.60 82.81 ± 22.58 90.92 ± 11.58 104.20 ± 15.34 120.43 ± 10.93∗
Data are represented here is the mean ± SD (n = 3) and the significance difference is denoted as ∗p < 0.05.
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Fig. 3. SEM morphology of JFP films (left-magnification of 100 × ) and (right-magnification of 1000 × ).
Fig. 4. (a). In vitro degradation (erosion) rate of JFP/HMP film in SGF 2 h and SIF 4 h. The values are mean ± SD (n = 3) and the significance difference is denoted as ∗∗∗p < 0.01. (b). In vitro release profile of methylene blue from the hard capsule in digestive fluids. (c). The degradation of capsule in simulated digestive fluids. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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4. Conclusions
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Vicente, Seed extracts of Gleditsia triacanthos: functional properties evaluation and incorporation into galactomannan films, Food Res. Int. 43 (2010) 2031–2038. [41] T.H. McHugh, R. Avena-Bustillos, J. Krochta, Hydrophilic edible films: modified
This study investigated the pectinous polysaccharide extraction from achenes of jelly fig to prepare the film and colon-specific capsule. The films were first characterized by Fourier transform infrared (FT-IR) spectroscopy, color test, thickness test, moisture content, and mechanical properties. The results indicated that the JFP films derived in this study possessed good mechanical and physical properties to be used as a basis material in the CDDS development. The results showed that film prepared from pure JFP exhibited good stability in simulated digestive fluids. The result of the in vitro release study also demonstrated that the JFP composite hard capsule could pass through the simulated gastrointestinal fluids without significant degradation and successfully released the model drug in the SCF fluid. Therefore, JFP could be a promising ingredient for developing colon-targeted capsules. CRediT authorship contribution statement Thangavel Ponrasu: Formal analysis, Validation, Writing - review & editing. Jhao-Syuan Gu: Investigation, Visualization, Writing - original draft. Jia-Jiuan Wu: Conceptualization. Yu-Shen Cheng: Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Writing - review & editing. Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, “Evaluation of jelly fig polysaccharide as a shell composite ingredient of colon-specific drug delivery”. Acknowledgments The funding of this work was supported by the Ministry of Science and Technology, Taiwan (MOST107-2622-E-224-002-CC3, and MOST 106-2622-E-224-007-CC3). References [1] A.B. Meneguin, B.S.F. Cury, R.C. Evangelista, Films from resistant starch-pectin dispersions intended for colonic drug delivery, Carbohydr. Polym. 99 (2014) 140–149. [2] K.L. Deore, N.A. Thombre, P.S. Gide, Formulation and development of tinidazole microspheres for colon targeted drug delivery system, J. Pharm. Res. 6 (2013) 158–165. [3] M. Chourasia, S. Jain, Pharmaceutical approaches to colon targeted drug delivery systems, J. Pharm. Pharmaceut. Sci. 6 (2003) 33–66. [4] P.M. Treuting, M.J. Arends, S.M. Dintzis, Upper gastrointestinal tract, Comparative Anatomy and Histology, second ed., Elsevier, 2017, pp. 191–211. [5] K.A. Elkhodairy, S.A. Afifi, A.S. Zakaria, A promising approach to provide appropriate colon target drug delivery systems of vancomycin HCL: pharmaceutical and microbiological studies, BioMed Res. Int. (2014) 2014. [6] S. Nalinbenjapun, C. Ovatlarnporn, Chitosan-5-aminosalicylic acid conjugates for colon-specific drug delivery: methods of preparation and in vitro evaluations, J. Drug Deliv. Sci. Technol. (2019) 101397. [7] E. Moghimipour, F.A. Dorkoosh, M. Rezaei, M. Kouchak, J. Fatahiasl, K.A. Angali, Z. Ramezani, M. Amini, S. Handali, In vivo evaluation of pH and time-dependent polymers as coating agent for colonic delivery using central composite design, J. Drug Deliv. Sci. Technol. 43 (2018) 50–56. [8] L. Yang, Biorelevant dissolution testing of colon-specific delivery systems activated by colonic microflora, J. Contr. Release 125 (2008) 77–86. [9] R. Sureshkumar, M. Munikumar, G. Ganesh, N. Jawahar, D. Nagasamyvenkatesh, V. Senthil, L. Raju, M. Samantha, Formulation and evaluation of pectin-hydroxypropyl methylcellulose coated curcumin pellets for colon delivery, Asian J. Pharm.: Free full text articles from Asian J Pharm 3 (2014). [10] K.E. Uhrich, S.M. Cannizzaro, R.S. Langer, K.M. Shakesheff, Polymeric systems for controlled drug release, Chem. Rev. 99 (1999) 3181–3198. [11] M. Gautam, D. Santhiya, In-situ mineralization of calcium carbonate in pectin based edible hydrogel for the delivery of protein at colon, J. Drug Deliv. Sci. Technol. 53
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T. Ponrasu, et al. procedure for water vapor permeability and explanation of thickness effects, J. Food Sci. 58 (1993) 899–903. [42] W. Li, W. Hao, Z. Xiaohua, H. Yinchen, L. Wangwang, Y. Gongming, J. Aimin, Pectin-chitosan complex: preparation and application in colon-specific capsule, Int. J. Agric. Biol. Eng. 8 (2015) 151–160. [43] H.J. Bae, D.S. Cha, W.S. Whiteside, H.J. Park, Film and pharmaceutical hard capsule formation properties of mungbean, waterchestnut, and sweet potato starches, Food Chem. 106 (2008) 96–105. [44] C. Lara-Espinoza, E. Carvajal-Millán, R. Balandrán-Quintana, Y. López-Franco, A. Rascón-Chu, Pectin and pectin-based composite materials: beyond food texture, Molecules 23 (2018) 942.
[45] C. Lofgren, Microstructure and Gelation Behaviour of High Methoxyl and Low Methoxyl Pectin Gels and Their Mixtures, (2005). [46] M. Haghighi, K. Rezaei, General analytical schemes for the characterization of pectin-based edible gelled systems, Sci. World J. (2012) 2012. [47] E.A. Günter, O.V. Popeyko, Calcium pectinate gel beads obtained from callus cultures pectins as promising systems for colon-targeted drug delivery, Carbohydr. Polym. 147 (2016) 490–499. [48] A. Rascón-Chu, G.H. Gomez-Rodriguez, E. Carvajal-Millan, A.C. Campa-Mada, Pectin in drug delivery applications, Natural Polysaccharides in Drug Delivery and Biomedical Applications, Elsevier, 2019, pp. 249–262.
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Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Evaluation of jelly fig polysaccharide as a shell composite ingredient of colon-specific drug delivery Thangavel Ponrasua, Jhao-Syuan Gua, Jia-Jiuan Wub, Yu-Shen Chenga,∗ a b
Department of Chemical and Materials Engineering, National Yunlin University of Science and Technology, Douliu, Yunlin, 64002, Taiwan Department of Nutrition, China Medical University, No. 91, Hsueh-Shih Road, Taichung, 404, Taiwan
ARTICLE INFO
ABSTRACT
Keywords: Ficus pumila var. awkeotsang Pectin Polysaccharide Hard capsule Colon-targeted delivery
The polysaccharide extracted from the achenes of jelly fig (Ficus pumila var. awkeotsang) was characterized as low methoxyl pectin and was blended with commercial high methoxyl pectin in film casting to evaluate the suitability of the combinations for developing a composite encapsulant for colon target drug delivery. The physiochemical properties of the casted films were characterized by Fourier Transform Infrared (FT-IR) spectroscopy, color measurement, thickness test, water content analysis, and mechanical strength measurement. Among all tested blending ratios, the film prepared from pure jelly fig polysaccharide has higher tensile strength and better performance of anti-erosion in simulated gastrointestinal fluids. Hence, for evaluation of applicability, the composite hard capsules were prepared by using jelly fig polysaccharide as the main component blending with xanthan gum and resistant starch. The results of the in vitro release study showed that jelly fig polysaccharide composite capsules were stable without significant deformation and releasing of encapsulated substance in the simulated gastrointestinal fluids during the test period. Then, the capsules were degraded in simulated colonic fluid in 2 h and the release rate was increased from 15% to 90%. Therefore, pectinous polysaccharide extracted from jelly fig could be a useful material for the preparation of a colon-targeted capsule.
1. Introduction Colon-specific drug delivery systems (CDDS) play a vital role in increasing the bioavailability of drugs in the colon through oral administration [1,2]. CDDS has a great impact to improve the treatment of local diseases affecting the colon and minimizing systemic side effects [3]. The delivery of drugs specifically to the colon without being absorbed first in the upper gastrointestinal tract [4] allowing high concentrations of drugs to reach colon with minimal body resorption. This system has been applied to reduce the delay of delivering the drugs to achieve high concentration, dosing frequency, and achieve sustained release during the treatment of inflammatory disease of the gut [5]. CDDS through oral admission can be classified into different types according to the functional mechanisms including time-dependent, pHresponsive, osmotically controlled, and bacterial triggered [6,7]. Nevertheless, time-dependent, pH-responsive, and osmotically controlled release systems are not always reliable among different cases. Since the colon contains more than 400 species of microorganisms that can produce a variety of enzymes capable of effectively degrade undigested carbohydrates reaching the large intestine [8], the bacterial triggered mechanism for drug release has become a popular research
∗
topic in CDDS. Successful delivery of a drug to the colon requires suitable protection for the drug from degradation or release in the stomach and sustained release in the colon [9]. Therefore, proper care must be taken while formulating drugs and dosage for CDDS to overcome the obstacles caused by the gastrointestinal tract during the drug delivery. The desired properties of CDDS can be achieved by using some polymers either alone or in a combination [10]. Some nature polymers like pectin [11], chitosan [6], alginates [12], and gum arabica [13] are inexpensive and available in various structures. Jelly fig (Ficus pumila var. awkeotsang) is a native plant of Taiwan and its achenes are a rich source of pectinous polysaccharide which can be easily extracted by warm water with great yield [14]. The pectinous polysaccharide extracted from the achenes of jelly fig is mainly composed of low methyl pectin (LMP) because of the release of pectinesterase (PE) during water extraction [15]. Pectin is a type of polysaccharide found in the primary cell wall and intercellular tissues of the plant, especially in the fruits [16]. Pectin is a natural heteropolysaccharide composed of galacturonic acid with a varying degree of methyl esterification (DM) and other neutral sugars. The DM varies depending on the plant type, tissue part, and maturity [17]. When DM
Corresponding author. E-mail address: [email protected] (Y.-S. Cheng).
https://doi.org/10.1016/j.jddst.2020.101679 Received 17 January 2020; Received in revised form 16 March 2020; Accepted 17 March 2020 1773-2247/ © 2020 Elsevier B.V. All rights reserved.
Please cite this article as: Thangavel Ponrasu, et al., Journal of Drug Delivery Science and Technology, https://doi.org/10.1016/j.jddst.2020.101679
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is higher than 50%, it is known as high methoxyl pectin (HMP) or DM lower than 50%, it is called as low methoxyl pectin (LMP) [18]. HMP can form a gel under acidic conditions in the presence of high sugar concentrations [19]. On the other hand, LMP forms gels by interaction with divalent cations, according to the “egg-box” model [20]. LMP has been suggested as potential material for CDDS due to its partial degradation by side-chain hydrolysis when exposure to the acidic conditions of the stomach (around pH 2) and by beta elimination or de-esterification of polygalacuronan at conditions of the small intestine (around pH 6). However, pectin is a water-soluble natural polysaccharide and tends to swell and erosion in aqueous solution. The swollen and eroded pectin based matrix is prone to release the drug prematurely in the upper digestive tract and thus limiting the capability of pectin to serve as a carrier of CDDS. Therefore, many different formulations have been developed to prevent pectin-based matrix from releasing the drug in the upper gastrointestinal tract, including crosslinking with multi-valent cations, mixing with viscous polymer, coacervation with a positively charged polyelectrolyte, or coating with pH resistant or low aqueous soluble polymer. Blending pectins with other polysaccharides showed various synergic effects that could be useful for the preparation of microcapsules [21,22]. For example, pectin/alginate composites were first prepared and exhibited an excellent gelling characteristic in drug delivery through the synergistic interaction between galacturonic acid chains of pectin and the glucuronic acid moiety of alginate. The intermolecular binding of pectin and alginate improved molecular interaction [23]. Chitosan and pectin molecules can form polyelectrolyte complexes through electrostatic interaction between amino groups of chitosan and the carboxyl groups of pectin. These polyelectrolyte complexes were widely used in food, pharmaceutical industry and in CDDS due to the possess the ability to protect the drug in the upper intestinal tract [24]. The mixture of LMP and HMP was also found to have strong synergistic effects that occurred in the gelation behavior [25]; however, there is no previous study described the combination of jelly fig polysaccharide and commercial HMP as an encapsulant for the CDDS. Therefore, in this present work, the jelly fig polysaccharide (JFP) was mixed with different ratios of commercial HMP to prepare the encapsulant for the CDDS. In order to evaluate the colonic delivery performance, hard capsules were prepared and their physicochemical properties such as thickness, color, moisture content, water vapor permeability, mechanical strength, morphology, in vitro degradation and release behavior were thoroughly investigated.
Table 1 Film formulation based on the desinged ratio between JFP and HMP. Film #
1 2 3 4 5
Ratio of the total polysaccharides
Final concentration of polysaccharide in the mixture (% w/v)
JFP
HMP
JFP
HMP
100% 87.5% 75% 62.5% 50%
0 12.5% 25% 37.5% 50%
0.6 0.525 0.45 0.375 0.3
0 0.075 0.15 0.225 0.3
knife mill and stored in a moisture-proof cabinet until further use. 2.3. Preparation of JFP and JFP/HMP films Five mixtures with a concentration of total polysaccharides at 0.6% (w/v) were prepared by solubilizing jelly fig polysaccharide (JFP) and high-methoxyl pectin (HMP) at different ratios in deionized water and 0.3% (w/v) of glycerol was added as the plasticizer (Table 1). Then, the mixture was homogenized at 300 rpm for 45 min at 60 °C, air bubbles were removed by ultrasonic shock. The films were cast by adding calcium ions to the mixture for the crosslinked mixture and transferred to Teflon dishes. Then, the dishes were kept for drying at 40 °C for 24 h in a convection oven. 2.4. Fourier transform infrared (FT-IR) spectrometer analysis FT-IR spectra of prepared films were obtained with a spectrometer (Spectrum One and Autoimagic system, Perkin Elmer). The spectra were scanned over the wavenumber range of 4000 to 450 cm−1 at ambient temperature. The most important two characteristic peaks were found to be 1615 cm−1 and 1744 cm−1 which corresponding respectively to the carboxylic group and methyl esters of carboxylic acids. These peak areas were used to determine the degree of methoxylation (DM) using the following equation [26]. (1)
DM = A1744 /(A1615 + A1744) × 100% 2.5. Thickness measurement
Thickness was measured using the Film Thickness Gauge (MET-CM, SEAT) at the four corners and the middle portion of each specimen. The mean thickness of each type of film was determined from an average measurement of five films at five different positions of each film sample [27].
2. Materials and methods 2.1. Materials Jelly fig achenes were purchased from the local market (Nantou, Taiwan). All chemicals used in this study were analytical grade obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Pepsin from porcine gastric, pancreatin from porcine pancreas, and pectinase from Aspergillus niger were purchased from Sigma-Aldrich, Inc. (Saint Louis, MO, USA). Resistant starch, xanthan gum, high methoxy pectin used were food-grade purchased from Emperor Chemical Co., Ltd. (Taipei, Taiwan).
2.6. Color measurment The color of the prepared films was determined with a color reader (KONICA MINOLTA, CR-20) which was calibrated and operated according to the official manual. The CIELAB color parameters (lightness and darkness, L∗; red and green, a∗; yellow and blue, b∗) were employed and the for the films was calculated to present differences of color parameters of sample films. The numerical value represents the average of five measurements (one at the center and four around the perimeter) on each film. The total difference in color (ΔE∗) was calculated according to the following equations [28]:
2.2. Extraction and precipitation the polysaccharides from jelly fig Jelly fig achenes were extracted with deionized water at a ratio of 1:40 (w/v) at 50 °C for 40 min. After the extraction was completed, the jelly fig achenes were removed by filtration through a cheesecloth. Four volumes of ethanol (95%) was added to the filtrate to precipitate the polysaccharides. The mixture was kept at room temperature over the time to completely precipitated the polysaccharides. Then, the precipitate was collected in a glass plate and dried at 80 °C in a convection oven for approximately 24 h. The dried samples were powdered using a
E=
(LR
Ls )2 + (aR
as )2 + (bR
bs )2
(2)
2.7. Moisture content analysis The prepared film samples of 3 cm average diameter were dried at 105 °C in a convection oven for 24 h and then the moisture content was 2
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determined. The initial weight (W0) and dry weight (Wd) of each film samples were accurately weighed and the water content (WC) was calculated by the following equation:
WC = (W 0
Wd )/ W 0 × 100%
2.12. Preparation of hard capsule The formulation of the hard capsule was achieved by optimizing the concentration of jelly fig polysaccharide, thickener, and stabilizer. For each hard capsule, JPF (1.2%, w/v), resistant starch (0.8%, w/v), xanthan gum (0.2%, w/v) and glycerol were solubilized in 50 mL of deionized water. Then this dispersion was homogenized for 45 min at 60 °C, 300 rpm and air bubbles formed in the mixture were removed by vacuum ultrasonic approach. The capsules were prepared by dipping a stainless steel rod into the mixture solution and dried at 40 °C for 12 h in a convection oven. Finally, the capsules were removed from the rod carefully, cut, capped and sealed for the further study.
(3)
2.8. Water vapor permeability (WVP) analysis The water vapor permeability (WVP) was determined using the cup method standardized by ASTM E96-80 (ASTM, 1987). The film samples were mounted over the cups, in order to maintain a 75% relative humidity (RH) gradient across the film, anhydrous CaCl2 (0% RH) was placed inside the cup and a saturated NaCl solution (75% RH) was added to the glass chamber. Under steady-state conditions, the osmotic membrane is weighed at the same time and regular intervals for 24 h. The water vapor permeability of the samples was determined in triplicate with the following equation:
WVP = W × L /(A × t × p)
2.13. In vitro release profile of hard capsule To determine the release behavior of the capsule, methylene blue was used as a model drug. After filling with methylene blue, the capsule was sealed and immersed simultaneously in SGF for 2 h, SIF for 4 h and finally in SCF for 5 h. The capsules were thoroughly washed in every step before transferring into subsequential simulation fluid. The whole experiment was performed at 37 °C in a shaker incubator at 50 rpm (LM-570R). The aliquots (2 mL) were withdrawn and replaced by the same simulation fluid periodically at a regular time interval to measure the release content. The release of methylene blue was observed for 9 h at a predetermined time interval of 30 min. The drug release was measured at 660 nm using a UV/Vis spectrophotometer (LKU-5200, LINKO).
(4)
where w is the weight of the water permeated through the film (g), L is the thickness of the film (mm), A is the permeation area (m2), t is the time of permeation (h), and Δp is the water vapor pressure difference between the two sides of the film (kPa). 2.9. Mechanical strength measurement The mechanical strength of the films was tested in terms of tensile strength and elongation. The tensile strength (TS) and elongation (E) at the break of each film was measured with Tinius Olsen Benchtop Tester (H5KT, Atec, Inc., USA) based on the ASTM Standard Method D882-12 (ASTM, 2012). Tensile strength (TS) is the maximum stress that the material can withstand while being stretched or pulled before failing or breaking. The elongation (E) at the breaking point is the change percentage of the original length of a material. The samples were cut into the size of 8 cm × 2.5 cm and incubated for 48 h at 25 °C with 50% relative humidity before the measurement. The initial grip separation and cross-head speed were set at 4 cm and 20 mm/min, respectively. Three replications of the measurements were performed for the statistical analysis.
2.14. Statistics All the experiments were performed in triplicate and the data are expressed as means ± standard deviation (SD). Data were subjected to analysis of students t-test using GraphPad Prism version 5 (GraphPad Software Inc. San Diego CA, California, USA). The values of ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 were considered as statistically significant. 3. Results and discussion 3.1. Film castsing and physiochemical characterization
2.10. Scanning electron microscopy (SEM) analysis
The jelly fig polysaccharide (JFP) was extracted from jelly fig achenes and ground into fine powder after drying. The yield of jelly fig polysaccharide was 82 ± 5 mg/g. The prepared polysaccharide was employed to cast the films. Preliminary experiments were conducted to determine the concentration of JFP in each film-forming solution. The study revealed that film-forming can be achieved using 0.6% w/v of JFP. However, JFP films were found to be brittle in nature and did not have flexibility. Thus, 50% (w/w, based on total polysaccharide weight) glycerol was added to the film-forming solutions to improve the flexibility of the films. In addition, HMP has been commonly used as a low-cost gelling and thickening agent and the mixture of LMP and HMP was reported to have strong synergistic effects on the gelation behavior [25], therefore, HMP was introduced into the JFP film preparation at the different ratios to test the effect of adding high-methoxyl pectin on the film formation and resistance in simulated gastrointestinal. The jelly fig polysaccharide and JFP/HMP films were analyzed by FTIR spectroscopy (Fig. 1 a) and their spectra were compared against two commercial pectin standards. The fingerprint region of FTIR (between the wavenumbers of 950 cm−1 and 1200 cm−1) is considered as specific for carbohydrates. The position and intensity of the bands are also specific for every polysaccharide. This region allows the identification of major chemical groups in polysaccharides. The characteristic peak observed at 2936 cm−1 is attributes C–H bond of the methylene
The surface morphology of the prepared films was examined by SEM (S3000H, HITACHI, Japan). The films were fixed on the specimen stage by using a double-sided adhesive tape and then sprayed with a layer of platinum for the clear surface visualization. The accelerating voltage used in this process was 10 kV. The photomicrographs were magnified (100× and 1000 x) and recorded. 2.11. In vitro degradation/erosion study The in vitro degradation test was performed to determine the suitability of the jelly fig polysaccharide complex for the colon-specific drug delivery system. The degradation study was performed in simulation solutions such as Simulated Gastric Fluid (SGF; pH 1.2), Simulated Intestinal Fluid (SIF; pH 6.0) and Simulated Colonic Fluid (SCF; pH 6.8) [29,30]. The film samples were cut into 30 mm × 30 mm size, weighed (W0), and then were degraded in vitro by simulated fluids. First, the samples were incubated in SGF for 2 h and washed with distilled water. Second, the samples were incubated in SIF for 4 h and washed with distilled water. Finally, the samples were incubated in SCF for 5 h. The experiment was conducted at 37 °C at a constant rotation of 50 rpm in an orbital shaking incubator (LM-570R, YIHDER TECHNOLOGY CO., LTD., Taiwan).
3
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Fig. 1. (a) FTIR spectra and (b) thickness of the JFP/HMP films. Data are represented here is the mean ± SD (n = 3) and the level of significance is denoted as ∗p < 0.05.
group (CH2) and the O–H bond is observed as a wideband at 3443 cm−1. However, the two most important characteristic peaks responsible for the degree of methylation were found at 1615 cm−1 and 1744 cm−1 respectively. The peak at 1615 cm−1 was produced by carboxylate (COO−), whereas the peak at 1744 cm−1 was methyl
esterified carboxyl. The structure of (COOMe) and the characteristic peak area of carboxylate and methyl esterified carboxyl groups are commonly used to determine the degree of methylation of the polysaccharide [31]. The degree of methylation for jelly fig polysaccharide was found to be 24.8%. As its degree of methylation values is less than
4
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significantly lower than the previously reported films like galactomannan films (0.235 ?? × ????⁄ ??2 × ℎ × ??????) [40], and polyester film (0.0091 ?? × ????⁄ ??2 × ℎ × ??????) [41]. This is probably due to the presence of calcium ions in the JFP/HMP film. The presence of calcium ions can crosslink the pectin molecule and form a network structure which lowers the WVP. Furthermore, the increased HMP ratio would also increase the density of film due to the decreased thickness. The films with higher bulk density will provide increased resistance to mass transfer and lower the WVP.
Table 2 Color measurements and appreance of JFP and JFP/HMP films. Film #
L∗
1 2 3 4 5
87.14 86.52 86.35 85.88 85.75
± ± ± ± ±
0.02 0.04 0.03 0.03 0.02
a∗
b∗
0.31 ± 0.01 0.22 ± 0.01 0.03 ± 0.01 - 0.13 ± 0.01 - 0.09 ± 0.01
5.11 4.23 4.19 3.38 2.99
ΔE∗ ± ± ± ± ±
0.02 0.03 0.03 0.02 0.01
11.18 11.56 11.72 12.04 12.13
± ± ± ± ±
0.01 0.04 0.03 0.03 0.03
3.4. Mechanical strength and film morphology
50%, the presence of pectin molecule in jelly fig polysaccharide is low methyl pectin.
Mechanical strength was characterized by assessing the tensile strength (TS) and elongation (E) of film materials. The mechanical properties of JFP and JFP/HMP films are presented in Table 3. The results revealed that the TS and E values obtained in our study were not significantly different from other biopolymer films such as pectinchitosan/carrageenan/starch mixture films (18,41 MPa/58.75%) [42] and gelatin films (19.5 MPa/122.0%) [43]. The addition of high methyl pectin in films caused significant differences in TS and E. Increasing the concentration of high methyl pectin in JFP films from 0% to 50% (w/ w), decreased the TS of these films from 22.94 MPa to 13.86 MPa. However, it increased the E from 67.43% to 120.43%. This could be attributed to the reducing intermolecular forces formed between the LMP molecules and therefore increase the mobility of polymer chains [44,45]. Scanning electron microscopy (SEM) was employed to investigate the microstructures of the surfaces of the JFP films and JFP/ HMP films. The SEM micrographs were given in Fig. 3. The microscopic view of the prepared films showed a relatively smooth and uniform surface morphology without cracks or breaks on the surfaces of the JFP films. However, JFP/HMP films showed slightly rough surface morphology compared to JFP film.
3.2. Thickness and color measurements of casted films The results showed that the JFP and JFP/HMP films had thicknesses within a range from around 57.8 to 93.3 μm at the casting condition applied in this study (Fig. 1b). The film thickness was reduced when the ratio of HMP in the film was increased. A similar result had also been reported by Christine Byun [32]. In the reference, the addition of calcium ions resulted in LMP films which were significantly thicker than HMP films. This could be attributed to the formation of intermolecular junction zones between LMP molecules by calcium ions, which makes the structure of LMP film is stiffer and less compressible than HMP film [33,34]. The color measure provided a convenient and non-destructive way to track the effects of variations on the process parameters on the resulting material. In addition, the color presentation is important because film opacity is a critical property governing the applicability of conditioned films, particularly if the film is to be used in industrial applications [35]. Color measurement by observation of CIELAB color space parameters for the films based on different blending ratios of JFP and HMP are presented in Table 2. The results showed that JFP films and JFP/HMP films were transparently opaque and slightly yellowish. With increasing JFP content, the value of a∗ was slightly decreased while the values of L∗ and b∗ increased. The positive value of b∗ indicated a yellow coloration associated with the natural color of JFP. The color properties of the JFP and JFP/HMP-based films were similar to the polysaccharide-based composite films reported by other studies [36–38], but the ΔE values of all prepared films were slightly higher than the films prepared from gelatin. These results agreed with visual observation. It was also observed that the opacity and transparency of JFP/HMP films depended on not only the concentration of polysaccharides but also the film thickness.
3.5. In vitro erosion behavior The erosion studies were carried out for prepared JFP film and JFP/ HMP films and the results were given in Fig. 4a. First, the film was incubated in SGF for 2 h, washed and transferred to SIF for 4 h. The results showed that the film made by pure JFP was around 50% eroded but the film was observable after the incubation in SGF and SIF. However, the films prepared from JFP/HMP mixtures were almost completely eroded and dissolved. This characteristic feature indicates that pure JFP can be used as a new material for a colon-specific drug delivery system. However, the JFP cannot be used exclusively to cast hard capsules because of its low viscoelasticity [46]. Therefore, composite capsules were formulated using JFP as the main ingredient blended with resistant starch and xanthan gum for the applicability evaluation of JFP in hard capsule preparation.
3.3. Moisture content and water vapor permeability Moisture content and water vapor permeability are frequently used as important measures in film preparation because moisture can pass through the film and subsequently change the properties of the film as well as the substance being packaged or encapsulated [39]. The moisture content of the JFP and JFP/HMP films are depicted in Fig. 2a and the results indicated that the addition of high-methyl pectin concentration from 0% to 50% (w/w) in JFP/HMP significantly increased the moisture content from 25.68% to 45.18%. Addition of high methyl pectin produced roughness and nonuniform surfaces in the JFP/HMP films. The rough surfaces of the films are capable of high interaction with water and hence increased moisture content was observed in JFP/ HMP films. WVP of the JFP and JFP/HMP films are provided in (Fig. 2b). The effect of high-methyl pectin in JFP/HMP films showed decreased WVP from 5.55 × 10−3 to 2.7 × 10−3 (?? × ????⁄ ??2 × ℎ × ??????). The WVP of the films developed in this study was
3.6. Hard capsule preparation and in vitro release profile Methylene blue was used as a model encapsulated substance to examine the release profile of the JFP composite hard capsule. The results (Fig. 4b) indicated that methylene blue was protected by the composite capsules in SGF during the test period with a minimal release (less than 2%) and the outer layer of the capsule remained fairly intact (Fig. 4c). Then, the capsule was removed from SGF and put into the SIF, the composite capsule remained integral over the test period without significant degradation but the release amount of methylene blue was slightly increased from ∼2% to ∼15%. The release of methylene blue in SIF could be attributed to the unsealed connection between the capsule cap and body (as indicated by the red arrow in the middle picture of Fig. 4c) that allows water molecules to penetrate the capsules
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Fig. 2. (a). Moisture content of JFP/HMP films. (b). Water vapor permeability of JFP/HMP films. Each value represented here is the mean ± SD (n = 3) and the significance difference is denoted as ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.01.
and causes the leak of the methylene blue. The resistance of simulated gastrointestinal fluids of JFP composite was in agreement with observation obtained in the film erosion test and the results reported in other LMP derived drug delivery systems [22,47,48]. Finally, the JFP composite capsule was removed from SIF and placed into the SGF. The rate of methylene blue release from the capsule was increased significantly from 15% to 90% in 2 h because of the substantial degradation of the composite capsule (as indicated by the red arrow in the picture on the right side of Fig. 4c). Based on the results of the in vitro release study, JFP can be a potential material for the preparation of colon-targeted hard capsules.
Table 3 Mechanical properties of JFP and JFP/HMP films. Film #
TS (MPa)
1 2 3 4 5
22.94 18.82 16.70 14.57 13.86
± ± ± ± ±
E (%) 2.58 4.82 3.71 3.49 1.07∗
67.43 ± 14.60 82.81 ± 22.58 90.92 ± 11.58 104.20 ± 15.34 120.43 ± 10.93∗
Data are represented here is the mean ± SD (n = 3) and the significance difference is denoted as ∗p < 0.05.
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Fig. 3. SEM morphology of JFP films (left-magnification of 100 × ) and (right-magnification of 1000 × ).
Fig. 4. (a). In vitro degradation (erosion) rate of JFP/HMP film in SGF 2 h and SIF 4 h. The values are mean ± SD (n = 3) and the significance difference is denoted as ∗∗∗p < 0.01. (b). In vitro release profile of methylene blue from the hard capsule in digestive fluids. (c). The degradation of capsule in simulated digestive fluids. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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4. Conclusions
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This study investigated the pectinous polysaccharide extraction from achenes of jelly fig to prepare the film and colon-specific capsule. The films were first characterized by Fourier transform infrared (FT-IR) spectroscopy, color test, thickness test, moisture content, and mechanical properties. The results indicated that the JFP films derived in this study possessed good mechanical and physical properties to be used as a basis material in the CDDS development. The results showed that film prepared from pure JFP exhibited good stability in simulated digestive fluids. The result of the in vitro release study also demonstrated that the JFP composite hard capsule could pass through the simulated gastrointestinal fluids without significant degradation and successfully released the model drug in the SCF fluid. Therefore, JFP could be a promising ingredient for developing colon-targeted capsules. CRediT authorship contribution statement Thangavel Ponrasu: Formal analysis, Validation, Writing - review & editing. Jhao-Syuan Gu: Investigation, Visualization, Writing - original draft. Jia-Jiuan Wu: Conceptualization. Yu-Shen Cheng: Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Writing - review & editing. Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, “Evaluation of jelly fig polysaccharide as a shell composite ingredient of colon-specific drug delivery”. Acknowledgments The funding of this work was supported by the Ministry of Science and Technology, Taiwan (MOST107-2622-E-224-002-CC3, and MOST 106-2622-E-224-007-CC3). References [1] A.B. Meneguin, B.S.F. Cury, R.C. Evangelista, Films from resistant starch-pectin dispersions intended for colonic drug delivery, Carbohydr. Polym. 99 (2014) 140–149. [2] K.L. Deore, N.A. Thombre, P.S. Gide, Formulation and development of tinidazole microspheres for colon targeted drug delivery system, J. Pharm. Res. 6 (2013) 158–165. [3] M. Chourasia, S. Jain, Pharmaceutical approaches to colon targeted drug delivery systems, J. Pharm. Pharmaceut. Sci. 6 (2003) 33–66. [4] P.M. Treuting, M.J. Arends, S.M. Dintzis, Upper gastrointestinal tract, Comparative Anatomy and Histology, second ed., Elsevier, 2017, pp. 191–211. [5] K.A. Elkhodairy, S.A. Afifi, A.S. Zakaria, A promising approach to provide appropriate colon target drug delivery systems of vancomycin HCL: pharmaceutical and microbiological studies, BioMed Res. Int. (2014) 2014. [6] S. Nalinbenjapun, C. Ovatlarnporn, Chitosan-5-aminosalicylic acid conjugates for colon-specific drug delivery: methods of preparation and in vitro evaluations, J. Drug Deliv. Sci. Technol. (2019) 101397. [7] E. Moghimipour, F.A. Dorkoosh, M. Rezaei, M. Kouchak, J. Fatahiasl, K.A. Angali, Z. Ramezani, M. Amini, S. Handali, In vivo evaluation of pH and time-dependent polymers as coating agent for colonic delivery using central composite design, J. Drug Deliv. Sci. Technol. 43 (2018) 50–56. [8] L. Yang, Biorelevant dissolution testing of colon-specific delivery systems activated by colonic microflora, J. Contr. Release 125 (2008) 77–86. [9] R. Sureshkumar, M. Munikumar, G. Ganesh, N. Jawahar, D. Nagasamyvenkatesh, V. Senthil, L. Raju, M. Samantha, Formulation and evaluation of pectin-hydroxypropyl methylcellulose coated curcumin pellets for colon delivery, Asian J. Pharm.: Free full text articles from Asian J Pharm 3 (2014). [10] K.E. Uhrich, S.M. Cannizzaro, R.S. Langer, K.M. Shakesheff, Polymeric systems for controlled drug release, Chem. Rev. 99 (1999) 3181–3198. [11] M. Gautam, D. Santhiya, In-situ mineralization of calcium carbonate in pectin based edible hydrogel for the delivery of protein at colon, J. Drug Deliv. Sci. Technol. 53
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