http://informahealthcare.com/ddi ISSN: 0363-9045 (print), 1520-5762 (electronic) Drug Dev Ind Pharm, Early Online: 1–7 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2013.858730
RESEARCH ARTICLE
Characterization of freeze-dried gallic acid/xyloglucan Namon Hirun, Tanatchaporn Sangfai, and Vimon Tantishaiyakul
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Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Nanotec-PSU Center of Excellence for Drug Delivery Systems, Prince of Songkla University, Hat-Yai, Thailand Abstract
Keywords
Background: Tamarind seed xyloglucan (TSX) is generally used for drug delivery systems. Gallic acid (GA) possesses various pharmacological activities. It has a good solubility and bioavailability but short half-life. Purpose: To prepare a sustained-release of GA to overcome its relatively short half-life. GA was blended with TSX and freeze-dried. The physicochemical properties of freeze-dried GA and freeze-dried GA/TSX were characterized, and the release profiles of GA from these freeze-dried samples were investigated. Method: All freeze-dried samples were characterized by PXRD, spectroscopic and thermal analyses. The dissolution studies were performed according to the United States Pharmacopeia (USP) XXX. Results: According to FTIR, FT-Raman and 13C CP/MAS NMR, the spectra of freeze-dried GA were similar to that of the anhydrous form. Nevertheless, DRIFTS and DSC were able to differentiate these two forms. The crystallinity of GA in the freeze-dried GA/TSX was the same as that of the freeze-dried GA. DSC indicates that there were interactions between GA and TSX. It was of interest that a freeze-dried sample with low amount of GA, 0.2% GA/1% TSX was mostly in an amorphous form. Moreover, all freeze-dried GA/TSX preparations demonstrated a sustainedrelease of GA compared to GA alone. The freeze-dried 1% GA/1% TSX provided the best sustained-release of GA of up to 240 min. Conclusions: TSX could change a crystal form of a small molecule to a mostly amorphous form. It was of importance that the freeze-dried GA/TSX could effectively retard the release of GA. These samples may be able to overcome the limitation for the therapeutic use of GA due to its short biological half-life.
Dissolution, DRIFTS, DSC, gallic acid, xyloglucan History Received 17 December 2012 Revised 6 June 2013 Accepted 17 October 2013 Published online 14 November 2013
Introduction Gallic acid (GA) is a strong antioxidant that has antimutagenic and anticarcinogenic activities1,2. Various polymorphic forms of GA have been reported in the literature, namely forms I, II, III, IV and an anhydrous form3,4 as well as a dry and crystal form5. GA is rapidly absorbed from the stomach and the small intestine within 1–2 h after intake6. It has a short biological half-life of about 1.06–1.19 h7. Generally, the short half-life of a drug is the main reason for its therapeutic inefficiency8,9. Therefore, generation of a sustained-release oral preparation of a drug which is able to extend its biological half-life has been extensively investigated10. Generally, a drug that has a low aqueous solubility at a biological temperature (i.e. less than 1 mg mL1) and a low percentage absorption (i.e. less than 20%) is unsuitable for administration as a sustained-release preparation8. GA has a good solubility (17.9 mg mL1 at 37 C) and good bioavailability7,11. Thus, it is appropriate to develop a sustained-release preparation of GA to overcome its relatively short half-life.
Address for correspondence: Vimon Tantishaiyakul, PhD, Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, NanotecPSU Center of Excellence for Drug Delivery Systems, Prince of Songkla University, Hat-Yai 90112, Thailand. E-mail: [email protected]
Biopolymers have been employed to protect and deliver bioactive compounds. These carriers can extend the absorption phase of bioactive compounds since the compounds have to be released from a polymeric matrix to allow absorption through the gastrointestinal wall12. Tamarind seed xyloglucan (TSX) is a natural polysaccharide generally used in the food industries and for drug delivery systems13. As previously reported by our group14, GA at certain amounts can form gels with TSX, thus GA could interact with TSX and possibly have a sustained release from a GA/TSX system. Since freeze drying is a method that can conserve polymer properties and produce no degradation of the polymer, all samples in this study, GA and GA/TSX, were dried by the freeze drying method. The crystallinity of freeze-dried GA and freeze-dried GA/TSX was investigated using powder X-ray diffraction (PXRD). The differences between these freeze-dried samples from the other polymorphic forms were examined using Fourier transform infrared spectroscopy (FTIR), diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), FT-Raman spectroscopy, cross polarization and magic angle spinning (CP/MAS) 13C NMR spectroscopy and differential scanning calorimetry (DSC). In addition, these methods were also used to explore the interaction between the GA and TSX. The thermal stability of the freeze-dried GA/TSX was explored using
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thermogravimetric analysis (TGA). Finally, the release of the GA from the freeze-dried GA/TSX was determined.
to tetramethylsilane (0 ppm), and solid adamantine was used as a secondary standard.
Materials and methods
Thermal analysis
GA was obtained from Fluka Chemie GmbH (Buchs, Switzerland) and was identified as GA form II based on its FTIR and PXRD pattern3. TSX (molecular weight of 202 kDa, and sugar composition of xylose:glucose:galactose:arabinose ¼ 36 : 45 : 16 : 3) was purchased from Megazyme International Ireland Ltd., Wicklow, Ireland. All other reagents used were of analytical grade.
The DSC studies were performed in a nitrogen atmosphere using a Q2000 TA Instruments (Lindon, UT). DSC scans were recorded at a heating rate of 10 C/min and 80 C/min. The TGA were performed under a nitrogen purge using a thermogravimetric analyzer (TGA7, Perkin-Elmer, Phoenix, AZ). Samples were heated at a rate of 10 C/min from 50 C to 500 C.
Sample preparation
GA release study
GA form IV was prepared by evaporating a methanolic solution of GA. The anhydrous preparation was prepared as previously described4. A stock solution of TSX was prepared by dispersing the required amount of TSX in water. Then, the dispersion was slowly homogenized with a mechanical stirrer for 4 h at 50 C. GA solutions were prepared by dissolving the appropriate amounts of GA in distilled water. Appropriate volumes of the GA solutions were then added into the TSX solution with vigorous stirring at 40 C to obtain 0.2, 0.4, 0.6, 0.8 and 1.0% (w/v) solutions of GA in 1.0% (w/v) TSX. All samples were then cooled to room temperature. Subsequently, the viscous mixtures (0.2GA/TSX, 0.4GA/TSX and 0.6GA/TSX) and the gel (0.8GA/TSX and 1GA/TSX) were lyophilized in a lyophilizer (Dura-StopÔ, FTS Systems, Inc., Stone Ridge, NY) to obtain the dry samples. These freeze-dried samples were used for instrumental characterization. For the GA release studies, the hot GA/TSX mixtures were filled in 24-well plates at a volume of 2.5 mL per well, then allowed to cool to room temperature and freeze-dried.
The dissolution of GA from freeze-dried GA/TSX mixtures and pure GA were examined using the rotating paddle apparatus. The paddle speed was maintained at 50 rpm. The samples were placed in 750 mL pepsin-free simulated gastric fluid at a pH of 1.0 (HCl 0.1 M). After 2 h, the pH was increased to pH 6.8 by adding 250 mL of 0.2 M tribasic phosphate buffer, the pH was adjusted with 2 N sodium hydroxide and/or 2 N HCl. This will simulate the conditions in the more basic small intestine according to USPXXX. At the appropriate time intervals, 4 mL aliquots were removed from the dissolution vessels and replaced with 4 mL of fresh medium. GA released profiles were determined using high performance liquid chromatography (HPLC) (Waters Inc., Milford, MA) equipped with a UV detector (UV-1575 intelligent UV/VIS detector; Jasco, Tokyo, Japan) and a C-18-reversed phase column (BDS HYPERSIL C18, 150 4.6 mm, 5 mm; Thermo Scientific, Cincinnati, OH). The mobile phase consisted of methanol and 0.7% phosphoric acid (15:85 v/v, pH 3). The flow rate and injection volume were 0.5 mL/min and 20 mL, respectively, with detection at 267 nm.
FTIR, DRIFTS and FT-Raman measurements FTIR spectra were measured using a Perkin Elmer Spectrum One FTIR spectrometer (Perkin-Elmer, Waltham, MA). The sample was ground with KBr in an agate mortar to a fine powder and then pressed into a disc under high pressure. The spectra were recorded from 4400 to 450 cm1 by averaging 16 scans at a 2 cm1 resolution. For the DRIFTS spectra collection, the samples were placed in a microsample cup for the PerkinElmer Spectrum One FTIR diffuse reflectance accessory using the supplied sample cup holder. The spectra were collected from 4400 to 450 cm1 by averaging 128 scans at 2 cm1 resolution. All reflectance spectra were converted to Kubelka-Munk (KM) units by the use of a PerkinElmer Spectrum for Windows version 5.02 software package. FT-Raman spectra were acquired on a Perkin-Elmer spectrum GX with excitation at 1064 nm using an Nd:YAG laser, resolution of 2.0 cm1. PXRD measurement PXRD patterns were obtained using an X-ray diffractometer (X’ Pert MPD, Philips, Netherlands) in the angular range of 5–90 (2y) with Cu-Ka radiation ( ¼ 0.154 nm) at a voltage of 40 kV and a current of 30 mA. 13
C CP/MAS NMR measurement
The NMR experiments were performed using a Bruker Avance 300 NMR spectrometer operating at 75.51 MHz for 13C using a standard 4 mm cross-polarization magic angle spinning probe (CP/MAS). The samples were spun at the magic angle at a rate of 10 620 Hz. The total number of scans was 10 000 to eliminate spinning side band interference. A contact time and a recycle delay between scans for all the samples were 5.0 ms and 3 s, respectively. The 13C chemical shifts were referenced with respect
Results and discussion Because the freeze drying process can transform a crystalline solid material to an amorphous state the freeze-dried GA alone and the freeze-dried GA/TSX were characterized to compare with the other polymorphic forms of GA. Although various GA polymorphs have been reported in the literature, this is the first time that freeze-dried GA has been investigated. FTIR and FT-Raman analysis Generally, the OH stretching of the alcoholic or phenolic group will be detected at the region from 3700 to 3000 cm1. The intermolecular hydrogen-bonded OH band is generally observed as a broad band. However, a sharp peak can be observed due to an intra-molecular hydrogen-bonded OH or a free O–H stretch. This sharp band will appear in combination with the broad intermolecular hydrogen-bonded OH peak. For GA, the OH groups at position 3, 4 and 5 on the benzene ring are partially overlapped with the OH stretching bands from the acidic group (COOH). The FTIR spectra for forms II and IV, the anhydrous and the freeze-dried GA are shown in Figure 1. The spectra of forms II and IV are comparable and agree with previous reports3. Those of the anhydrous and freeze-dried GA are also similar to each other and these forms are different from the previously reported for form III3. As shown in Figure 1, broad OH peaks were observed for the GA forms II and IV. However, both anhydrous and freezedried GA show a sharp peak at 3496 cm1. This sharp peak of the OH group may be caused by the non-hydrogen bonds or by the intra-molecular hydrogen bonds of this group. Due to the different conformational arrangements of the various GA forms, they are able to form inter- or intra-molecular hydrogen bonds that reflect the distinctive FTIR patterns of the OH groups. Furthermore, the GA forms II and IV show an intense peak of C ¼ O stretching at
Freeze-dried GA and freeze-dried GA/TSX
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DOI: 10.3109/03639045.2013.858730
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Figure 3. FT-Raman spectra of various GA forms.
Figure 1. FTIR spectra of various GA forms.
Figure 2. DRIFTS spectra of various GA forms.
1702 cm1. However, those for the anhydrous and freeze-dried GA show the C ¼ O bands at 1667 cm1. It is relatively difficult to distinguish anhydrous and freeze-dried GA by comparing their FTIR spectra. DRIFTS has been widely used to determine drug–polymer interactions and also for transformation of drug polymorph15,16. This technique was employed to analyze various forms of GA. As shown in Figure 2, the DRIFTS spectra of GA forms II and IV, the anhydrous and freeze-dried GA are different from each other. The prominent characteristic at the OH region for all forms are also comparable to those in the FTIR. The differences occur at the C ¼ O region. Form II shows a C ¼ O peak at about 1720 cm1 while a very low intensity at 1718 cm1 was observed for form IV. The anhydrous form showed an intense C ¼ O band at 1686 cm1 but the freeze-dried form displays a peak at 1682 cm1 and this
peak cannot be separated from the C ¼ C band at 1629 cm1. Thus using different methods of measurement, DRIFTS versus FTIR, is a practical way to distinguish between different forms of GA. The FT-Raman spectroscopy is regarded as a complementary technique to FTIR, and in some cases it has been shown to offer a better chance to differentiate different polymorphs. This method was also used in an attempt to differentiate various forms of GA. The FT-Raman spectra of these GA forms are displayed in Figure 3. The C ¼ O stretching band for the anhydrous and freezedried GA disappears, but that for the GA form II displays at 1689 cm1. These two types of spectra are similar to those reported for the dry and crystal form5. Nevertheless, this method could not distinguish between the anhydrous and freeze-dried GA. Since only the DRIFTS can differentiate the various GA forms, this technique was used to investigate the polymorphic forms of GA in the freeze-dried GA/TSX samples. The DRIFTS spectra of TSX, freeze-dried GA and GA/TSX at various ratios are shown in Figure 4. TSX shows a broad peak of OH stretching. The mixture that contains 0.2% GA also shows a broad peak at the OH region. A small sharp peak of OH stretching of GA at 3496 cm1 was observed for other GA/TSX mixtures (0.4GA/TSX, 0.6GA/TSX, 0.8GA/TSX and 1GA/TSX). This may indicate that this OH group of GA at 0.2% may be able to completely interact with TSX, so no sharp peak for the OH group is observed. The sharp peaks for the OH group from other mixtures are similar to those detected for the freeze-dried and anhydrous GA. The C ¼ O stretching of GA in the mixtures show similar features to the pure freeze-dried GA but not the anhydrous GA. PXRD analysis PXRD has been widely used to analyze for the various polymorphic forms of compounds17,18. In this study, PXRD were also employed to investigate the changes in crystallinity of the various polymorphic forms of GA and freeze-dried GA and freeze-dried GA/TSX. As shown in Figure 5, TSX is amorphous but GA is a crystalline solid. The PXRD profiles of the GA forms II, IV and the anhydrous form are the same as found in a previous report3. The freeze-dried GA was present as a mixture of two different crystalline forms. The intense peaks at 25.2 and 27.5 (2y) were indicative of the GA anhydrous form (major), while the peaks at 18.4 and 36.0 (2y) indicated the presence of form IV (minor) in the freeze-dried sample. It should be noted that the intensity of the crystalline reflection at 16.3 (2y) of the anhydrous GA is lower than that of the freeze-dried GA. The differences in
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Figure 4. DRIFTS spectra of freeze-dried samples of TSX, GA and various compositions of GA/TSX.
intensity may be caused by the presence of GA form IV which also shows a peak at 16.3 (2y). In addition, the presence of GA form IV may cause a difference in the DRIFT spectra of the freeze-dried GA and the anhydrous GA in some regions. For the freeze-dried GA/TSX samples, 0.2GA/TSX exhibits a PXRD characteristic of an amorphous halo (Figure 5). This may due to the strong interactions between GA and TSX as mentioned above in the DRIFTS analysis for this sample. The other mixtures, 0.4GA/TSX, 0.6 GA/TSX, 0.8 GA/TSX and 1 GA/TSX, show crystalline GA peaks. The major peaks of these samples are similar to those obtained in the freeze-dried GA samples, in terms of intensity and position. These crystalline peaks exist in combination with the amorphous materials of TSX or some part of the GA. 13
C CP/MAS NMR
A 13C CP/MAS NMR of GA was previously reported19 and agreed with the GA form II as shown in Figure 6. The spectrum of the anhydrous form is similar to that of the freeze-dried form. Hence, this method cannot differentiate between the freeze-dried and the anhydrous forms. The mixture of freeze-dried 1GA/TSX shows broad peaks at almost the same positions as for TSX and the freeze-dried GA. These broad peaks demonstrate the characteristics of the amorphous solid that are possibly contributed from TSX and some part of the GA that are amorphous20. This corresponds to the PXRD analyses and indicates the amorphous property in these mixtures. Nevertheless, the solid-state NMR cannot provide conclusive evidence for a GA-TSX interaction. Thermal analysis The DSC thermograms of the GA forms II, IV, the anhydrous and freeze-dried are shown in Figure 7. Two endothermic peaks for
Figure 5. Powder X-ray diffraction patterns of various GA forms, freezedried TSX and various compositions of freeze-dried GA/TSX.
the GA monohydrate form II and IV were detected. The first one between 125 and 140 C and the second one at 275.7 C. This first endothermic peak was not detected in the anhydrous GA and freeze-dried GA samples. According to the previous report3, two endothermic peaks for form II (a broad peak between 90 and 115 C and a peak at 268.1 C) and for form IV (a broad peak between 100 and 115 C and a peak at 268.5 C) were detected. The reported endothermic peaks for these forms are comparable to those obtained in this study, the differences in the melting points detected are probably due to the use of a different heating rate. Endothermic melting peaks for the anhydrous and freezedried GA are observed at 277.0 and 262.3 C, respectively. Thus, DSC can differentiate between these two forms. Thermal analyses were also used to investigate the interaction between GA and TSX. As shown in Figures 8 and 9, TSX exhibits a broad, endothermic decomposition peak at about 322.2 C (Figure 8). The TGA curve (Figure 9) demonstrates a 10% weight loss of water starting at 58 C of TSX. The second stage starts at 320 C due to the degradation of TSX as previously reported21–23. The GA form II melts with decomposition as indicated by the mass loss (Figure 9). This agrees with a previous report on the thermal decomposition of GA24. The thermogram of 0.2GA/TSX
Freeze-dried GA and freeze-dried GA/TSX
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Figure 6. Solid state GA/TSX.
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C NMR spectra of TSX, GA and freeze-dried
Figure 8. DSC thermograms of freeze-dried samples at the heating rate of 10 C/min.
presented a broad peak at 246 C (Figure 8) that did not comply with the melting point of crystalline GA. This fusion temperature could be attributed to the presence of impurities in the sample, the residual crystalline GA or multiple amorphous forms, as previously described for the appearance of endotherm in X-ray amorphous samples25. These were consistent with weight losses observed for this sample in Figure 9. As shown in Figure 8, a depression of the crystalline GA and TSX melting points was observed for all freeze-dried mixtures with a GA percentage higher than 0.2%. This was attributed to the GA–TSX interactions. A broad, endothermic decomposition of TSX was observed that accompanied with the melting of GA. With increased amounts of GA, to 0.8 and 1%GA, in the GA/TSX mixtures, two endothermic peaks were observed. This may be attributed to the melting of the GA domains that were in close contact with and far-off the TSX as previously described for the drug and polymer blends26. Figure 7. DSC thermograms of various forms of GA at the heating rate of 80 C/min.
Drug release The release profiles of pure GA and GA from the freeze-dried GA/TSX in a simulated gastrointestinal condition are shown in Figure 10. A one-way analysis of variance (ANOVA) with a post
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are statistically higher than that of the freeze-dried 0.6GA/TSX (p50.02). Nevertheless, the releases of these three samples were not statistically different after 45 min (p40.085). The release of GA from the freeze-dried 0.8GA/TSX is different from those of the three samples but not after 90 min (p40.06). Furthermore, the release of GA from the 1GA/TSX is lower than from the other four samples. Nevertheless, it demonstrates the same release as the others after 240 min (p40.05). As previously reported by our group14, at 37 C, GA at higher than 0.69% in 1%TSX can form a gel. In this study, with the increase in the amount of GA, the samples could possibly form a gel in the dissolution medium, reflecting the slow release of GA from these samples. In conclusion, it is of interest that the DRIFTS rather than the FTIR, FT-Raman and 13C CP/MAS NMR can differentiate between various polymorphic forms of GA. The freeze-dried GA has been shown to have a different form compared to other polymorphic forms reported in the literature based on the DRIFTS and the DSC thermograms. According to the PXRD patters, the crystalline forms of GA from the freeze-dried GA/TSX mixtures seem to be the same as in the pure freeze-dried GA except for the freeze-dried form with low amounts of GA, 0.2%GA in 1%TSX which is mostly in an amorphous state. Furthermore, the freezedried GA/TSX samples can provide an optimal sustained release profile for oral administration.
Declaration of interest
Figure 9. TGA of freeze-dried samples at the heating rate of 10 C/min.
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper. This work was supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program through Grant Nos PHD/0259/2549 and PHD/0045/2552 and the Nanotechnology Center (NANOTEC), NSTDA, Ministry of Science and Technology, Thailand, through its program of Center of Excellence Network.
References
Figure 10. Dissolution profiles of various compositions of freeze-dried GA/TSX.
hoc least significant difference (LSD) test was used for comparisons the released profiles of different samples. The release of pure GA is very fast and complete within 15 min. The release of GA from the GA/TSX freeze-dried samples demonstrated a sustained release pattern. As mentioned above, GA in 0.2GA/TSX was mostly in an amorphous form but a crystalline form was detected for GA in the 0.4GA/TSX sample. Generally, a drug in an amorphous state will have a higher dissolution than that in a crystalline form27. The release of GA from the freeze-dried 0.2GA/TSX is slightly higher than that for the freeze-dried 0.4GA/TSX but not statistically different. Both release profiles
1. Inoue M, Suzuki R, Koide T, et al. Antioxidant, gallic acid, induces apoptosis in HL-60 RG cells. Biochem Biophys Res Commun 1994; 204:898–904. 2. Ji B-C, Hsu W-H, Yang J-S, et al. Gallic acid induces apoptosis via caspase-3 and mitochondrion-dependent pathways in vitro and suppresses lung xenograft tumor growth in vivo. J Agric Food Chem 2009;57:7596–604. 3. Clarke HD, Arora KK, Wojtas L, Zaworotko MJ. Polymorphism in multiple component crystals: forms III and IV of gallic acid monohydrate. Cryst Growth Des 2011;11:964–6. 4. Hirun N, Saithong S, Pakawatchai C, Tantishaiyakul V. 3,4,5Trihydroxybenzoic acid. Acta Crystallographica Section E 2011;E 67:787. 5. Billes F, Mohammed-Ziegler I, Bombicz P. Vibrational spectroscopic study on the quantum chemical model and the X-ray structure of gallic acid, solvent effect on the structure and spectra. Vib Spectrosc 2007;43:193–202. 6. Lafay S, Gil-Izquierdo A. Bioavailability of phenolic acids. Phytochem Rev 2008;7:301–11. 7. Shahrzad S, Aoyagi K, Winter A, et al. Pharmacokinetics of gallic acid and its relative bioavailability from tea in healthy humans. Hum Nutr Metab Res Commun 2001;131:1207–10. 8. Collett J, Moreton C. Modified-release peroral dosage forms. In: Aulton ME, ed. Pharmaceutics the scienc of dosage form design. 2nd ed. New York: Elsevier Science; 2002:290–5. 9. Kong X, Jin L, Wei M, Duan X. Antioxidant drugs intercalated into layered double hydroxide: structure and in vitro release. Appl Clay Sci 2010;49:324–9. 10. Amighi K, Timmermans J, Puigdevall J, et al. Peroral sustainedrelease film-coated pellets as a means to overcome physicochemical and biological drug-related problems. I. In vitro development and evaluation. Drug Dev Ind Pharm 1998;24:509–15. 11. Lu L-L, Lu X-Y. Solubilities of gallic acid and its esters in water. J Chem Eng Data 2007;52:37–9.
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RESEARCH ARTICLE
Characterization of freeze-dried gallic acid/xyloglucan Namon Hirun, Tanatchaporn Sangfai, and Vimon Tantishaiyakul
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Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Nanotec-PSU Center of Excellence for Drug Delivery Systems, Prince of Songkla University, Hat-Yai, Thailand Abstract
Keywords
Background: Tamarind seed xyloglucan (TSX) is generally used for drug delivery systems. Gallic acid (GA) possesses various pharmacological activities. It has a good solubility and bioavailability but short half-life. Purpose: To prepare a sustained-release of GA to overcome its relatively short half-life. GA was blended with TSX and freeze-dried. The physicochemical properties of freeze-dried GA and freeze-dried GA/TSX were characterized, and the release profiles of GA from these freeze-dried samples were investigated. Method: All freeze-dried samples were characterized by PXRD, spectroscopic and thermal analyses. The dissolution studies were performed according to the United States Pharmacopeia (USP) XXX. Results: According to FTIR, FT-Raman and 13C CP/MAS NMR, the spectra of freeze-dried GA were similar to that of the anhydrous form. Nevertheless, DRIFTS and DSC were able to differentiate these two forms. The crystallinity of GA in the freeze-dried GA/TSX was the same as that of the freeze-dried GA. DSC indicates that there were interactions between GA and TSX. It was of interest that a freeze-dried sample with low amount of GA, 0.2% GA/1% TSX was mostly in an amorphous form. Moreover, all freeze-dried GA/TSX preparations demonstrated a sustainedrelease of GA compared to GA alone. The freeze-dried 1% GA/1% TSX provided the best sustained-release of GA of up to 240 min. Conclusions: TSX could change a crystal form of a small molecule to a mostly amorphous form. It was of importance that the freeze-dried GA/TSX could effectively retard the release of GA. These samples may be able to overcome the limitation for the therapeutic use of GA due to its short biological half-life.
Dissolution, DRIFTS, DSC, gallic acid, xyloglucan History Received 17 December 2012 Revised 6 June 2013 Accepted 17 October 2013 Published online 14 November 2013
Introduction Gallic acid (GA) is a strong antioxidant that has antimutagenic and anticarcinogenic activities1,2. Various polymorphic forms of GA have been reported in the literature, namely forms I, II, III, IV and an anhydrous form3,4 as well as a dry and crystal form5. GA is rapidly absorbed from the stomach and the small intestine within 1–2 h after intake6. It has a short biological half-life of about 1.06–1.19 h7. Generally, the short half-life of a drug is the main reason for its therapeutic inefficiency8,9. Therefore, generation of a sustained-release oral preparation of a drug which is able to extend its biological half-life has been extensively investigated10. Generally, a drug that has a low aqueous solubility at a biological temperature (i.e. less than 1 mg mL1) and a low percentage absorption (i.e. less than 20%) is unsuitable for administration as a sustained-release preparation8. GA has a good solubility (17.9 mg mL1 at 37 C) and good bioavailability7,11. Thus, it is appropriate to develop a sustained-release preparation of GA to overcome its relatively short half-life.
Address for correspondence: Vimon Tantishaiyakul, PhD, Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, NanotecPSU Center of Excellence for Drug Delivery Systems, Prince of Songkla University, Hat-Yai 90112, Thailand. E-mail: [email protected]
Biopolymers have been employed to protect and deliver bioactive compounds. These carriers can extend the absorption phase of bioactive compounds since the compounds have to be released from a polymeric matrix to allow absorption through the gastrointestinal wall12. Tamarind seed xyloglucan (TSX) is a natural polysaccharide generally used in the food industries and for drug delivery systems13. As previously reported by our group14, GA at certain amounts can form gels with TSX, thus GA could interact with TSX and possibly have a sustained release from a GA/TSX system. Since freeze drying is a method that can conserve polymer properties and produce no degradation of the polymer, all samples in this study, GA and GA/TSX, were dried by the freeze drying method. The crystallinity of freeze-dried GA and freeze-dried GA/TSX was investigated using powder X-ray diffraction (PXRD). The differences between these freeze-dried samples from the other polymorphic forms were examined using Fourier transform infrared spectroscopy (FTIR), diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), FT-Raman spectroscopy, cross polarization and magic angle spinning (CP/MAS) 13C NMR spectroscopy and differential scanning calorimetry (DSC). In addition, these methods were also used to explore the interaction between the GA and TSX. The thermal stability of the freeze-dried GA/TSX was explored using
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thermogravimetric analysis (TGA). Finally, the release of the GA from the freeze-dried GA/TSX was determined.
to tetramethylsilane (0 ppm), and solid adamantine was used as a secondary standard.
Materials and methods
Thermal analysis
GA was obtained from Fluka Chemie GmbH (Buchs, Switzerland) and was identified as GA form II based on its FTIR and PXRD pattern3. TSX (molecular weight of 202 kDa, and sugar composition of xylose:glucose:galactose:arabinose ¼ 36 : 45 : 16 : 3) was purchased from Megazyme International Ireland Ltd., Wicklow, Ireland. All other reagents used were of analytical grade.
The DSC studies were performed in a nitrogen atmosphere using a Q2000 TA Instruments (Lindon, UT). DSC scans were recorded at a heating rate of 10 C/min and 80 C/min. The TGA were performed under a nitrogen purge using a thermogravimetric analyzer (TGA7, Perkin-Elmer, Phoenix, AZ). Samples were heated at a rate of 10 C/min from 50 C to 500 C.
Sample preparation
GA release study
GA form IV was prepared by evaporating a methanolic solution of GA. The anhydrous preparation was prepared as previously described4. A stock solution of TSX was prepared by dispersing the required amount of TSX in water. Then, the dispersion was slowly homogenized with a mechanical stirrer for 4 h at 50 C. GA solutions were prepared by dissolving the appropriate amounts of GA in distilled water. Appropriate volumes of the GA solutions were then added into the TSX solution with vigorous stirring at 40 C to obtain 0.2, 0.4, 0.6, 0.8 and 1.0% (w/v) solutions of GA in 1.0% (w/v) TSX. All samples were then cooled to room temperature. Subsequently, the viscous mixtures (0.2GA/TSX, 0.4GA/TSX and 0.6GA/TSX) and the gel (0.8GA/TSX and 1GA/TSX) were lyophilized in a lyophilizer (Dura-StopÔ, FTS Systems, Inc., Stone Ridge, NY) to obtain the dry samples. These freeze-dried samples were used for instrumental characterization. For the GA release studies, the hot GA/TSX mixtures were filled in 24-well plates at a volume of 2.5 mL per well, then allowed to cool to room temperature and freeze-dried.
The dissolution of GA from freeze-dried GA/TSX mixtures and pure GA were examined using the rotating paddle apparatus. The paddle speed was maintained at 50 rpm. The samples were placed in 750 mL pepsin-free simulated gastric fluid at a pH of 1.0 (HCl 0.1 M). After 2 h, the pH was increased to pH 6.8 by adding 250 mL of 0.2 M tribasic phosphate buffer, the pH was adjusted with 2 N sodium hydroxide and/or 2 N HCl. This will simulate the conditions in the more basic small intestine according to USPXXX. At the appropriate time intervals, 4 mL aliquots were removed from the dissolution vessels and replaced with 4 mL of fresh medium. GA released profiles were determined using high performance liquid chromatography (HPLC) (Waters Inc., Milford, MA) equipped with a UV detector (UV-1575 intelligent UV/VIS detector; Jasco, Tokyo, Japan) and a C-18-reversed phase column (BDS HYPERSIL C18, 150 4.6 mm, 5 mm; Thermo Scientific, Cincinnati, OH). The mobile phase consisted of methanol and 0.7% phosphoric acid (15:85 v/v, pH 3). The flow rate and injection volume were 0.5 mL/min and 20 mL, respectively, with detection at 267 nm.
FTIR, DRIFTS and FT-Raman measurements FTIR spectra were measured using a Perkin Elmer Spectrum One FTIR spectrometer (Perkin-Elmer, Waltham, MA). The sample was ground with KBr in an agate mortar to a fine powder and then pressed into a disc under high pressure. The spectra were recorded from 4400 to 450 cm1 by averaging 16 scans at a 2 cm1 resolution. For the DRIFTS spectra collection, the samples were placed in a microsample cup for the PerkinElmer Spectrum One FTIR diffuse reflectance accessory using the supplied sample cup holder. The spectra were collected from 4400 to 450 cm1 by averaging 128 scans at 2 cm1 resolution. All reflectance spectra were converted to Kubelka-Munk (KM) units by the use of a PerkinElmer Spectrum for Windows version 5.02 software package. FT-Raman spectra were acquired on a Perkin-Elmer spectrum GX with excitation at 1064 nm using an Nd:YAG laser, resolution of 2.0 cm1. PXRD measurement PXRD patterns were obtained using an X-ray diffractometer (X’ Pert MPD, Philips, Netherlands) in the angular range of 5–90 (2y) with Cu-Ka radiation ( ¼ 0.154 nm) at a voltage of 40 kV and a current of 30 mA. 13
C CP/MAS NMR measurement
The NMR experiments were performed using a Bruker Avance 300 NMR spectrometer operating at 75.51 MHz for 13C using a standard 4 mm cross-polarization magic angle spinning probe (CP/MAS). The samples were spun at the magic angle at a rate of 10 620 Hz. The total number of scans was 10 000 to eliminate spinning side band interference. A contact time and a recycle delay between scans for all the samples were 5.0 ms and 3 s, respectively. The 13C chemical shifts were referenced with respect
Results and discussion Because the freeze drying process can transform a crystalline solid material to an amorphous state the freeze-dried GA alone and the freeze-dried GA/TSX were characterized to compare with the other polymorphic forms of GA. Although various GA polymorphs have been reported in the literature, this is the first time that freeze-dried GA has been investigated. FTIR and FT-Raman analysis Generally, the OH stretching of the alcoholic or phenolic group will be detected at the region from 3700 to 3000 cm1. The intermolecular hydrogen-bonded OH band is generally observed as a broad band. However, a sharp peak can be observed due to an intra-molecular hydrogen-bonded OH or a free O–H stretch. This sharp band will appear in combination with the broad intermolecular hydrogen-bonded OH peak. For GA, the OH groups at position 3, 4 and 5 on the benzene ring are partially overlapped with the OH stretching bands from the acidic group (COOH). The FTIR spectra for forms II and IV, the anhydrous and the freeze-dried GA are shown in Figure 1. The spectra of forms II and IV are comparable and agree with previous reports3. Those of the anhydrous and freeze-dried GA are also similar to each other and these forms are different from the previously reported for form III3. As shown in Figure 1, broad OH peaks were observed for the GA forms II and IV. However, both anhydrous and freezedried GA show a sharp peak at 3496 cm1. This sharp peak of the OH group may be caused by the non-hydrogen bonds or by the intra-molecular hydrogen bonds of this group. Due to the different conformational arrangements of the various GA forms, they are able to form inter- or intra-molecular hydrogen bonds that reflect the distinctive FTIR patterns of the OH groups. Furthermore, the GA forms II and IV show an intense peak of C ¼ O stretching at
Freeze-dried GA and freeze-dried GA/TSX
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Figure 3. FT-Raman spectra of various GA forms.
Figure 1. FTIR spectra of various GA forms.
Figure 2. DRIFTS spectra of various GA forms.
1702 cm1. However, those for the anhydrous and freeze-dried GA show the C ¼ O bands at 1667 cm1. It is relatively difficult to distinguish anhydrous and freeze-dried GA by comparing their FTIR spectra. DRIFTS has been widely used to determine drug–polymer interactions and also for transformation of drug polymorph15,16. This technique was employed to analyze various forms of GA. As shown in Figure 2, the DRIFTS spectra of GA forms II and IV, the anhydrous and freeze-dried GA are different from each other. The prominent characteristic at the OH region for all forms are also comparable to those in the FTIR. The differences occur at the C ¼ O region. Form II shows a C ¼ O peak at about 1720 cm1 while a very low intensity at 1718 cm1 was observed for form IV. The anhydrous form showed an intense C ¼ O band at 1686 cm1 but the freeze-dried form displays a peak at 1682 cm1 and this
peak cannot be separated from the C ¼ C band at 1629 cm1. Thus using different methods of measurement, DRIFTS versus FTIR, is a practical way to distinguish between different forms of GA. The FT-Raman spectroscopy is regarded as a complementary technique to FTIR, and in some cases it has been shown to offer a better chance to differentiate different polymorphs. This method was also used in an attempt to differentiate various forms of GA. The FT-Raman spectra of these GA forms are displayed in Figure 3. The C ¼ O stretching band for the anhydrous and freezedried GA disappears, but that for the GA form II displays at 1689 cm1. These two types of spectra are similar to those reported for the dry and crystal form5. Nevertheless, this method could not distinguish between the anhydrous and freeze-dried GA. Since only the DRIFTS can differentiate the various GA forms, this technique was used to investigate the polymorphic forms of GA in the freeze-dried GA/TSX samples. The DRIFTS spectra of TSX, freeze-dried GA and GA/TSX at various ratios are shown in Figure 4. TSX shows a broad peak of OH stretching. The mixture that contains 0.2% GA also shows a broad peak at the OH region. A small sharp peak of OH stretching of GA at 3496 cm1 was observed for other GA/TSX mixtures (0.4GA/TSX, 0.6GA/TSX, 0.8GA/TSX and 1GA/TSX). This may indicate that this OH group of GA at 0.2% may be able to completely interact with TSX, so no sharp peak for the OH group is observed. The sharp peaks for the OH group from other mixtures are similar to those detected for the freeze-dried and anhydrous GA. The C ¼ O stretching of GA in the mixtures show similar features to the pure freeze-dried GA but not the anhydrous GA. PXRD analysis PXRD has been widely used to analyze for the various polymorphic forms of compounds17,18. In this study, PXRD were also employed to investigate the changes in crystallinity of the various polymorphic forms of GA and freeze-dried GA and freeze-dried GA/TSX. As shown in Figure 5, TSX is amorphous but GA is a crystalline solid. The PXRD profiles of the GA forms II, IV and the anhydrous form are the same as found in a previous report3. The freeze-dried GA was present as a mixture of two different crystalline forms. The intense peaks at 25.2 and 27.5 (2y) were indicative of the GA anhydrous form (major), while the peaks at 18.4 and 36.0 (2y) indicated the presence of form IV (minor) in the freeze-dried sample. It should be noted that the intensity of the crystalline reflection at 16.3 (2y) of the anhydrous GA is lower than that of the freeze-dried GA. The differences in
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Figure 4. DRIFTS spectra of freeze-dried samples of TSX, GA and various compositions of GA/TSX.
intensity may be caused by the presence of GA form IV which also shows a peak at 16.3 (2y). In addition, the presence of GA form IV may cause a difference in the DRIFT spectra of the freeze-dried GA and the anhydrous GA in some regions. For the freeze-dried GA/TSX samples, 0.2GA/TSX exhibits a PXRD characteristic of an amorphous halo (Figure 5). This may due to the strong interactions between GA and TSX as mentioned above in the DRIFTS analysis for this sample. The other mixtures, 0.4GA/TSX, 0.6 GA/TSX, 0.8 GA/TSX and 1 GA/TSX, show crystalline GA peaks. The major peaks of these samples are similar to those obtained in the freeze-dried GA samples, in terms of intensity and position. These crystalline peaks exist in combination with the amorphous materials of TSX or some part of the GA. 13
C CP/MAS NMR
A 13C CP/MAS NMR of GA was previously reported19 and agreed with the GA form II as shown in Figure 6. The spectrum of the anhydrous form is similar to that of the freeze-dried form. Hence, this method cannot differentiate between the freeze-dried and the anhydrous forms. The mixture of freeze-dried 1GA/TSX shows broad peaks at almost the same positions as for TSX and the freeze-dried GA. These broad peaks demonstrate the characteristics of the amorphous solid that are possibly contributed from TSX and some part of the GA that are amorphous20. This corresponds to the PXRD analyses and indicates the amorphous property in these mixtures. Nevertheless, the solid-state NMR cannot provide conclusive evidence for a GA-TSX interaction. Thermal analysis The DSC thermograms of the GA forms II, IV, the anhydrous and freeze-dried are shown in Figure 7. Two endothermic peaks for
Figure 5. Powder X-ray diffraction patterns of various GA forms, freezedried TSX and various compositions of freeze-dried GA/TSX.
the GA monohydrate form II and IV were detected. The first one between 125 and 140 C and the second one at 275.7 C. This first endothermic peak was not detected in the anhydrous GA and freeze-dried GA samples. According to the previous report3, two endothermic peaks for form II (a broad peak between 90 and 115 C and a peak at 268.1 C) and for form IV (a broad peak between 100 and 115 C and a peak at 268.5 C) were detected. The reported endothermic peaks for these forms are comparable to those obtained in this study, the differences in the melting points detected are probably due to the use of a different heating rate. Endothermic melting peaks for the anhydrous and freezedried GA are observed at 277.0 and 262.3 C, respectively. Thus, DSC can differentiate between these two forms. Thermal analyses were also used to investigate the interaction between GA and TSX. As shown in Figures 8 and 9, TSX exhibits a broad, endothermic decomposition peak at about 322.2 C (Figure 8). The TGA curve (Figure 9) demonstrates a 10% weight loss of water starting at 58 C of TSX. The second stage starts at 320 C due to the degradation of TSX as previously reported21–23. The GA form II melts with decomposition as indicated by the mass loss (Figure 9). This agrees with a previous report on the thermal decomposition of GA24. The thermogram of 0.2GA/TSX
Freeze-dried GA and freeze-dried GA/TSX
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Figure 6. Solid state GA/TSX.
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C NMR spectra of TSX, GA and freeze-dried
Figure 8. DSC thermograms of freeze-dried samples at the heating rate of 10 C/min.
presented a broad peak at 246 C (Figure 8) that did not comply with the melting point of crystalline GA. This fusion temperature could be attributed to the presence of impurities in the sample, the residual crystalline GA or multiple amorphous forms, as previously described for the appearance of endotherm in X-ray amorphous samples25. These were consistent with weight losses observed for this sample in Figure 9. As shown in Figure 8, a depression of the crystalline GA and TSX melting points was observed for all freeze-dried mixtures with a GA percentage higher than 0.2%. This was attributed to the GA–TSX interactions. A broad, endothermic decomposition of TSX was observed that accompanied with the melting of GA. With increased amounts of GA, to 0.8 and 1%GA, in the GA/TSX mixtures, two endothermic peaks were observed. This may be attributed to the melting of the GA domains that were in close contact with and far-off the TSX as previously described for the drug and polymer blends26. Figure 7. DSC thermograms of various forms of GA at the heating rate of 80 C/min.
Drug release The release profiles of pure GA and GA from the freeze-dried GA/TSX in a simulated gastrointestinal condition are shown in Figure 10. A one-way analysis of variance (ANOVA) with a post
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are statistically higher than that of the freeze-dried 0.6GA/TSX (p50.02). Nevertheless, the releases of these three samples were not statistically different after 45 min (p40.085). The release of GA from the freeze-dried 0.8GA/TSX is different from those of the three samples but not after 90 min (p40.06). Furthermore, the release of GA from the 1GA/TSX is lower than from the other four samples. Nevertheless, it demonstrates the same release as the others after 240 min (p40.05). As previously reported by our group14, at 37 C, GA at higher than 0.69% in 1%TSX can form a gel. In this study, with the increase in the amount of GA, the samples could possibly form a gel in the dissolution medium, reflecting the slow release of GA from these samples. In conclusion, it is of interest that the DRIFTS rather than the FTIR, FT-Raman and 13C CP/MAS NMR can differentiate between various polymorphic forms of GA. The freeze-dried GA has been shown to have a different form compared to other polymorphic forms reported in the literature based on the DRIFTS and the DSC thermograms. According to the PXRD patters, the crystalline forms of GA from the freeze-dried GA/TSX mixtures seem to be the same as in the pure freeze-dried GA except for the freeze-dried form with low amounts of GA, 0.2%GA in 1%TSX which is mostly in an amorphous state. Furthermore, the freezedried GA/TSX samples can provide an optimal sustained release profile for oral administration.
Declaration of interest
Figure 9. TGA of freeze-dried samples at the heating rate of 10 C/min.
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper. This work was supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program through Grant Nos PHD/0259/2549 and PHD/0045/2552 and the Nanotechnology Center (NANOTEC), NSTDA, Ministry of Science and Technology, Thailand, through its program of Center of Excellence Network.
References
Figure 10. Dissolution profiles of various compositions of freeze-dried GA/TSX.
hoc least significant difference (LSD) test was used for comparisons the released profiles of different samples. The release of pure GA is very fast and complete within 15 min. The release of GA from the GA/TSX freeze-dried samples demonstrated a sustained release pattern. As mentioned above, GA in 0.2GA/TSX was mostly in an amorphous form but a crystalline form was detected for GA in the 0.4GA/TSX sample. Generally, a drug in an amorphous state will have a higher dissolution than that in a crystalline form27. The release of GA from the freeze-dried 0.2GA/TSX is slightly higher than that for the freeze-dried 0.4GA/TSX but not statistically different. Both release profiles
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