Piperine as Placebo: Stability of Gelatin Capsules without Cross-linker

Subscriber access provided by University of Massachusetts Amherst Libraries

Article

Piperine as Placebo: Stability of Gelatin Capsules without Cross-linker Utkarsh Bhutani, Anshaj Ronghe, and Saptarshi Majumdar ACS Appl. Bio Mater., Just Accepted Manuscript • Publication Date (Web): 26 Sep 2018 Downloaded from http://pubs.acs.org on September 26, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Bio Materials

Piperine as Placebo: Stability of Gelatin Capsules without Cross-linker Utkarsh Bhutani, Anshaj Ronghe, Saptarshi Majumdar* Email: [email protected] Department of Chemical Engineering, Indian Institute of Technology Hyderabad, Sangareddy, 502285, Telangana, India

Abstract Gelatin has been the biomaterial of choice for decades now. Its low cost, renewable, nontoxic and biodegradable properties make it one of the most desirable materials for controlled release applications. However, the usage of gelatin is limited by its poor mechanical/thermal stability and high water solubility. Chemical cross-linkers and hydrophobic modifications of gelatin have solved this problem but they lead to the problem of toxicity and/ or high processing cost. This research attempts to employ a nontoxic hydrophobic drug molecule to curb early degradation of gelatin in an aqueous environment. We report the design of non-cross-linked gelatin capsules with high dissolution resistance in an aqueous medium. Piperine, a hydrophobic drug (Solubility: 40mg/L in water) was coated on the gelatin capsules to enhance its stability in an aqueous environment. The hydrophobic piperine molecules repelled the water molecules to intensify its dissolution resistance. This stabilization was used to control the release of naproxen sodium, encapsulated inside the gelatin matrix. Piperine, in this case, acts as a placebo i.e it has zero therapeutic effect but its presence was necessary to control the early degradation of gelatin matrix. The deposition of piperine was done using the solvent evaporation method where ethanol was used as the solvent. The wettability studies revealed the hydrophobic nature of surface after the deposition of piperine while SEM analysis showed the presence of long cylindrical (fiber-like) structures over the gelatin surface. Further investigation (FTIR/ATR and molecular dynamics) revealed that the long fiber structures were due to the crystallization of piperine over the surface of gelatin. This crystallization was triggered by the intermolecular association (hydrogen bond) of ethanol and piperine. These observations enabled us to optimize the piperine loading protocol over the gelatin capsules that helped in achieving a zero order naproxen release for 32 hours. Keywords: Gelatin; Piperine; Placebo; Naproxen sodium; zero order release Introduction Research on Polymers has been endless so far, and biopolymers being a subset is no less as an everdeveloping front. Biopolymer research has advanced at a brisk pace with a plethora of applications, of which controlled release remains the eminent one. There biodegradable, nontoxic, biocompatible and renewable natures make them the most beneficial class of polymers in sustained release applications13 . Gelatin is one such biopolymer that has all the characteristics of the biopolymer family and has been widely accepted and used in applications like drug delivery, wound dressings etc4-9. The usage of gelatin is often limited by its poor mechanical and thermal stability followed by its high hydrosolubility. In order to overcome these pitfalls, gelatin is often cross-linked with glutaraldehyde, carbodiimides, formaldehyde etc. These crosslinkers form covalent bonds with gelatin residues and improve its mechanical properties and increase its stability in aqueous medium10-12. Though crosslinkers optimize the material characteristics but are associated with the short-term release of toxic byproducts13-16. Glutaraldehyde (GTA) is reported to form Schiff's base with an amino group of lysine. It is also reported to autopolymerize via aldol condensation reaction13. Studies have revealed that GTA byproducts are cytotoxic (killed the surrounding fibroblast cells) and were reported to 1 ACS Paragon Plus Environment

ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 20

release up to six months after a GTA cross-linked tendon implant13. Genipin (naturally extracted cross-linker) has been reported to have a lower acute toxicity than glutaraldehyde but not deemed as a fully safe 17-21. It is also an expensive chemical when compared to glutaraldehyde and others in the same group. To avoid these toxic cross-linkers, reagent-free methods are practiced, namely electron beam/gamma-ray and UV radiation methods22. The downside of these methods is the low penetration depth of UV rays through thick layers of gelatin, while gamma radiation requires long exposure times (2-7 KGy) to achieve high dose. To further improve the limiting properties of gelatin it is often associated plasticizing agents like glycerol23 D-sorbitol23 PEG24-25 or it is reinforced with other biopolymers like chitosan26, silk fibroin27, sodium alginate (SA)28-29, or even with inorganic materials like silica gel30-31 and clay minerals32. Other prominent covalent associations include the formation of conjugates with dendrimers. Star-shaped polyamidoamine dendrimer-gelatin conjugates have been reported33. Dendrimers have been investigated and found to have toxic behaviors although functionalized PAMAM dendrimers are proven to have lower cytotoxicity34. Gelatin- cyclodextrin and gelatin-cyclodextrin-GTA35 blends have been studied and observed to improve the physical properties of gelatin. Cyclodextrins are again associated with nephro toxicities36 and the complexation of cyclodextrins with GTA is expected to be even more harmful. Recently, gelatin methacrylate (modified gelatin) has been used to form hydrogels and implemented in tissue engineering applications37. The degree of methacryloylation can result in modification of gelatin mechanical strength and gelling behavior. However, the quantification of groups involved in the methacryloylation process is still an unresolved issue. Gelatin methacrylate is expensive, which also limits its usage. Thus, numerous methodologies have been adopted to improve the physical strength and hydrosolubility of gelatin. Majority of them are associated with the covalent modification of gelatin or blending it with other polymers. The toxicity associated with these methods and expensive synthesis procedures are issues that are still unresolved and need to be addressed. Thus, cross-linker free polymeric biomaterials remain as one of the main challenges, which should be achieved without tampering with the basic molecular nature and structure of FDA approved biopolymers38. In the quest to resolve these issues, we had earlier worked on non-crosslinked sodium alginate-gelatin hydrogels and explored the role SA viscosity in the stabilization of hydrogel matrix against degradation39. Two natural materials; soya nuggets40 and cardamom husks41 were also introduced and transformed into drug carriers. These were very novel concepts introduced in the controlled release arena. Both (soya and cardamom husks), were devoid of chemical cross-linkers and were capable of encapsulating and releasing drugs (hydrophobic & hydrophilic) at a controlled rate.

Schematic 1: (a) Preparation of gelatin capsule (b) Solvent Evaporation method (ethanol evaporation) (c) Piperine coated gelatin capsule

2 ACS Paragon Plus Environment

Page 3 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The present research focuses on the design of gelatin capsules that are cross-linker free and devoid of undue chemical modifications, but still capable of resisting an early dissolution in an aqueous environment. To achieve this goal, the surface of gelatin was coated with a hydrophobic molecule i.e. piperine, which repelled the water molecules from its surface (Schematic 1). The idea of using piperine was an outcome of our previous research41. Piperine was observed to stabilize the SA and gelatin matrix inside the cardamom husk. This gave us an important clue that its hydrophobicity could be used in stabilizing biopolymers like gelatin against high hydrosolubility. Piperine is a hydrophobic drug (an alkaloid) extracted from black pepper. Piperine enhances the bioavailability of anticancerous drugs like curcumin42 but as an individual molecule, it processes poor bioavailability and minimal therapeutic benefits. Piperine enhanced the surface hydrophobicity of gelatin capsules which enabled us to control the release of naproxen sodium (active component) from the gelatin matrix. The surface of piperine was further stabilized using a coating of sodium alginate. SA solves two purposes, firstly it prevents the detachment of any loose piperine from capsule’s surface and also protects the gelatin capsule at pH 1.2 as gelatin is susceptible to degradation at acidic pH (pH 1.2). Piperine here can be termed as a placebo inside the gelatin capsule. It closely mimics the role of a cross-linker in absence of real crosslinker that introduces covalent interactions. The study also focusses on the role of hydrogen bonds in the crystallization and deposition of piperine over the gelatin surface. MD (Molecular dynamics) studies add an additional dimension to this research as it could complement our experimental findings in order to establish a more uniform protection of gelatin capsule. Materials and Methods Materials Gelatin Type A 175 Bloom powder was used for the preparation of gelatin capsules, while high viscosity sodium alginate (SA, 1000–1500 cps, 1% in water) was used to coat the gelatin capsules. Naproxen sodium and Piperine were used as the model drugs. Phosphate buffer saline (PBS, pH 7.4) and 0.1 N HCl (pH 1.2) were prepared and used for drug release studies. Gelatin, SA, naproxen sodium, piperine and the chemicals used in the preparation of PBS were purchased from Alfa Aesar ( Thermo Fischer Scientific). Absolute ethanol and acetone were used as organic solvents for drug loading and were purchased from Hychem laboratories, Hyderabad, India. Methods Preparation of Gelatin capsules Gelatin Type A (650 mg) was mixed with naproxen sodium (30 mg) and the powdered mixture was poured into a capsule mold. 1 ml of deionized water was poured over the mixture and was allowed to form a gel. The mold was stored at 20oC for 24 h. At a temperature below 30oC gelatin transforms from a random coil to a triple helix43 by forming physical cross-links. Once the gelatin networks were stabilized the capsules were coated with piperine. Piperine solutions (10 & 5mg/ml) were prepared in ethanol. The gelatin/naproxen capsules were dipped in 5ml of piperine solutions of desired concentrations at 20oC. Once the ethanol evaporates, it forms a coating of piperine over the gelatin capsules (solvent evaporation method). The piperine coated capsules were further dried for 12 h before they were coated with high viscosity SA. High viscosity SA solutions (2% w/v) solution were prepared at 80oC. The piperine coated capsules were then dipped in 2% SA solutions for 10 min and then dried again at 20oC for 24 h. The low-temperature drying was preferred to prevent the degradation of gelatin capsules by freshly coated SA, as SA is hydrophilic in nature and may contain water molecules. Contact Angle Measurements



Page 4 of 20

To measure the hydrophobicity induced by piperine on the gelatin surface, gelatin and naproxen sodium containing gelatin films were prepared. 6.5 % w/v gelatin solutions were prepared with naproxen sodium (6 mg/ml). 5 ml of this solution (30 mg naproxen sodium) was poured into Petri dish (60 mm) and allowed to form stable films at 20oC. 5 ml of piperine solutions with desired concentrations were poured over the films and ethanol was allowed to dry at 20oC for 24 h. The wettability of gelatin films (with naproxen) coated with hydrophobic drug piperine was studied using the sessile drop method (Rame-Hart, USA; Model: 290-F4). Water droplets (3 µl) and ~2 mm in diameter were used for contact angle measurements. The films were cut uniformly and placed on a glass slide before the measurement. The images were processed using ImageJ software. Loading Efficiency: Piperine The gelatin capsules with the deposited piperine (w/o SA) were immersed in 20 ml ethanol solution. The solubility of piperine in ethanol is around 60mg/ml. The ethanol solution with the capsule was stirred for 6 hours to further ensure the complete solubility of piperine. The solutions were then analyzed using UV-vis spectroscopy (Lambda 35, Perkin Elmer) at 342 nm (λmax for piperine). The loading efficiency was calculated by the equation (1) given below: % =

ℎ 100

Scanning Electron Microscopy (SEM) The deposition of piperine over gelatin capsules was confirmed using the SEM analysis. The piperine deposited (after solvent evaporation) gelatin capsules were put through the SEM (Phenom world Pro X) analysis. The samples were analyzed at 15 kV acceleration voltage using a charge reduction sample holder. Fourier Transform Infrared Spectroscopy/ Attenuated Total Reflectance (FTIR/ATR) The chemical interactions within the gelatin capsules and the interactions of the hydrophobic drug piperine with the solvent were confirmed using the FTIR/ATR (Bruker Tensor 37, MIRacle Single Reflection Horizontal ATR accessory) analysis. The prepared gelatin capsules were cut and were used during the FTIR-ATR analysis. The deposited piperine was removed from the surface of the gelatin capsule and analyzed separately. To further confirm the interactions of piperine and ethanol (99% and 97% (diluted with water)), the deposited piperine was also analyzed using the Kbr pellet. 250 mg of Kbr and 5mg of deposited piperine were mixed and allowed to form a pellet, which was later subjected to FTIR analysis. The pure SA, gelatin, naproxen and piperine powders were also examined. 256 scans were done with a resolution of 4 cm-1. The spectra were recorded in the range of 400-4000 cm-1. Simulation Details GROMACS version 5.0 was used for all atomistic simulations. The GROMOS43a1 force field, which is most suitable for proteins, carbohydrates, acids and several other biomolecules in general, is employed. This force field provides a great reliability in reproducing the solvation free energies as compared to other force fields. Initial configuration consisted of 10 piperine molecules and 100 ethanol molecules randomly arranged in a PBC (Periodic Boundry Conditions) box. Two copies of this system were generated. One system was equilibrated at a temperature of 293K (20oC) and the other system was equilibrated at a temperature of 333K (60oC). NVT equilibration was used for total time of 40ns with the time step of 2 fs for constant volume equilibration. The v-scale thermostat was used in equilibrating the system. Lincs algorithm44 was used to constraint the bonds and outputs were saved every 100ps. Cut-offs of 1.0 nm were used for both van der Wall and Columbic interactions. Finally, a production MD (molecular dynamics) run of 40 ns with a time step of 1fs was given to both 4 ACS Paragon Plus Environment

Page 5 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the systems. The outputs were saved after every 100ps. Configurations of both the systems were noted at 40ns. The number of hydrogen bonds was calculated over entire production molecular dynamics run of 40ns. A similar protocol was followed for a system where ethanol was replaced by acetone. The analysis for acetone was performed at 293K. Stability and Controlled Release Experiments (In-vitro Release Studies) The in-vitro drug release studies were carried out in PBS (pH 7.4) and 0.1N HCl (pH 1.2)45. These solutions mimic the environment of the human stomach (pH 1.2) and intestine (pH 7.4). 100 ml of PBS and 0.1N HCl was used for the study. The prepared capsules were transferred to these solutions at 37oC and stirred at 100 rpm for 24- 48 h. 3 ml aliquots were collected at regular intervals for the analysis. An equivalent amount of PBS and 0.1N HCl was added back to maintain the sink conditions. The drug release was quantified using UV visible spectroscopy (Perkin Elmer Lambda 35). The absorbance values were measured at 272 nm (λmax for naproxen). The experiments were done in triplicate and the standard deviations were calculated. The drug release was also analyzed using a carefully defined Release factor (RF). The release factor was calculated using the equation (2) given below: ! = ! !" ! !"

#

$

The release factor was defined keeping in mind the characteristic features of a controlled release i.e. the zero order kinetics. The release fraction (RFN) and the time fraction (TFN) correspond to the amount of drug released during the time frame up to which the zero order kinetics was followed i.e. R2 ~0.99. The corresponding expressions for RFN and TFN are given below: !" =

!" =

% & ' ) % ℎ (

ℎ *ℎ ℎ ℎ & + * , - % ℎ

The RFN (equation. 3) and TFN (equation. 4) values were normalized with respect to the best case in terms of release amount and time up to which the zero order kinetics was recorded. Considering this normalization the RF was termed as NRF i.e the normalized release factor. R2 value assures the smoothness of the release profile, which is also an important factor to be controlled. Statistical Analysis The paired t-test was performed to analyze the difference between two treatment means. Treatment here refers to the experiments that were performed on non-coated and piperine & sodium alginate coated (separate & together) gelatin capsules. The null hypothesis was stated as ‘the means of the measurement variable are equal for the two treatments i.e. the non-coated and coated gelatin capsules have the same effect. Results were identified as statistically significant at p < 0.05. The experiments were carried out in triplicates and represented as a mean ± standard deviation to ensure the reproducibility Results and Discussion Contact Angle Measurements The central theme of this research is to restrict the degradation of gelatin capsules in an aqueous medium in absence of chemical cross-linkers. The methodology adopted to achieve this target was to 5 ACS Paragon Plus Environment


Page 6 of 20

coat the gelatin capsule with the hydrophobic drug piperine such that it repels the water molecules from its surface and enhances its stability against dissolution. To test the hydrophobicity induced on the gelatin surface, contact angle measurements were performed. Gelatin films containing naproxen sodium were coated with piperine (20, 10, 5, & 2.5mg/ml) and were employed for wettability analysis. To our surprise, the 20mg/ml coated films showed the poorest hydrophobicity (79.03o) with contact angles lower than the gelatin (Fig. 1a) and gelatin/naproxen (Fig. 1b) containing films (Fig. 1c). The hydrophobic nature was expected to increase with piperine concentration, however, an opposite trend was observed. The contact angle increased from 90.050 (Fig. 1d) (10 mg/ml) to 96.210 (Fig. 1e) (5mg/ml) and 103.710 (Fig. 1f) in 2.5 mg/ ml piperine loaded samples. It was anticipated that lower concentration of piperine leads to the formation of a uniform mesh over the film (Fig. 2a), leading to an enhanced hydrophobicity. The formation of uniform mesh (fibers) over the surface of gelatin capsules might be a result of piperine crystallization. The crystallization of drug molecules on different substrates has been reported previously46. A lower concentration of piperine may restrict the rate of crystallization leading to thin fibers and increased hydrophobicity. To further investigate the crystallization of piperine and understand the wettability studies, SEM and FTIR-ATR studies were performed. These studies were important to understand the chemical interactions involved and the morphology of piperine deposited over gelatin capsules.

Fig. 1: Contact angle measurements (a) Gelatin Film (b) Gelatin/Naproxen Film (c) Gelatin/Piperine (20mg/ml) (d) Gelatin/Piperine (10 mg/ml) (e) Gelatin/Piperine (5 mg/ml) (f) Gelatin/Piperine (2.5 mg/ml) Loading Efficiency: Piperine The amount of piperine actually deposited on the gelatin capsules decreased with a decrease in piperine concentration in the loading solution. The piperine amount decreased from 21.42 mg (20mg/ml) to 18.79 mg (10mg/ml), which further reduced to 3.15 mg (2.5 mg/ml) (Table.1). The LE (%) was found the maximum for the case of 10 mg/ml i.e. 37.49% (Table. 1). 10 mg/ml piperine 6 ACS Paragon Plus Environment

Page 7 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


loaded samples were further tested for drug release. To further understand the deposition, these samples were put through the SEM analysis. Table 1. LE (%): Piperine Piperine (mg/ml) 20 10 5 2.5

Amount Given: Piperine (mg) 100 50 25 12.5

Amount Loaded (mg) 21.42±0.52 18.79±2.09 7.13±0.72 3.15±0.57

LE (%) 21.42±0.552 37.49±4.18 28.52±2.86 25.22±4.55

Scanning Electron Microscopy (SEM) The layering of piperine over the gelatin capsule was done to prevent its early dissolution in an aqueous medium. Piperine, hydrophobic in nature was expected to repel the water molecules and enhance the stability of gelatin in absence of chemical cross-linkers. However, the SEM analysis revealed that piperine formed a fibrous structure instead of a continues film. The fibers could be seen with the naked eye as well (Fig. 2a).

Fig. 2: Digital Images (a) Piperine coated over gelatin using ethanol at 200C (b) Piperine coated over gelatin using ethanol at 600C It was observed that the fiber mesh on the surface of gelatin became denser with the decrease in the concentration of piperine (Fig. 3b, c, d & e). These results were in agreement with the wettability studies (Fig. 1e). The dense fibers led to the closure of open surfaces (Fig. 1d, e, f) that elevated the hydrophobicity of the film. The striking feature here was the tendency of increase in surface to volume ratio of piperine molecules over a hydrophilic surface (gelatin) i.e. the formation of fibers (long cylindrical structures). To explain this phenomenon, the chemical structure of piperine and ethanol were studied and it was found that piperine had 3 hydrogen bond acceptor sites (0 donor sites) while in ethanol the -OH group acts both as an acceptor and a donor. Piperine gains initial bond stability by forming hydrogen bonds with ethanol, and it was suspected that hydrogen bonding may assist the crystallization of piperine, leading to the formation of long cylindrical structures. The hydrogen bonding between piperine and ethanol influenced the rate of crystallization and morphology. The concentration of piperine also plays an important role in the crystallization process. A lower concentration of piperine is expected to restrict the crystallization, which resulted in thin fibers of piperine (Fig. 3c, d) while a high concentration of piperine fastens the crystallization process, leading to thick fibers (Fig. 3b). The micro-nano structures (fibers), as a result of piperine crystallization, were responsible for the surface hydrophobicity observed during contact angle measurements.



Page 8 of 20

To further confirm the role of hydrogen bonds in the formation of fibrous structures, the gelatin films were subjected to piperine loading at 60oC instead of 20oC. The higher temperature was expected to disrupt the hydrogen bonding between piperine and ethanol. The results were in accordance with our expectation. Fibrous structures were not observed in this case and a film kind of deposition was observed with few aberrations on the surface (Fig. 2b, 3f).

Fig. 3: SEM (a) Gelatin/Naproxen Capsule Surface (b) Gelatin/Piperine (20mg/ml) (c) Gelatin/Piperine (10 mg/ml) (d) Gelatin/Piperine (5 mg/ml) (e) Gelatin/Piperine (2.5 mg/ml) (f) Gelatin/Piperine (10mg/ml/60oC) (g) Gelatin/Piperine (10mg/ml)/Acetone (h) Gelatin/Piperine (10mg/ml)/Benzene To reconfirm our hypothesis, ethanol was replaced with acetone and benzene. Unlike ethanol, acetone and benzene are devoid of hydrogen bond donor sites and were presumed to show no hydrogen bond formation with piperine. The piperine deposition using these solvents was done at 20oC and the morphology again revealed the absence of fibrous structure (Fused fiber morphology) (Fig. 3g, h). The outcomes of the SEM analysis enabled us to anticipate the role of hydrogen bonding in the transition of piperine morphology from a fibrous (crystallized structure) to a non-fibrous (film like) structure over the gelatin capsules. Our prediction regarding the role of hydrogen bonding in this transition was confirmed by FTIR/ATR and MD simulation studies. Fourier Transform Infrared Spectroscopy/ Attenuated Total Reflectance (FTIR/ATR) SEM results revealed the transition in the morphology of piperine from fibrous to a nonfibrous one. The formation of long cylindrical structures was the result of piperine crystallization over the surface of gelatin. The conjecture for this transition and crystallization was associated with the intermolecular hydrogen bonding between piperine and ethanol molecules. The above argument was further supported by FTIR/ATR studies. The ATR analysis of piperine deposited on gelatin revealed the presence of a peak at 3465 cm-1 (Fig. 4a). This peak was absent in the pure piperine (Fig. 4a). Comparing the spectra with pure ethanol, a peak shift is observed from 3325 cm-1 in pure ethanol to 3465 cm-1 in deposited piperine. The presence of this peak (3465 cm-1) clearly indicates the formation of hydrogen bond formation between the -OH group of ethanol (donor) and the hydrogen bond acceptor sites of piperine (Fig. 4a & Schematic 2). However, this peak was drastically reduced in the case where piperine was loaded using ethanol at 60oC (Fig. 4b). High temperature shattered the hydrogen bonding between piperine and ethanol and was confirmed by the ATR spectra. The absence of intermolecular hydrogen bonding was also encountered in acetone and benzene samples (Fig. 4b). 8 ACS Paragon Plus Environment

Page 9 of 20

Benzene/Piperine Acetone/Piperine Ethanol/Piperine 60 Degree Celsius Ethanol/Piperine 20 Degree Celsius

Pure Piperine Ethanol Ethanol/Piperine 20 Degree Celsius

(b)

(a) Aromatic -CH No Peak around 3500 cm-1

Peak absent

3325 -OH

CH2/CH3

Peak Shift

3465

-1

% (T)

around 3500 cm

% (T)

Region Similar to

H2 Bond Interaction

pure Piperiene

(Piperine and Ethanol)

4000

3500

3000

2000 Wavenumber(cm-1)

1500

1000

4000

3500

3000

2000

1500

1000

Wavenumber(cm-1)

(c) 1.0 0.8

% (T)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.6 0.4 0.2

Reduction in Intensity

99% Ethanol/Piperine 97% Ethanol/Piperine

0.0 4500

4000

3500

3000

2500

2000

-1

1500

1000

500

0

Wavenumber(cm )

Fig.4: FTIR/ATR: (a) IR spectra for Ethanol, piperine, and piperine deposited using ethanol (200C) (b) IR spectra for piperine deposited using acetone, benzene and piperine deposited using ethanol at 600C (c) Piperine deposited over the gelatin capsule at 20oC using 99.9 % and 97% Ethanol To further confirm the intermolecular association of ethanol and piperine, the concentration of ethanol was changed from 99.9% to 97% ethanol (diluted with water), keeping the piperine concentration as constant. The deposited piperine (10mg/ml) was then removed from the capsule and subjected to FTIR (Kbr pellet, 256 scans). No peak shift was observed but a reduction in the intensity of the peak was observed for piperine deposited using 97% Ethanol (Fig. 4c) This reduction in the intensity also proves the intermolecular interaction between piperine and ethanol. Intermolecular hydrogen bonding is affected by concentration and temperature change. Intermolecular hydrogen bonding should be altered by dilution and temperature change. Dilution reduces the possibility of an intermolecular association. At lower concentrations, the possibility of intermolecular interactions become less and the intensity of the corresponding absorption band also becomes less. Since, the evaporation of ethanol initiates the deposition of piperine on the gelatin surface, the ethanol-ethanol interactions are minimal due to its vaporization. The hydrogen bonded ethanol-piperine interactions were clearly confirmed by the presence of a new peak at 3465 cm-1 (Fig. 4a). The confirmation of hydrogen bonding substantiates our SEM analysis. The transition of piperine from fibrous structures (Cylindrical) to nonfibrous structures is due to the presence and absence of hydrogen bonding between piperine-ethanol molecules. The FTIR-ATR spectra for pure gelatin39, SA39 and naproxen47 were also studied (Fig. 5). The hydrogen bond interactions between gelatin and water lead to a slight shift in the amide region of gelatin capsule (3288 cm-1) (Fig. 5). To confirm the interaction of naproxen and gelatin, ATR analysis 9 ACS Paragon Plus Environment


was performed for a cross-section of the capsule (Fig. 5). There was a shift in the amide peak of gelatin (3288 cm-1) after the incorporation of naproxen into the matrix (3244 cm-1). This peak shift is due to the hydrogen bond interactions between the amide groups of gelatin and the carboxyl groups of naproxen. However, the individual peaks of naproxen remained intact and no new peaks were observed during the ATR analysis (Fig. 5). This further confirms the chemical stability of naproxen inside the gelatin matrix. The DSC analysis has been previously studied for gelatin and gelatinnaproxen dosage forms47 that has clearly shown the absence of any probable chemical interaction between naproxen and gelatin. The SA coated capsules show spectra similar to the pure SA (Fig.5).

Schematic 2: Hydrogen bonding interactions between piperine and ethanol

Gelatin (G) 3280

Gelatin-Water H2 Bond Interactions

G-Capsule 3288

Naproxen(N)

% (T)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 20

3344 Peak shift after Naproxen inclusion in capsule

Naproxen and Gelatin Peaks

G/N Capsule 3244

SA SA Coating confirmed by ATR-FTIR

SA-G-N Capsule

1000

1500

2500 3000 Wavenumber (cm-1)

3500

4000

Fig. 5 FTIR-ATR: Pure Gelatin; Gelatin capsule; Pure Naproxen; Gelatin/Naproxen Capsule; Pure Sodium alginate; Sodium alginate coated capsule The ATR analysis asserted the chemical interactions between piperine and ethanol molecules. However, a much clear understanding regarding the formation of cylindrical structures i.e. the increase in surface to volume ratio was developed after the molecular dynamics studies. Molecular Modelling


Page 11 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The formation of fibrous structure motivated us to further investigate the molecular arrangement of piperine in different environments (ethanol 20 & 60 oC and Acetone) through molecular dynamics simulations. The average number of the hydrogen bond between piperine-ethanol and ethanol-ethanol decreased with the increase in temperature (Table. 2), exactly complimenting the results of ATR (Fig. 4a) and as expected no hydrogen bonds were observed between piperine-acetone (Fig. 4b). The noticeable observation here was the arrangement of piperine molecules in each case. As stated previously, piperine is a hydrogen bond acceptor and it forms hydrogen bonds in presence of ethanol at 20oC. The configurations at 40 ns (Fig. 6a, b) for the piperine-ethanol system at 20oC and 60oC gives an indication that piperine has a tendency to come together (hydrophobic nature) and at 20oC it stabilizes itself by forming hydrogen bonds at the surface with ethanol (Fig. 6a). Table 2: Average number of Hydrogen bond between a given pair calculated using MD simulations Interaction Pair Piperine-Piperine Piperine-Ethanol Ethanol-Ethanol Piperine-Acetone

Average No of H2 Bond Average No of H2 Bond (20oC) (60oC) 0 0 1.227±0.402 0.764±0.33 2.853±0.656 1.47±.0.129 0 -

Ethanol molecules can be seen accumulating around the piperine molecules at 20oC while they remain scattered at 60oC (Fig. 6b). This large surface stabilization induced at 20oC leads to the crystallization of piperine and formation of long fibrous (higher surface to volume ratio) structures. The absence of hydrogen bond induced surface stabilization in acetone (Fig. 6c) leads to the formation of non-fibrous structures as seen in SEM (Fig. 3g).

Fig.6: MD Simulations (a) Snapshots of the MD run of the piperine (Red) in ethanol (Green) system at 20oC and T = 40 ns (b) Snapshots of the MD run of the piperine (Red) in ethanol (Green) system at 60oC and T = 40 ns (c) Snapshots of the MD run of the piperine (Red) in Acetone (Green) system at 20oC and T = 40 ns The findings so far gave us three aspects that control the deposition of pieprine over the gelatin capsules: (i) Lower concentration of piperine forms dense fibers and induces higher hydrophobicity (ii) High-temperature loading with ethanol (60oC) disrupts the fiber morphology and a film like deposition of piperine was observed (iii) Replacing ethanol with acetone also damages the fiber forming characteristic of piperine. Thus, ethanol at elevated temperature and acetone were used for further designing of gelatin capsules. There are other solvents like benzene that lack hydrogen bond donor capability, but since the research targets application in the area of drug release, FDA approved solvents were chosen (ethanol and acetone). Stability and Controlled Release Experiments (In-vitro Release Studies) 11 ACS Paragon Plus Environment


The drug release experiments were strategically designed to identify the role of piperine in maximizing the stability of gelatin capsules in an aqueous medium. The hydrophobic nature of piperine enhanced the dissolution times of gelatin capsules in PBS (pH =7.4), that resulted in a controlled release of naproxen sodium (Zero order release with the total initial loading of 30 mg in each case). R2 = 0.99

100 110

(a)

R2 = 0.98 R2 = 0.98

80

80 70 60 50 40 30

G/N (E1) G/N/P (E4) G/N/P (10mg/ml)/SA (E6) G/N/SA (E2)

20 10

Cumulative Release(%)

90

Cumulative Release (%)

(b)

90

100

0

100

500

1000

2000

2500

3000

Time(min)

(c)

90

1500

2

R = 0.98

70 60 50 40 30

G/N (E1) G/N/P (E4) G/N/P (10mg/ml)/SA (E6) G/N/SA (E2)

20 10

0 3500

0 0

110 100

80

90

70

80



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 20

60 50 40 30 20

G/N/P / T=60 Degree (E8) G/N/P / T=60 Degree/SA (E9) G/N/P/5mg/ml (Acetone)/SA (E10)

10 0 0

500

1000

1500

2000

2500

3000

200

400

600

800

1000

1200

1400

Time(min)

(d) R2 = 0.99

R2 = 0.99

R2 = 0.98

70 60 50 40 30 20

G/N/P / T=60 Degree (E8) G/N/P / T=60 Degree/SA (E9) G/N/P/5mg/ml (Acetone)/SA (E10)

10 0 3500 0

500

1000

1500

2000

Time(min)

Time (min)

Fig.7: Controlled Release (%) Study (p

Piperine as Placebo: Stability of Gelatin Capsules without Cross-linker

Recommend Documents