Clove Oil Emulsified Buccal Patch of Serratiopeptidase for Controlled Release in Toothache

The objective of this work was to formulate and characterize clove oil emulsified buccal patch of serratiopeptidase to offer their combinational controlled release actions in dental pain. The buccal patches were prepared by two methods 1. Adding serratiopeptidase and clove oil in alcoholic solution of hydroxypropyl methylcellulose (HPMC) and Eudragit. 2. Emulsification of clove oil in aqueous serratiopeptidase solution and then dispersed in mixture of polymeric solution of HPMC and Eudragit. Scanning electron microscopy (SEM) of patches showed uniform distribution of w/o emulsion of serratiopeptidase and clove oil; and regular surface of patches. Entrapment efficiencies of serratiopeptidase and clove oil were found to be significant. In vitro and in vivo release studies showed the controlled release of serratiopeptidase and clove oil for 24 h. Fourier transform infrared spectroscopy (FTIR) and Differential scanning calorimetry (DSC) studies confirmed no interaction of serratiopeptidase and clove oil with polymers and excipients. It showed that buccal patch of emulsified clove oil with serratiopeptidase can be used for their controlled delivery in toothache or dental pain. *Corresponding author: Pravin K Shende, SPP-School of Pharmacy and Technology Management, NMIMS University, Mumbai, India, Tel: +91-2242332000; Fax: +91-22-26185422; E-mail: shendepravin94@gmail.com Received December 16, 2015; Accepted April 15, 2016; Published April 22, 2016 Citation: Shende PK, Gaud RS, Bakal R, Yeole Y (2016) Clove Oil Emulsified Buccal Patch of Serratiopeptidase for Controlled Release in Toothache. J Bioequiv Availab 8: 134-139. doi:10.4172/jbb.1000283 Copyright: © 2016 Shende PK, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Ingredient F1 F2 F3 F4 F5 HPMC 500 mg 600 mg 400 mg 450 mg 500 mg Ethanol 8 ml 8 ml 8 ml 8 ml 4 ml Serratiopeptidase 10 mg 10 mg 10 mg 10 mg 10 mg Ethanol 4 ml 4 ml 4 ml 4 ml 4 ml Clove oil 0.06 ml 0.06 ml 0.06 ml 0.06 ml 2 ml Eudragit L100 500 mg 400 mg 600 mg 550 mg 500 mg Tween 80 0.05 ml Water 4 ml Ratio w/w (HPMC:Eudragit L 100) 1:1 3:2 2:3 4.5:5.5 1:1 Table 1: Composition of buccal patch formulation. Citation: Shende PK, Gaud RS, Bakal R, Yeole Y (2016) Clove Oil Emulsified Buccal Patch of Serratiopeptidase for Controlled Release in Toothache. J Bioequiv Availab 8: 134-139. doi:10.4172/jbb.1000283 J Bioequiv Availab ISSN: 0975-0851 JBB, an open access journal Volume 8(3): 134-139 (2016) 135 dispersing aqueous serratiopeptidase solution in clove oil (ratio 1:1 v/v) and Tween 80 mixture using Silverson homogenizer (Remi industries, India) at room temperature. This emulsion was added gradually in alcoholic solution of HPMC under continuous stirring. Then Eudragit L100 was slowly added to form homogenous mixture. This mixture was transferred in a petri dish and kept for drying to obtain buccal patch. The formulation was abbreviated as F5 (Table 1). Characterization of buccal patch Thickness: Thicknesses of the patches were measured using vernier caliper (Unique Enterprises, Mumbai) at different areas on the film and the average was calculated [8]. Patch weight: Three patches of each formulation (F1, F2, F3, F4 and F5) were taken and weighed individually on digital balance and results were analyzed for mean standard deviation. Moisture content: The prepared patch formulations were weighed individually and kept in a desiccator containing calcium chloride at room temperature for 24 h. The patches were weighed again after fixed interval, until they showed constant weights. The percent moisture content was calculated by using following formula [2]. % Moisture content = Initial weight Final weight / Final weight × 100 Folding endurance: Three patches of each formulation (1 × 1 cm2) were cut by using stainless steel cutter. Folding endurance was estimated by frequently folding a small strip of patch at the same position till it breaks. The patch folded at the same place without breaking and will propose the value of folding endurance [9]. Results were analyzed for mean standard deviation. Swelling index: The pre-weighed patch formulations of (1 × 1 cm2) diameter were placed in 20 ml of phosphate buffer pH 6.8 at ambient temperature. After 1 h, the patches were removed and excess of water wiped with tissue paper and weighed. The swelling index can be determined by using the formula [10]. Swelling Index = Final weight Initial weight / Initial weight × 100 Scanning electron microscopy (SEM): The size and shape of plain serratiopeptidase and surface morphology of buccal patch formulations were assessed by SEM (S-4800, Type II Hitachi, Japan) using the gold sputtering technique. The sample was placed in gold palladium coated stubs and photographs were obtained at proper resolution [11]. Entrapment efficiency: Buccal patch formulations (F1, F2, F3, F4 and F5) equivalent to 10 mg of serratiopeptidase and clove oil were dissolved in phosphate buffer pH 6.8. The contents of clove oil and serratiopeptidase were analyzed by UV-Vis spectrophotometer (UV1800, Shimadzu, Japan) at 280 nm and 291 nm, respectively. The encapsulation efficiencies of clove oil and serratiopeptidase were calculated by following formula. % Drug entrapment efficiency = (Initial weight Final weight / Initial weight) × 100 Ex-vivo mucoadhesive force: The mucoadhesive strength of buccal patches was determined on goat buccal mucosa (obtained from local market) by measuring the force of detachment or the force of adhesion [12]. The goat buccal mucosa was fixed on the internal side of a beaker. The patch was wetted with 100 μL of phosphate buffer pH 6.8 and pasted to the rabbit buccal tissue by applying a light force with a fingertip for 1 min. The beaker was filled with 300 mL phosphate buffer pH 6.8 and kept at 37 ± 1°C. Then, the beaker was magnetically stirred at 50 rpm stirring rate to simulate the buccal cavity environment. The time required for detachment of these patches from the buccal mucosa was recorded. The experiment was performed in triplicate and the results were analyzed for mean and SD. In vitro release studies of serratiopeptidase and clove oil: In vitro release studies of patches were carried out by Franz-diffusion cells using dialysis membrane (Himedia, India). 15 ml of phosphate buffer pH 6.8 solutions was used as the receptor medium at 37°C for 50 rpm. The patches were placed in contact with the dialysis membrane. At scheduled intervals, 2 ml of aliquots were withdrawn for the analysis of clove oil and serratiopeptidase using UV-Vis spectrophotometer (UV 1800, Shimadzu, Japan) at 280 nm and 291 nm, respectively. The experiment was repeated for triplicate reading [13]. Aliquots were replaced with fresh buffer solution at the various intervals to maintain sink condition. On the basis of results of encapsulation efficiency, folding endurance, mucoadhesion test and in vitro release studies of buccal patches, the formulation F5 was selected for further studies like in vivo drug release, ATR-FTIR, DSC and XRPD. In vivo release studies of serratiopeptidase and clove oil: Wistar rats were used for determination of in vivo release of clove oil and serratiopeptidase from formulation F5. Animals were housed in a room maintained at 25 ± 1.0oC for 12 h. They were allowed free access to food and water. Formulation F5 was applied in the buccal cavity of size (0.5 cm × 0.5 cm) bilaterally in light ether anesthetized animal. At scheduled time intervals 1, 2, 3, 4 and 24 h, the blood samples were collected in Eppendorf tubes by retero-orbital plexus method. The plasma was separated by centrifugation for 20 min at 3000 rpm and kept in refrigerator for 24 h. The release of serratiopeptidase and clove oil from formulation F5 was determined by UV-Vis spectrophotometer (UV1800, Shimadzu, Japan) at 280 and 291 nm, respectively [14]. Anti-inflammatory activity: Healthy Wistar rats weighing 120-150 g were used for anti-inflammatory activity of the formulation F5 by the carrageenan induced paw edema method. The animals were housed in a group of 6 in a single clean polypropylene cage. They were housed in an environmentally controlled room temperature 25 ± 1.0oC and relative humidity 50-60%. Under 12 h light dark cycles, the animals were fed on standard pelletized laboratory animal diet. The rats were fasted with free access to water for 12 h prior to the test. Paw edema was induced by 1% carrageenan solution in normal saline by injecting 0.1 ml (s.c.) into the right sub plantar region of the hind-paw. The control group received no treatment while formulation F5 was placed in buccal cavity of treatment group. The paw volume up to tibiotarsal articulation was measured at 0 h (V0), 1, 3, 5 and 24 h (VT) after carrageenan injection using a plethysmometer. Differences in paw volume between V0 and VT were taken as a measure of edema [15,16]. Analgesic study: The analgesic activity of the formulation F5 was evaluated by hot-plate technique [16]. Wistar rats weighing between 120-150 g were used for determination of analgesic activity. A solution of serratiopeptidase dose 5 mg/kg was prepared in normal saline water. The patch was stuck in the upper oral mucosa. The animals were placed on electrical heated surface of hot plate maintained at temperature 55°C to 56°C and latency time until either licking or jumping occurs was recorded at 0, 30, 60, 90 and 120 min. ATR-FTIR: ATR-FTIR of plain serratiopeptidase, clove oil and formulation F5 were obtained by FTIR spectrophotometer (Agilent, USA) The wavelength was used in the range of 400-4000 cm-1 [17]. Citation: Shende PK, Gaud RS, Bakal R, Yeole Y (2016) Clove Oil Emulsified Buccal Patch of Serratiopeptidase for Controlled Release in Toothache. J Bioequiv Availab 8: 134-139. doi:10.4172/jbb.1000283 J Bioequiv Availab ISSN:


Introduction
Buccal drug delivery shows an attractive alternative route to oral delivery systems due to its advantages like prevention of GI irritation, minimum systemic toxicity and evasion of first pass metabolism. Since buccal mucosa has rich blood supply and acts as excellent site for absorption, it is well accepted by patient and easy for self-medication [1,2]. Serrapeptase is known as serratiopeptidase and is a proteolytic enzyme isolated from the non-pathogenic Enterobacterium serratia sp. E-15. The serratiopeptidase acts by hydrolyzing the histamine, bradykinin and serotonin and uses for pain and inflammatory conditions such as arthritis and minor muscle trauma. It can absorb through intestines and transported directly in to blood stream [3,4]. Clove oil is an essential oil and is obtained from the dried flowers, buds, leaves, stem of the tree Syzygium aromaticum or Eugenia cariophyllata and Eugenia aromaticum. It has been used as anesthetic, anti-inflammatory and analgesic agents for toothaches [5]. Clove oil acts by denaturing the proteins and reacting with cell membrane phospholipids changing their permeability and inhibiting the Gram-negative, Gram-positive bacteria and yeast. To overcome first pass metabolism, gastrointestinal irritation and systemic toxicity of serratiopeptidase; and burning sensation, nerve and tissue damage of concentrated clove oil, the buccal patch was an interesting delivery system in dental pain [6]. So, the objective of this work was to formulate and characterize clove oil emulsified buccal patch of serratiopeptidase to investigate their combinational controlled release actions like analgesic and anti-inflammatory in dental pain.

Materials
Serratiopeptidase was obtained from Advanced Enzyme Technologies, Thane, India. Eudragit L100 was gifted by Evonik Roehm Pharma Polymer, Mumbai, India. Clove oil, Tween 80, ethanol and HPMC K4M were purchased from S.D. Fine Chemicals, Mumbai, India. All other chemicals used were of analytical grades.

Abstract
The objective of this work was to formulate and characterize clove oil emulsified buccal patch of serratiopeptidase to offer their combinational controlled release actions in dental pain. The buccal patches were prepared by two methods 1. Adding serratiopeptidase and clove oil in alcoholic solution of hydroxypropyl methylcellulose (HPMC) and Eudragit. 2. Emulsification of clove oil in aqueous serratiopeptidase solution and then dispersed in mixture of polymeric solution of HPMC and Eudragit. Scanning electron microscopy (SEM) of patches showed uniform distribution of w/o emulsion of serratiopeptidase and clove oil; and regular surface of patches. Entrapment efficiencies of serratiopeptidase and clove oil were found to be significant. In vitro and in vivo release studies showed the controlled release of serratiopeptidase and clove oil for 24 h. Fourier transform infrared spectroscopy (FTIR) and Differential scanning calorimetry (DSC) studies confirmed no interaction of serratiopeptidase and clove oil with polymers and excipients. It showed that buccal patch of emulsified clove oil with serratiopeptidase can be used for their controlled delivery in toothache or dental pain. dispersing aqueous serratiopeptidase solution in clove oil (ratio 1:1 v/v) and Tween 80 mixture using Silverson homogenizer (Remi industries, India) at room temperature. This emulsion was added gradually in alcoholic solution of HPMC under continuous stirring. Then Eudragit L100 was slowly added to form homogenous mixture. This mixture was transferred in a petri dish and kept for drying to obtain buccal patch. The formulation was abbreviated as F5 ( Table 1).

Characterization of buccal patch
Thickness: Thicknesses of the patches were measured using vernier caliper (Unique Enterprises, Mumbai) at different areas on the film and the average was calculated [8].
Patch weight: Three patches of each formulation (F1, F2, F3, F4 and F5) were taken and weighed individually on digital balance and results were analyzed for mean standard deviation.

Moisture content:
The prepared patch formulations were weighed individually and kept in a desiccator containing calcium chloride at room temperature for 24 h. The patches were weighed again after fixed interval, until they showed constant weights. The percent moisture content was calculated by using following formula [2]. % Moisture content = Initial weight -Final weight / Final weight × 100 Folding endurance: Three patches of each formulation (1 × 1 cm 2 ) were cut by using stainless steel cutter. Folding endurance was estimated by frequently folding a small strip of patch at the same position till it breaks. The patch folded at the same place without breaking and will propose the value of folding endurance [9]. Results were analyzed for mean standard deviation.

Swelling index:
The pre-weighed patch formulations of (1 × 1 cm 2 ) diameter were placed in 20 ml of phosphate buffer pH 6.8 at ambient temperature. After 1 h, the patches were removed and excess of water wiped with tissue paper and weighed. The swelling index can be determined by using the formula [10].
Swelling Index = Final weight -Initial weight / Initial weight × 100 Scanning electron microscopy (SEM): The size and shape of plain serratiopeptidase and surface morphology of buccal patch formulations were assessed by SEM (S-4800, Type II Hitachi, Japan) using the gold sputtering technique. The sample was placed in gold palladium coated stubs and photographs were obtained at proper resolution [11].
Entrapment efficiency: Buccal patch formulations (F1, F2, F3, F4 and F5) equivalent to 10 mg of serratiopeptidase and clove oil were dissolved in phosphate buffer pH 6.8. The contents of clove oil and serratiopeptidase were analyzed by UV-Vis spectrophotometer (UV1800, Shimadzu, Japan) at 280 nm and 291 nm, respectively. The encapsulation efficiencies of clove oil and serratiopeptidase were calculated by following formula. % Drug entrapment efficiency = (Initial weight -Final weight / Initial weight) × 100 Ex-vivo mucoadhesive force: The mucoadhesive strength of buccal patches was determined on goat buccal mucosa (obtained from local market) by measuring the force of detachment or the force of adhesion [12]. The goat buccal mucosa was fixed on the internal side of a beaker. The patch was wetted with 100 μL of phosphate buffer pH 6.8 and pasted to the rabbit buccal tissue by applying a light force with a fingertip for 1 min. The beaker was filled with 300 mL phosphate buffer pH 6.8 and kept at 37 ± 1°C. Then, the beaker was magnetically stirred at 50 rpm stirring rate to simulate the buccal cavity environment. The time required for detachment of these patches from the buccal mucosa was recorded. The experiment was performed in triplicate and the results were analyzed for mean and SD.
In vitro release studies of serratiopeptidase and clove oil: In vitro release studies of patches were carried out by Franz-diffusion cells using dialysis membrane (Himedia, India). 15 ml of phosphate buffer pH 6.8 solutions was used as the receptor medium at 37°C for 50 rpm. The patches were placed in contact with the dialysis membrane. At scheduled intervals, 2 ml of aliquots were withdrawn for the analysis of clove oil and serratiopeptidase using UV-Vis spectrophotometer (UV 1800, Shimadzu, Japan) at 280 nm and 291 nm, respectively. The experiment was repeated for triplicate reading [13]. Aliquots were replaced with fresh buffer solution at the various intervals to maintain sink condition.
On the basis of results of encapsulation efficiency, folding endurance, mucoadhesion test and in vitro release studies of buccal patches, the formulation F5 was selected for further studies like in vivo drug release, ATR-FTIR, DSC and XRPD.
In vivo release studies of serratiopeptidase and clove oil: Wistar rats were used for determination of in vivo release of clove oil and serratiopeptidase from formulation F5. Animals were housed in a room maintained at 25 ± 1.0ºC for 12 h. They were allowed free access to food and water. Formulation F5 was applied in the buccal cavity of size (0.5 cm × 0.5 cm) bilaterally in light ether anesthetized animal. At scheduled time intervals 1, 2, 3, 4 and 24 h, the blood samples were collected in Eppendorf tubes by retero-orbital plexus method. The plasma was separated by centrifugation for 20 min at 3000 rpm and kept in refrigerator for 24 h. The release of serratiopeptidase and clove oil from formulation F5 was determined by UV-Vis spectrophotometer (UV1800, Shimadzu, Japan) at 280 and 291 nm, respectively [14].
Anti-inflammatory activity: Healthy Wistar rats weighing 120-150 g were used for anti-inflammatory activity of the formulation F5 by the carrageenan induced paw edema method. The animals were housed in a group of 6 in a single clean polypropylene cage. They were housed in an environmentally controlled room temperature 25 ± 1.0ºC and relative humidity 50-60%. Under 12 h light dark cycles, the animals were fed on standard pelletized laboratory animal diet. The rats were fasted with free access to water for 12 h prior to the test. Paw edema was induced by 1% carrageenan solution in normal saline by injecting 0.1 ml (s.c.) into the right sub plantar region of the hind-paw. The control group received no treatment while formulation F5 was placed in buccal cavity of treatment group. The paw volume up to tibiotarsal articulation was measured at 0 h (V 0 ), 1, 3, 5 and 24 h (V T ) after carrageenan injection using a plethysmometer. Differences in paw volume between V 0 and V T were taken as a measure of edema [15,16].
Analgesic study: The analgesic activity of the formulation F5 was evaluated by hot-plate technique [16]. Wistar rats weighing between 120-150 g were used for determination of analgesic activity. A solution of serratiopeptidase dose 5 mg/kg was prepared in normal saline water. The patch was stuck in the upper oral mucosa. The animals were placed on electrical heated surface of hot plate maintained at temperature 55°C to 56°C and latency time until either licking or jumping occurs was recorded at 0, 30, 60, 90 and 120 min. which contributed to the successful encapsulation and stabilization of the drugs in the patch.

Mucoadhesive strength
The mucoadhesion strength of formulations was in the range of 3.20 ± 0.12 to 9.80 ± 0.14 g as shown in Figures 3-5. It was observed that the longest adhesion time and the convenient bioadhesion strength reflect the different behavior of Eudragit which, in addition to its role as filmforming polymer, possesses non-neglectable mucoadhesive properties that enhanced mucoadhesion [13]. This study was sustained on the assumption that ionizable polymers exhibit the best mucoadhesive characteristics. Formulation F2 demonstrated low strength which can

DSC:
Thermal analyses of serratiopeptidase and formulation F5 were carried out using DSC (Shimadzu, Japan) with a scanning temperature 10°C/min [18].

Thickness
The thickness of the patches ranges between 0.41 ± 0.004 mm to 0.56 ± 0.005 mm. Each formulation has a uniform thickness. The thickness of patches was in the order of F4>F3>F2>F1 might be due to increase in Eudragit L100 concentration. Formulation F5 showed thickness 0.48 ± 0.006 mm was more due to entrapment of emulsion droplets in the patch.

Patch weight
The patch weight was in the order of F2>F3>F4>F1>F5 as shown in Table 2. Mass difference of the formulated patches was increased in F2 due to high concentration of hydrophilic polymer, HPMC.

Moisture content
Moisture content of the formulation was in the order of F2>F3>F4>F1>F5 due to the presence of hydrophilic nature of HPMC and hydrophobic nature of Eudragit L100. The high moisture content of the formulation F2 was perhaps associated with the major proportion of water retained in HPMC.

Folding endurance
Formulated buccal patches showed good folding endurance in order of F5>F1>F3>F2>F4 and no visible cracks. Formulations showed good film properties as shown in Table 2.

Swelling index
The percentage swelling index of different formulations was in the order of F2>F5>F1>F4>F3. The formulations F2 and F5 showed higher swelling index due to increase in wettability of higher HPMC concentration and the presence of matrix network structure to the movement of water molecules in w/o emulsion, respectively. The effect of Eudragit in swelling of the patches was negligible due to its hydrophobic nature (Table 2).

SEM
SEM studies showed the uniform distribution of serratiopeptidase and clove oil in formulations F1-F4; and their w/o emulsion throughout the patch formulation F5. The particle size of serratiopeptidase and emulsion droplets were 23 µm and 1 µm, respectively as shown in Figures 1 and 2. It was also observed homogenous, clear and regular surface of patches.

Entrapment efficiency
The % entrapment efficiencies of serratiopeptidase in formulated buccal patches were found in the order of F5>F4>F1>F3>F2 while clove oil was in the order of F5>F1>F4>F3>F2. The less values of standard deviation designated that serratiopeptidase and clove oil were homogeneously distributed throughout the formulated patches. This might be due to adhesive forces of drug and polymer was stronger than cohesive forces between drug molecules and polymer structures, endurance, mucoadhesion test and in vitro release studies of buccal patches, the formulation F5 was selected for further studies like in vivo release, ATR-FTIR, DSC and XRPD.

In vivo drug release study
In vivo release study showed that maximum drugs released was found in combinational effect serratiopeptidase and clove oil as compared to formulation F5. It indicates that increase in bioavailability of serratiopeptidase in emulsion formulation F5.

Analgesic activity
The results of the hot plate test are presented in Figures 7-9. All be due to the high swellability of HPMC producing a non-adhesive mucilage layer near the polymer/mucosa interfacial surface and this sentence was supported by the result of the swelling index.

In vitro release studies of serratiopeptidase and clove oil
In vitro release profiles of serratiopeptidase and clove oil from formulated buccal patches were shown in Figures 6 and 7, respectively. Serratiopeptidase and clove oil released from formulation F5, showed highest release 74.32% and 80.65%, respectively. Serratiopeptidase and clove oil patches showed % release was in the order of F5>F2>F1>F4>F3 for 24 h in controlled manner. The formulation F5 ratio (1:1 w/w) of HPMC and Eudragit showed the highest percent release with an initial burst release for 4 h followed by controlled release for 24 h. As an inert nature of polymer, Eudragit, solvent penetration into the patch was the rate limiting factor for the release of the drug. At the start of the study, drug available at and near the surface of the patch dissolved quickly. When the dissolution process progresses, there was a greater resistance to the penetration of the solvent inside of the matrix film, due to the hydrophobicity of the Eudragit and the decreasing length of the solvent front. The drug immediately dissolved by dissolution medium and diffused from the interface between the patch surface and the surrounding media. Later slow down diffusion process led to controlled release of drug from mucoadhesive [14]. This outcome suggested that delivery of serratiopeptidase and clove oil as a controlled release formulation was possible with formulated patches with interpolymer complexes of Eudragit and HPMC wherein emulsion droplets were uniformly distributed.
On the basis of results of encapsulation efficiency, folding    drugs along with the formulation significantly (P<0.05) delayed response at selected time points after treatment. The formulation showed maximum effects when compared with serratiopeptidase and clove oil. This might be due to controlled release of serratiopeptidase and clove oil in animal treated with F5.

Anti-inflammatory effect
The increase in volume displaced by paw edema in control rats was observed. The serratiopeptidase and F5 showed significant reduction in volume displaced by paw edema in rats compared to control group ( Figure 10).

FTIR
FTIR spectra of serratiopeptidase and clove oil and formulation F5 were shown in Figure 11. The characteristic peaks of serratiopeptidase were shown due to presence of aromatic NH 2 (3325 cm -1 ), aliphatic NH (1032 cm -1 ), aliphatic OH (1017 cm -1 ) and aromatic C=C (1649 cm -1 ). Clove oil spectrum exhibited peaks in the region of 3300-3600 cm -1 . The presence of -OH stretching and absorption peaks in the region of 1600-1680 cm -1 assigned to alkene CC and aromatic ring present. The spectrum of formulation F5 showed absorption bands on serratiopeptidase and clove oil, there is no change in the absorption bands due to emulsification. The FTIR spectra concluded that there was no chemical incompatibility between drugs and excipients used in the buccal patch formulation.

DSC
DSC thermograms of serratiopeptidase showed endothermic peak    at 240ºC matching to the melting point of the serratiopeptidase as shown in (Figure 12) while clove oil exhibited a broad exothermic peak at 162ºC and endothermic peak at 282ºC due to the thermal oxidative temperature and boiling point of eugenol, respectively. This indicated that the characteristic peaks of serratiopeptidase and clove oil appeared in the formulation F5 showing that there was no possible interaction between the drugs and the exicipients in the mucoadhesive buccal patch formulation.

XRPD
The comparison of plain serratiopeptidase and emulsified buccal patch formulation F5 showed a drastic change as shown in Figure 13. Formulation F5 showed reserve of amorphous nature of serratiopeptidase which was confirmed by this study. XRPD pattern of serratiopeptidase showed peaks at 11.2, 19.5 and 37.5 but this pattern was not observed in formulation F5.

Conclusion
The serratiopeptidase and clove oil buccal patch was prepared by emulsification method using HPMC and Eudragit L100 used in different ratio of 1:1, 3:2, 2:3, 4.5:5.5 and 1:1 exhibited significant results of bioadhesive, mucoadhesive properties, encapsulation efficiency and in vitro and in vivo release studies. The clove oil emulsified buccal patch of serratiopeptidase offered their combinational controlled release actions and is good alternative to avoid hepatic first pass metabolism.