Bromelain Loading and Release from a Hydrogel Formulated Using Alginate and Arabic Gum.

An ideal wound dressing ensures a moist environment around the wound area and absorbs exudates from the wound surface. Topical application of bromelain to incised wounds has been shown to reprogram the wound microenvironment to promote effective tissue repair. Combining the characteristics of hydrogels and bromelain is therefore of great interest. Herein, we describe the development of a hydrogel, formulated using alginate and Arabic gum, for bromelain loading and release. The hydrogel formulation was evaluated using response surface methodology, considering the pH value and the concentration of alginate and Arabic gum. Bromelain loading and release were evaluated based on passive diffusion. Differential scanning calorimetry and Fourier transform infrared spectroscopy were performed to confirm bromelain immobilization in the hydrogel. The final hydrogel formulation had a swelling ratio of 227 % and incorporated 19 % of bromelain from a bromelain solution. Bromelain immobilization in the hydrogel was the result of hydrogen bond formation and was optimal at 4 °C after 4 h of contact. This evidence suggests that bromelain entrapment into a hydrogel is a promising strategy for the development of wound dressings that support the debridement of burns and wounds.

Effect of Oral Administration of Bromelain on Postoperative Discomfort After Third Molar Surgery.

INTRODUCTION: The purpose of this prospective randomized controlled clinical trial was to evaluate the effect of oral administration of bromelain on discomfort after mandibular third molar surgery. MATERIALS AND METHODS: Eighty-four consecutive patients requiring surgical removal of a single mandibular impacted third molar under local anesthesia were randomly assigned to receiving no drug (control group, Group A), postoperative 40 mg bromelain every 6 hours for 6 days (Group B), preoperative 4 mg dexamethasone sodium phosphate as a submucosal injection (Group C), and preoperative 4 mg dexamethasone sodium phosphate as a submucosal injection plus postoperative 40 mg bromelain every 6 hours for 6 days (Group D). Standardized surgical and analgesic protocols were adopted. Maximum interincisal distance and facial contours were measured at baseline and on postoperative days 2 and 7. Pain was measured objectively by counting the number of analgesic tablets required. Patient perception of the severity of symptoms was assessed with a follow-up questionnaire (PoSSe scale). RESULTS: On postoperative day 2, there was a statistically significant reduction in facial edema in both Groups C and D compared with the control group, but no statistically significant differences were observed between Group B and the control group. At evaluation on postoperative day 7, Group D showed a statistically significant reduction in postoperative swelling compared with the control group. The combined use of bromelain and dexamethasone (Group D) induced a statistically significant reduction in the total number of analgesic tablets taken after surgery compared with the control group. The treatment groups had a limited, nonsignificant effect on trismus when compared with the control group. CONCLUSIONS: Bromelain used singly showed moderate anti-inflammatory efficacy, reducing postoperative swelling, albeit not to any significant extent compared with no drug administration. The combined use of bromelain and dexamethasone sodium phosphate yielded the best results in terms of control of postoperative discomfort.

Perioperative Bromelain Therapy after Wisdom Teeth Extraction - A Randomized, Placebo-Controlled, Double-Blinded, Three-Armed, Cross-Over Dose-Finding Study.

Reduction in postoperative edema and inflammatory reactions is the key to the posttraumatic regeneration process. Use of bromelain is well established in this indication, but there is some controversy with regard to the optimal dosing of this drug. The aim of our study was therefore to investigate the efficacy of dosage-dependent therapy with bromelain in patients after wisdom teeth extraction by comparing the registered dosage 1000 FIP (Fédération Internationale Pharmaceutique) against higher dosages of 3000 FIP and 4500 FIP. A total of 75 patients were randomized to one of the three dosage arms, and 68 of these patients were finally analyzed in the modified intention-to-treat population. Patients involved underwent two surgery sessions: one study period being conducted under treatment with bromelain and the other with placebo. Postoperative swelling determined by a 3D face scanning system was defined as the primary endpoint; further efficacy parameters were maximum swelling, pain, difficulty in swallowing, and use of analgesics. A superiority of treatment with 3000 FIP and 4500 FIP versus 1000 FIP could not be demonstrated. The analysis of pooled bromelain treatments versus placebo did, however, show a clear trend in favor of bromelain for all assessments. Adverse events did not occur more frequently under bromelain therapy compared with placebo. This study thus clearly supports the clinical relevance of treatment of postoperative conditions with bromelain, and the recommended daily dose was sufficiently effective in this trial and indication. Copyright © 2016 John Wiley & Sons, Ltd.

Poly(N-Isopropylacrylamide)-co-Acrylamide Hydrogels for the Controlled Release of Bromelain from Agroindustrial Residues of Ananas comosus.

This works reports the purification of bromelain extracted from Ananas comosus industrial residues by ethanol purification, its partial characterization from the crude extract as well as the ethanol purified enzyme, and its application onto poly(N-isopropylacrylamide)-co-acrylamide hydrogels. Bromelain was recovered within the 30-70 % ethanol fraction, which achieved a purification factor of 3.12-fold, and yielded more than 90 % of its initial activity. The resulting purified bromelain contained more than 360 U · mg(-1), with a maximum working temperature of 60 °C and pH of 8.0. Poly(N-isopropylacrylamide)-co-acrylamide hydrogels presented a swelling rate of 125 %, which was capable of loading 56 % of bromelain from the solution, and was able to release up to 91 % of the retained bromelain. Ethanol precipitation is suitable for bromelain recovery and application onto poly(N-isopropylacrylamide)-co-acrylamide hydrogels based on its processing time and the applied ethanol prices.

Perioperative bromelain reduces pain and swelling and improves quality of life measures after mandibular third molar surgery: a randomized, double-blind, placebo-controlled clinical trial.

PURPOSE: The purpose of the present study was to compare the effect of oral bromelain (4 × 250 mg) versus oral diclofenac sodium (4 × 25 mg) on pain, swelling, trismus, and quality of life (QOL) after surgical removal of impacted lower third molars. PATIENTS AND METHODS: A randomized, double-blind, placebo-controlled study was planned. The sample included patients requiring extraction under local anesthesia of a single partial bony impacted mandibular third molar. The patients were randomly distributed into 1 of 3 groups: bromelain, diclofenac, and placebo. Treatment started 1 day before surgery and continued for 4 days. The predictor variable was the type of the drug given to the patients. The outcome variables were pain, swelling, and trismus, which were measured at 1, 3, and 7 days postoperatively. The other study variables included QOL measures to assess the patients' perception regarding the effect of surgery on their well-being and daily activities. A validated questionnaire was used to measure QOL. The data were analyzed using analysis of variance, multiple measures analysis of variance, or Pearson's χ(2) test, as appropriate. P < .05 was considered significant. RESULTS: A total of 45 subjects requiring surgical removal of a single impacted mandibular third molar under local anesthesia were included in the present study. The bromelain and diclofenac groups both showed a significant reduction in pain compared with the placebo group at all intervals (P < .05). Diclofenac also resulted in a significant reduction of swelling at 3 and 7 days, and bromelain resulted in an insignificant reduction. A nonsignificant reduction in trismus occurred in both treatment groups compared with the placebo group. Both treatment groups also showed a significant difference in the effect on QOL in most subscales and total scores (P < .05). The effect was comparable between the 2 treatment groups for all parameters and at all intervals. CONCLUSIONS: The results of our study have shown that oral bromelain is an effective therapy to improve the QOL after surgical removal of impacted lower third molars, with an effect on the postoperative sequelae comparable to that of preemptive diclofenac sodium.

Halloysite nanotube-embedded microparticles for intestine-targeted co-delivery of biopharmaceuticals.

Microparticles (MPs) with pH-responding macropores have recently proved their significance for the delivery of vulnerable biomolecules for oral drug administration. The previous MP systems were proven to provide enhanced protection against the gastric environment, however, their application is hindered due to insufficient loading efficiencies and deficient penetration capabilities of encapsulated drugs across the mucus barrier. Here, we report a new co-delivery approach based on amine-functionalized halloysite nanotube (HNT)-embedded MPs (amine-HNT-MPs) with pH-responding macropores specifically designed to deal with the mucus barrier at the absorption site. The mean diameter and polydispersity index of the pored MPs were measured by a particle size analyzer to be 37.6 ± 1.3 µm and 1.15, respectively. The drug loading capacity of the co-delivery system was shown to be 50-times higher than previously reported pored MPs. Fluorescence microscopy analysis of sulforhodamine B (into a hollow interior of HNTs)/ fluorescent nanoparticles (into a hollow interior of MPs)-encapsulated MPs confirmed biphasic release behavior due to pH-dependent pore closing/opening in the simulated gastrointestinal (GI) digestive conditions. To verify the protective effect of the co-delivery system, bromelain and lactase were loaded into HNTs and MPs, respectively, and found to exhibit 94.5 ± 3.3% (bromelain) and 70 ± 14.1% (lactase) functional activity in simulated GI tract conditions. The considerable improvement in the stability of the encapsulated enzymes against gastric conditions are attributed to the efficient pore sealing of the co-delivery system after the encapsulation of enzymes and maintenance of these closed pores in the gastric environment. Furthermore, the mucolytic enzyme (i.e. bromelain)-encapsulated co-delivery system was found to enhance mucopenetration of the encapsulated drug from histological analysis using ex vivo porcine intestine tissue. Therefore, the new microencapsulation design proposed in this study provides a promising solution to the major issues hampering the wide-spread application of MPs in the development of oral drug formulations for biopharmaceuticals and vaccines.

Bacterial Nanocellulose Loaded with Bromelain: Assessment of Antimicrobial, Antioxidant and Physical-Chemical Properties.

Bacterial nanocellulose (BNC) has desirable properties for wound healing such as high purity, good shape retention, and high water binding capacity. Bromelain is a protease found in pineapple tissues and has been applied in several fields, it has anti-inflammatory and anticancer properties, promotes cell apoptosis, amongst others. In this work, a BNC based device for the controlled release of bromelain was developed. BNC were submersed in sterilized bromelain solution and incubated at 25 °C under 100 rpm for 24 h. Physical-chemical properties, protein concentration, antioxidant and antimicrobial activities were measured. Results demonstrate that BNC could improve bromelain antimicrobial activity 9 times. Those findings allow concluding that bromelain is a promising molecule to be incorporated into BNC's. The BNC's characteristics seem to represent a new promising delivery system of the loaded biomolecule, and protected from external actions.

Oral immunogenicity of the plant proteinase bromelain.

Bromelain is a natural mixture of proteolytic enzymes derived from pineapple stem that has been shown to have anti-inflammatory activity when administered orally. Although most proteins given orally without adjuvant (e.g., food) result in tolerance, we previously reported that long-term oral exposure to bromelain stimulated the development of high serum anti-bromelain antibody titers. The purpose of these studies was to further investigate the mechanisms responsible for the immunogenicity of oral bromelain. Results showed that repeated exposure was required for development of anti-bromelain antibodies, with strong antibody responses in all mice that received at least 12 doses of bromelain either orally or intragastrically over 3-6 weeks. Proteolytic activity was required for strong oral immunogenicity in the absence of conventional adjuvant, with strong serum antibody responses generated against proteolytically active bromelain and trypsin, but not against ovalbumin, lysozyme, or inactivated bromelain. Significantly higher anti-bromelain antibody titers were seen in IL-10-deficient versus wild-type mice, suggesting that simultaneous treatments that decrease IL-10 activity may further enhance systemic antibody responses following oral exposure. The antibodies generated did not affect the proteolytic activity of bromelain. The data demonstrate that proteolytically active antigens such as bromelain can stimulate both systemic and mucosal immune responses following repeated oral exposure. Further studies of the mechanisms involved in generation of immune responses following oral exposure to proteolytically active antigens can lead to a better understanding of mechanisms of oral tolerance and to the development of novel adjuvants for oral vaccines.

Hyaluronic acid grafted PLGA copolymer nanoparticles enhance the targeted delivery of Bromelain in Ehrlich's Ascites Carcinoma.

Rapidly increasing malignant neoplastic disease demands immediate attention. Several dietary compounds have recently emerged as strong anti-cancerous agents. Among, Bromelain (BL), a protease from pineapple plant, was used to enhance its anti-cancerous efficacy using nanotechnology. In lieu of this, hyaluronic acid (HA) grafted PLGA copolymer, having tumor targeting ability, was developed. BL was encapsulated in copolymer to obtain BL-copolymer nanoparticles (NPs) that ranged between 140 to 281nm in size. NPs exhibited higher cellular uptake and cytotoxicity in cells with high CD44 expression as compared with non-targeted NPs. In vivo results on tumor bearing mice showed that NPs were efficient in suppressing the tumor growth. Hence, the formulation could be used as a self-targeting drug delivery cargo for the remission of cancer.

Bromelain nanoparticles protect against 7,12-dimethylbenz[a]anthracene induced skin carcinogenesis in mouse model.

Conventional cancer chemotherapy leads to severe side effects, which limits its use. Nanoparticles (NPs) based delivery systems offer an effective alternative. Several evidences highlight the importance of Bromelain (BL), a proteolytic enzyme, as an anti-tumor agent which however has been limited due to the requirement of high doses at the tumor site. Therefore, we illustrate the development of BL loaded poly (lactic-co-glycolic acid) NPs that show enhanced anti-tumor effects compared to free BL. The formulated NPs with a mean particle size of 130.4 ± 8.81 nm exhibited sustained release of BL. Subsequent investigation revealed enhanced anti-tumor ability of NPs in 2-stage skin tumorigenesis mice model. Reduction in average number of tumors (∼ 2.3 folds), delay in tumorigenesis (∼ 2 weeks), percent tumorigenesis (∼ 4 folds), and percent mortality rate as well as a reduction in the average tumor volume (∼ 2.5 folds) in mice as compared to free BL were observed. The NPs were found to be superior in exerting chemopreventive effects over chemotherapeutic effects at 10 fold reduced dose than free BL, validated by the enhanced ability of NPs (∼ 1.8 folds) to protect the DNA from induced damage. The effects were also supported by histopathological evaluations. NPs were also capable of modulating the expression of pro-apoptotic (P53, Bax) and anti-apoptotic (Bcl2) proteins. Therefore, our findings demonstrate that developed NPs formulation could be used to improve the efficacy of chemotherapy by exerting chemo-preventive effects against induced carcinogenesis at lower dosages.

Therapeutic efficacy of bromelain in impacted third molar surgery: a randomized controlled clinical study.

OBJECTIVE: Bromelain is the name given to a family of proteolytic enzymes obtained from Ananas comosus, the pineapple plant. It is considered a potent anti-inflammatory and antiedematous substance. Surgery of impacted third molars in the outpatient setting is one of the procedures most often associated with postoperative pain and swelling. The aim of this study was to evaluate the efficacy of bromelain in reducing postoperative pain and swelling. METHOD AND MATERIALS: 80 patients were recruited to the study from patients attending the Maxillofacial Surgery Unit of the Istituto Stomatologico Italiano, Milan, Italy, for impacted third molar surgery. At time 0 when surgery was performed, patients started simultaneous antibiotic and analgesic therapy. On the following day, patients were divided into two groups. Patients in group 1 were prescribed bromelain. Patients in group 2 were prescribed only the analgesic if required. Three parameters were evaluated: pain, edema, and erythema. The first evaluation visit was performed 3 hours after surgery, the second 48 hours after surgery, and the final evaluation 7 days after surgery. RESULTS: Postoperative pain, edema, and erythema were significantly lower in the study group than in the control group. Analgesic consumption, both in terms of days of treatment and number of tablets taken, was slightly lower in the study group. Only one adverse event was recorded, which occurred in a patient in the control group. CONCLUSION: The present study demonstrates an important anti-inflammatory and anti-edematous effect of bromelain. Statistical analysis shows that in the group treated with bromelain the inflammatory response was significantly less than in the control group.

Anti-cancer activity of bromelain nanoparticles by oral administration.

Oral administration of anti-cancer drugs is an effective alternative to improve their efficacy and reduce undesired toxicity. Bromelain (BL) is known as an effective anti-cancer phyto-therapeutic agent, however, its activity is reduced upon oral administration. In addressing the issue, BL was encapsulated in Poly(lactic-co-glycolic acid) (PLGA) to formulate nanoparticles (NPs). Further, the NPs were coated with Eudragit L30D polymer to introduce stability against the gastric acidic conditions. The resultant coated NPs were characterized for BL entrapment, proteolytic activity and mean particle size. The stability and release pattern of NPs were evaluated under simulated gastrointestinal tract (GIT) pH conditions. Cytotoxicity studies carried out in human cell lines of diverse origin have shown significant dose advantage (-7-10 folds) with NPs in reducing the IC50 values compared with free BL. The cellular uptake of NPs in MCF-7, HeLa and Caco-2 cells monolayer was significantly enhanced several folds as compared to free BL. Altered expression of marker proteins associated with apoptosis and cell death (P53, P21, Bcl2, Bax) also confirmed the enhanced anti-carcinogenic potential of formulated NPs. Oral administration of NPs reduced the tumor burden of Ehrlich ascites carcinoma (EAC) in Swiss albino mice and also increased their life-span (160.0 ± 5.8%) when compared with free BL (24 ± 3.2%). The generation of reactive oxygen species, induction of apoptosis and impaired mitochondrial membrane potential in EAC cells treated with NPs confirmed the suitability of Eudragit coated BL-NPs as a promising candidate for oral chemotherapy.