Cytokinetic Study of MCF-7 Cells Treated with Commercial and Recombinant Bromelain

Fouz, Nour,Amid, Azura,Hashim, Yumi Zuhanis Has-Yun Asian Pacific Journal of Cancer Prevention 2013 Asian Pacific journal of cancer prevention Vol.14 No.11

Background: Breast cancer is a leading cause of death in women. The available chemotherapy drugs have been associated with many side effects. Bromelain has novel medicinal qualities including anti-inflammatory, anti-thrombotic, fibrinolytic and anti-cancer functions. Commercially available bromelain is obtained through tedious methods; therefore, recombinant bromelain may provide a cheaper and simpler choice with similar quality. Materials and Methods: This study aimed to assess the effects of commercial and recombinant bromelain on the cytokinetic behavior of MCF-7 breast cancer cells and their potential as therapeutic alternatives in cancer treatment. Cytotoxic activities of commercial and recombinant bromelain were determined using (sulforhodamine) SRB assay. Next, cell viability assays were conducted to determine effects of commercial and recombinant bromelain on MCF-7 cell cytokinetic behavior. Finally, the established growth kinetic data were used to modify a model that predicts the effects of commercial and recombinant bromelain on MCF-7 cells. Results: Commercial and recombinant bromelain exerted strong effects towards decreasing the cell viability of MCF-7 cells with $IC_{50}$ values of 5.13 ${\mu}g/mL$ and 6.25 ${\mu}g/mL$, respectively, compared to taxol with an $IC_{50}$ value of 0.063 ${\mu}g/mL$. The present results indicate that commercial and recombinant bromelain both have anti-proliferative activity, reduced the number of cell generations from 3.92 to 2.81 for commercial bromelain and to 2.86 for recombinant bromelain, while with taxol reduction was to 3.12. Microscopic observation of bromelain-treated MCF-7 cells demonstrated detachment. Inhibition activity was verified with growth rates decreased dynamically from 0.009 $h^{-1}$ to 0.0059 $h^{-1}$ for commercial bromelain and to 0.0063 $h^{-1}$ for recombinant bromelain. Conclusions: Commercial and recombinant bromelain both affect cytokinetics of MCF-7 cells by decreasing cell viability, demonstrating similar strength to taxol.

Bromelain-loaded polyvinyl alcohol–activated charcoal-based film for wound dressing applications

Yugesh Ramdhun,Mohanta Monalisha,Thirugnanam Arunachalam,Ritvesh Gupta,Devendra Verma 한국고분자학회 2023 Macromolecular Research Vol.31 No.5

The present research focuses on bromelain-loaded polyvinyl alcohol (PVA)–activated charcoal (AC) film with desired mechanical strength and biocompatible properties for wound dressing application. The developed films were investigated physicochemically, mechanically, and biologically. The drug release studies were performed in a phosphate-buffered solution (pH 7.4). The homogenous dispersion of bromelain in the PVA–AC film and their morphology were analyzed through field emission scanning electron micrograph, Fourier transform infrared spectrometry, and Raman spectroscopy. The decrease in the hydroxyl group peak intensity suggests the excellent miscibility of bromelain in the PVA–AC matrix. The medium-range viscosities of PVA-based films revealed formulation stability. It was also observed that the wettability of the films was reduced with the addition of bromelain and AC. It is due to the interaction between the hydroxyl groups of PVA and bromelain, which leaves all of the hydrophobic bromelain groups unbounded and exposed to the surface. Further, the tensile strength of developed films was enhanced by 19% with the AC reinforcement. The swelling rate, degradation, and water vapor transmission rate were decreased with the incorporation of AC and bromelain. The films were slightly hemolytic (less than 3% hemolysis) and showed moderate protein adsorption. The in vitro drug release studies demonstrated a consistent release of bromelain for 48 h. The bromelain-loaded PVA–AC exhibits good antibacterial against S. aureus and reasonable cell viability with L929 cells. Further, a scratch assay showed 99.4% wound closure after 24 h bromelain-loaded PVA–AC films. Therefore, the developed films can be a potent candidate for wound healing applications.

단백질 분해 효소의 안정화를 통한 화장품분야의 응용

이찬무(Chan-Mu Lee),장건희(Geon-Hee Jang),김광수(Kwang-Soo Kim) 한국화장품미용학회 2013 한국화장품미용학회지 Vol.3 No.1

Bromelain is a protein-digesting (proteolytic) enzyme complex found in the fruit and, in higher concentrations, in the stem of the pineapple (Ananas comosus). It is able to hydrolyze or break down a wide variety of protein types in a range of acid and alkaline environments. Using micro capsule technology, we were able to stabilize bromelain. It features high stabilization for skin care and personal care products. When we stabilized bromelain using the microencapsulation method, it maintained its enzyme activity at about 68% after 6 months, in 45℃ conditions. We measured spherical stabilized bromelain from 300 to 1,000㎛, utilizing SEM (scanning electron microscopy). Papain, a protein-digesting enzyme which is derived from the papaya is usually utilized in many cosmetic products, however, when compared to stabilized bromelain, with regards to the activity of GDU(Gelatin Digestion Unit), stabilized bromelain showed superior protein digestive activity. After applying the skin care product containing stabilized bromelain for 7 days, we found in our results that it removes dead skin cells and cleans the skin surface. Therefore, this stabilization technology might lead to stabilized bromelain being widely used in various cosmetic products. Bromelain was previously proved by many scientists to proteolytically remove certain cell surface molecules that affects lymphocyte migration and activation 3-9). In addition, bromelain markedly affects the creation of various cytokines and inflammatory effects.

Aqueous Two-phase Extraction of Bromelain from Pineapple Peels (‘Phu Lae’ cultv.) and Its Biochemical Properties

Sunantha Ketnawa,Phanuphong Chaiwut,Saroat Rawdkuen 한국식품과학회 2011 Food Science and Biotechnology Vol.20 No.5

This study examines the extraction of bromelain from pineapple peels by using an aqueous two-phase system (ATPS). Bromelain from the crude extract predominantly partitioned to the polyethylene glycol rich phase. The best condition for bromelain partitioning was found to be 18%PEG6000-17% MgSO_4, which increased purity 3.44-fold with an activity recovery of 206%. Protein patterns and activity staining showed the molecular weight of bromelain to be about 29 kDa. The bromelain showed the highest relative activity at pH 8.0 and at 60^oC. When increasing NaCl concentrations (up to 1.5%, w/v), its activity continuously decreased. The bromelain (0-0.3 units) was applied to hydrolyze collagen. The β, α1, α2 components of collagen extensively degraded into small peptides when treated with the bromelain. This study showed that the ATPS can be employed to isolate the bromelain from pineapple peels and the bromelain extract could be used as a meat tenderizing agent.

Bromelain-Induced Apoptosis in GI-101A Breast Cancer Cells

Sivanesan Dhandayuthapani,Honey Diaz Perez,Alexandra Paroulek,Panneerselvam Chinnakkannu,Umadevi Kandalam,Mark Jaffe,Appu Rathinavelu 한국식품영양과학회 2012 Journal of medicinal food Vol.15 No.4

Bromelain is a proteolytic enzyme extracted from the stems and the immature fruits of pineapple that was found to be antitumorigenic in different in vitro models. Bromelain has been reported to promote apoptosis, particularly in breast cancer cells, with the up-regulation of c-Jun N-terminal kinase and p38 kinase. Our study was designed to determine if bromelain could induce apoptosis in GI-101A breast cancer cells. GI-101A cells were treated with increasing concentrations of bromelain for 24 hours. The effect of bromelain for inducing cell death via activation of the apoptosis mechanism in GI-101A cells was further determined by using caspase-9 and caspase-3 assays along with the M30-Apoptosense assay to measure cytokeratin 18 (CK18) levels in the cytoplasm of the cultured cancer cells. A dose-dependent increase in the activities of caspase-9 and caspase-3 coinciding with elevation of CK18 levels was found in bromelain-treated cells compared with control cells. Furthermore, the apoptosis induction by bromelain was confirmed by DNA fragmentation analysis and 4,60-diamino-2-phenylindole dihydrochloride fluorescence staining of the nucleus. Our results indicate an increase in apoptosisrelated cell death in breast cancer cells with increasing concentrations of bromelain.

제주산 파인애플 유래 Bromelain관련 유전자 (BL1)를 이용반 형질전환 상추의 특성

정유진,김기훈,최장선,이순열,노일섭,박진희,강권규,Jung, Yu-Jin,Kim, Gi-Hyun,Choi, Jang-Sun,Lee, Soon-Youl,Nou, Il-Sup,Park, Jin-Heui,Kang, Kwon-Kyoo 한국식물생명공학회 2006 식물생명공학회지 Vol.33 No.1

파인애플 (Ananas comosus) 줄기에서 얻어지는 bromelain은 단백질 분해효소 중 cysteine protease의 복합체로 알려져 있다. 본 연구에서는 제주산 파인애플 줄기를 이용하여 bromelain 관련 유전자를 분리하였다. 분리된 BL1 유전자는 총 933개의 염기서열로 311개의 아미노산을 coding 하였다. 지금 까지 알려진 식물 유래 bromelain 관련 유전자와의 alignment 분석한 결과 BAA21929 유전자와 94%, T10516 유전자와 93% 및 P14518 유전자와 81%의 상동성을 보였다. BL1 유전자를 상추 게놈내에 도입하고자 NPTII 유전자 와 BL1 유전자로 제작한 pBI 121 BL 벡터를 Agrobacterium tumefacience LBA4404에 도입한 후, 상추잎 절편에 감염시켜 embryogenic callus 및 재분화 식물체를 육성하였다. 이들식물체로부터 T1세대를 육성하여 PCR 분석을 통해 왜래유전자의 도입 여부를 확인하였다. 또한 형질전환체의 발현여부는 Nothern blot분석 및 eno protease활성을 통해 형질전환체에서 BL1유전자가 안정적으로 상추세포내에서 발현되고 있음을 확인하였다. 따라서 본 실험에서 육성된 bromelain 관련 BL1 유전자가 도입한 형질전환 상추를 육종소재르 활용한다면 상업적으로 유용한 단백질을 분해하는 가수분해효소로써 건강 보조제, 사료첨가제 등에 널리 사용할 수 있을 것으로 생각되어진다. To clarify the roles of bromelain in plants, we isolated BL1 gene encoding bromelain from pineapple stem tissues and sequenced. The full length cDNA is 933 bp and encodes a polypeptide of 311 amino acid residues. The cDNA is most similar 94% at the amino acid level to bromelain previously isolated from pineapple (BAA21929). Explants of Lactuca sativa were co-cultivated with Agrobacterium tume-faciences LBA 4404 strains containing nptII and BL1 gene for transformation. Through initial selection of regenerated explants by culturing on a kanamycin and carbenicillin containing MS medium, multiple shoots were obtained after 2 months of culture. For a complementary step of selection, putative transgenic shoots were transferred to 1/2 Ms basal medium supplemented with 100 mg/L kanamycin and 500 mg/L carbenicillin. The selected shoots were obtained T1 generation seeds with emasculation, and tested with PCR analysis using 35S promoter and BL1 specific primers whether BL1 gene was introduced to genome of the plants. These results confirmed that produced the specific PCR bands in the putative transgenic lines. Additionally the Northern blot and endo protease activity showed that transcripts of BL1 gene were detected in transgenic lines. Theses results suggest that BL1 gene be successfully integrated and transcripted in the transgenic lettuce plants.

A Comparative Study of the Effects of Bromelain and Fresh Pineapple Juice on the Early Phase of Healing in Acute Crush Achilles Tendon Injury

Ayoola I. Aiyegbusi,Olaleye O. Olabiyi,Francis I.O. Duru,Cressie C. Noronha,Abayomi O. Okanlawon 한국식품영양과학회 2011 Journal of medicinal food Vol.14 No.4

Bromelain, an enzyme extracted from the stem of the pineapple plant, has been reported to reduce pain and swelling in acute soft tissue injuries, but no study has been done to compare its effect with that of fresh pineapple juice on the healing of acute tendon injuries. This study compared the effects of commercial bromelain and fresh pineapple juice on tenocyte proliferation and the malondialdehyde (MDA) level in the early stage of healing in a crush injury to the Achilles tendon of Sprague–Dawley rats. Twenty-four male rats were divided randomly into three groups of eight rats each; all the rats had induced crush injury to the Achilles tendon: Group 1 (control), no treatment; Group 2, oral bromelain treatment at a dosage of 7 mg/kg of body weight daily; and Group 3, fresh diluted pineapple juice at a dosage of 30 mg/kg of body weight. Treatment was given over the first 14 days post-injury. On day 15 post-injury, the animals were sacrificed, and the tendons were excised and processed for histological study and MDA assay. Results show a significant difference in the tenocyte population between the bromelain group and the control (P < .05), whereas pineapple juice also increased the tenocyte population, although not significantly (P = .36). Pineapple juice, however, significantly lowered the MDA level compared with both the control and bromelain-treated groups. Based on this study, 600 GDU bromelain given at a dosage of 7 mg/kg had a better effect on tenocyte proliferation than fresh pineapple juice given once daily in acute tendon injury.

꽃게(Ovalipes punctatus) 단백질 유래 항산화 기능성 펩타이드 제조 최적공정 확립 및 이화학적 특성

하유진,김도현,이병의,유선균 한국응용과학기술학회 2018 한국응용과학기술학회지 Vol.35 No.2

Swimming crab(Ovalipes punctatus) is produced in Korea and utilized as semi-processed food at streamed cooked state. Recently, protein hydrolysates have been known as having function such as antioxidant, suppression of hypertension, immunodulatory, alleviation of pain, and antimicrobial activity. This research was investigated to find the functional antioxidant from crab hydrolysates. To fine optimal protease enzyme, alcalase, bromelain, flavourzyme, neutrase, papain, and protamex were selected to evaluate the DPPH radical scavenging activity and finally bromelain to show the best activity was selected. The molecular weight of bromelain hydrolysates were distributed with range from 500 to 3,200 Da and 7 different molecules or more. The amino acids related to antioxidant capacity was about 42.54%. The processes optimization study used was the response surface methodology. The ranges of processes were the reaction temperature of 40 to 60 o C, pH 6 to 8, and enzyme concentration 1 to 3%(w/v). As a result, the optimization of process was determined at temperature of 55 o C, pH of 6.5, and enzyme concentration of 3%(w/v). In these conditions, degree of hydrolysates were maximum 71.60%. Therefore, we expect that those products are useful as functional food ingredients. 깨다시 꽃게(Ovalipes punctatus)는 갑각류로서 우리나라에서 잡히는 매끈 꽃겟속, 주름 꽃겟속, 톱날 꽃겟속, 민 꽃겟속, 두갈래 민꽃겟속 들 중에 하나이다. 대부분의 꽃게는 가공되지 않은 상태로 찜 또는 찜육 등 반 가공 형태로 산업화 되었지만 최근에 게로부터 생리활성을 나타내는 펩타이드를 생산하는 연구가 발표되고 있다. 본 연구는 항산화 기능성을 나타내는 펩타이드를 선별하고 생산 최저 공정 확립에 연구를 수행 하였다. 사용된 효소 alcalase, bromelain, flavourzyme, neutrase, papain, protamex들 중에서 bromelain으로 생산된 꽃게육 단백질 가수분해물이 가장 높은 활성을 보여 주었다. 꽃게육 단백질의 bromelain 가수분해물의 펩타이드들의 분자량 분포는 500-3,200 Da로서 7 종류의 이상의 펩타이드들로 구성되었다. 가수분해물의 구성아미노산 분포는 항산화 기능성에 관련된 소수성 아미 노산은 전체 42.54%를 차지하였다. 가수분해물의 최적 생산 수율 조건을 확립하기 위하여 공정 조건, 효소 반응 온도 40-60℃, pH 6-8, 효소의 농도 1–3%(w/v)로 표면반응 분석법을 수행한 결과 효소 반응온도 55℃, 반응 pH 6.5, 효소의 양은 3%(w/v)에서 결정되었다. 최적 조건에서 단백질 가수분해도는 최대 71.60%에 도달하였다.

Effects of Bromelain and Double Emulsion on the Physicochemical Properties of Pork Loin

Hyerin Shin,Hyo Tae Kim,Mi-Jung Choi,Eun-Young Ko 한국축산식품학회 2019 한국축산식품학회지 Vol.39 No.6

Our aim was to investigate the effects of bromelain embedded in double emulsion (DE) on physicochemical properties of pork loin. We evaluated DE characteristics such as size, zeta potential, and microscopy after fabrication. We marinated meat with distilled water (DW), 1% (w/v) bromelain solution, blank DE, and 1% (w/v) bromelain loaded in double emulsion (DE E) for 0, 24, 48, and 72 h at 4℃, and prepared raw meat for control. The marinated samples were assessed for color, water holding capacity, cooking loss, moisture content, pH, protein solubility, Warner-Bratzler shear force (WBSF) and gel electrophoresis. The droplet size of 1% (w/v) bromelain embedded in DE was increased compared with blank DE (p<0.05) and values of zeta potential decreased. The increase in lightness and color difference range of the DE-treated group was lower than that of the DW-treated group (p<0.05). Moreover, treatment by immersion in 1% (w/v) DE E resulted in the highest water holding capacity values (p<0.05) and lower cooking loss values than water base treatment (p<0.05). Results for myofibrillar protein solubility and WBSF showed a similar trend. 1% (w/v) DE E showed degradation of myosin heavy chain after 48 h in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thus, bromelain-loaded DE is useful for controlling and handling enzyme activity in food industry.

Antinociceptive and neuroprotective effects of bromelain in chronic constriction injury-induced neuropathic pain in Wistar rats

Ahmed Olalekan Bakare,Bamidele Victor Owoyele 대한통증학회 2020 The Korean Journal of Pain Vol.33 No.1

Background: The continuous search for a novel neuropathic pain drug with few or no side effects has been a main focus of researchers for decades. This study investigated the antinociceptive and neuroprotective effects of bromelain in sciatic nerve ligation-induced neuropathic pain in Wistar rats. Methods: Forty-eight Wistar rats randomly divided into eight groups comprised of six animals each were used for this study. Peripheral neuropathy was induced via chronic constriction of the common sciatic nerve. Thermal hyperalgesic and mechanical allodynia were assessed using a hotplate and von Frey filaments, respectively. The functional recovery and structural architecture of the ligated sciatic nerve were evaluated using the sciatic functional index test and a histological examination of the transverse section of the sciatic nerve. The neuroprotective effects of bromelain were investigated in the proximal sciatic nerve tissue after 21 days of treatment. Results: Bromelain significantly (P < 0.05) attenuated both the thermal hyperalgesia and mechanical allodynic indices of neuropathic pain. There were improvements in sciatic function and structural integrity in rats treated with bromelain. These rats showed significant (P < 0.05) increases in sciatic nerve nuclear transcription factors (nuclear factor erythroid-derived-2-related factors-1 [NrF-1] and NrF-2), antioxidant enzymes (superoxide dismutase and glutathione), and reduced membranelipid peroxidation compared with the ligated control group. Conclusions: This study suggest that bromelain mitigated neuropathic pain by enhancing the activities of nuclear transcription factors (NrF-1 and NrF-2) which increases the antioxidant defense system that abolish neuronal stress and structural disorganization.