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Bromelain isolated from pineapple ( Ananas comosus ) can be an excellent phytotherapeutic agent for cardiovascular treatment as it can inhibit platelet aggregation. However, if it is used orally, it can be easily degraded in an acidic pH environment due to enzymes secreted during the digestion process. Its instability under a certain condition will reduce its pharmacological activity and, as a result, will reduce its health benefit. Therefore bromelain needs to be encapsulated in a matrix such as alginate-carboxymethyl cellulose (CMC) microsphere cross-linked to Ca 2+ ion, which will act as a carrier agent. In this research, bromelain encapsulation is done by in situ encapsulation. Particle size analyzer (PSA), Fourier transform infrared spectrophotometer, and scanning electron microscope are used as characterization instruments to investigate the encapsulation of bromelain into the alginate-CMC microsphere. PSA analysis showed that the molecular size of the alginate-CMC microspheres was in the 496.05±2.72 and 629.65±8.70 nm. Encapsulation study using the Bradford method showed that the highest encapsulation ratio was achieved at alginate-CMC ratio of 1.5:0.5 (% w/v:% w/v). These results demonstrated that the alginate-CMC microsphere had potential to be an effective matrix for bromelain encapsulation.

Thrombus is blood congealment process (platelet) occurred in area of vein and is useful for preventing of bleeding occurrence. The considerable amount of thrombus in blood leads to blocked arteries and angina pectoris. The partial purification of bromelain originated from pineapple core ( Ananas comosus [L.] Merr) with the specific activity of 124.38 U/mg had inhibition activity to the platelet aggregation of 86.48%. On the other hand, the proteolytic activity of bromelain was relatively stable in the first 4 hours. However, the proteolytic activity significantly decreased in the next 4 hours due to the influence of gastric fluid (pH 1.2). To overcome the problem, bromelain must be encapsulated into alginate-hpmc nanospheres cross-linked by cation (CaCl 2 ). Based on the optimization result of the swelling index and the entrapment efficiency, the nanospheres with the composition of alginate-hpmc 1: 1 and 1:2 were the optimal formula and selected to encapsulate bromelain and be characterized by PSA and SEM. Alginate-hpmc nanospheres (1:1) had a particle size of 543.7±4.2 nm. The morphology of nanospheres were almost spherical and had a smooth surface. Moreover, the particle size of alginate-hpmc nanospheres (1:2) was 515.3±26.7 nm and the SEM micrographs showed the spherical nanospheres with slightly rough surface. The swelling degree, entrapment efficiency, PSA, and SEM data will relate to suitability of the nanospheres formulation to orally deliver bromelain.

The present study is aimed at isolating and purifying the bromelain enzyme extracted from the cores of the fruit of the pineapple plant (Ananas comusus). The process of purification of the crude enzyme extract included, in sequence, fractionation through ammonium sulfate precipitation, dialysis, and ion-exchange chromatography. Fractionation of the crude enzyme extract with ammonium sulfate resulted in an increase of the specific activity. The fraction with the highest specific activity was that obtained by precipitation with 50-80 % ammonium sulfate. For this fraction, the specific activity was 8.683 U/mg and the degree of purity of the enzyme was 160 times greater than that of the crude enzyme extract. Further purification by column chromatography using diethylaminoethyl cellulose (DEAE-C) as ion-exchange medium produced six fractions (out of 92) characterized by peaks in proteolytic activity. In particular, the highest proteolytic activity (10.530 U/mg) was displayed by the fifth such fraction. The platelet aggregation inhibitory activity of the DEAE-C-purified bromelian extract was assayed through the Born's method that uses platelet-rich plasma and asetosal as positive control. The aggregation percent of this bromelain extract was measured to be 49.70% and its inhibition percent 46.89 %.

Endophytic fungi may be a potent source of bioactive compounds and have a vast repertoire of diverse metabolites. One source of endophytic fungi host plants is a medicinal plant such as Brotowali or Tinospora crispa. Research corresponding to the antibacterial activity of endophytic fungi extracts isolated from T.crispa taken from several regions in West Java has never been reported yet. While antibacterial methods to evaluate their activity as well as to identify the chemical compounds is a TLC-bioautography assay. This research is aimed to investigate the components of endophytic fungal extracts and their antibacterial activity using the TLC-bioautography assay method. In this study, eighty isolates of endophytic fungi have been successfully isolated from plant tissues of T.crispa from several regions in West Java. Antibacterial activity by using TLC (dot-blot) plates revealed that the fungal extracts could inhibit S. aureus (77 extracts) and E. coli (35 extracts) with inhibition zones ranging from 8-30 mm. The endophytic fungi extracts that showed potent antibacterial activity against Gram-positive bacteria (S.aureus) were sixteen extracts, while nine extracts have good inhibition activity against Gram-negative bacteria (E.coli). Among the fungal extracts that have excellent ability against both Gram-positive and negative was namely TcBt1Bd-10 extract. Based on metabolites analysis using the TLC method, the possibility of the chemical compounds that played a role in antibacterial activity of the extracts, including phenolics, flavonoids, alkaloids, and terpenoids.

Bromelain is an enzyme belongs to the cysteine protease. In this study, bromelain isolated from pineapple core (Ananascomosus [L.] Merr) was purified by fractionation using ammonium sulfate followed by dialysis and then ion exchange chromatography. The fraction of bromelain obtained from each purification step showed an increase in specific activity. The highest specific activity of protease was found in 20-50% ammonium sulfate fraction of 104.018 U/mg with a purity level 3.2-fold compared to crude extract. Further purification by ion exchange chromatography using DEAE-Cellulose, the fraction of bromelain showed an increase in specific activity to 278.333 U/mg with a purity level 8.8-fold compared to crude extract. The determination of kinetics parameter of purified bromelain using Lineweaver-Burk plot gives Km value of 0.15 % (w/v) and Vmax of 0.056 U/min. This bromelain can be strongly inhibited by EDTA and PCMB. The addition of EDTA and PCMB at a concentration of 0.5 mM can decrease the activity of the enzyme up to 88.50% by showing the competitive and mix-inhibition types of inhibition, respectively. The antiplatelet activity of the bromelain fraction was tested in-vitro based on the Born method, by using plasma (PRP), acetosal as a positive control and ADP as an aggregator. The purified bromelain showed the ability of an antiplatelet agent with percentage of aggregation 29.51% and percentage of inhibition 68.91%.

Trastuzumab as intact IgG are well researched for theranostic agent in HER2 overexpressed breast cancer. However, due to the relatively large of molecules it is slowly moved and weak penetration of the target cells. Fragmentation of trastzumab has been developed by pepsin cleavages to get the F(ab')2 fragments. To observe the stability and accessibility of F(ab')2 structure in corporation with HER2 (human epidermal growth factor receptor-2), the structure of antibody modeling had been developed with 1IGT as a template. Molecular dynamics (MD) of the F(ab')2 structure simulation has been done in the aqueous phase with AMBER trajectories for 20 ns. Computational visualization by VMD (Visual Molecular Dynamics) were applied to identify binding site interaction details between trastuzumab F(ab')2 and HER2 receptor. The results of MD simulations indicated that the fragmentation of trastuzumab F(ab')2 did not change the structure and conformation of F(ab')2 as a whole, especially in the CDR (Complementarity Determining Region) area. SASA (solvent accessibility surface area) analysis on lysine residues showed that formation of conjugate DOTA-F(ab')2 predicted occur on outside of the CDR regions so its not interfered with binding affinity for the HER2 receptor. The molecular dynamic simulation of DOTA-F(ab')2 with HER2 receptor in aqueous system generated ΔGbinding more highly (15.5066 kkal/mol) than positive control HER2-Fab (-45.1446 kkal/mol).

The research aims to isolate, purify, and examine the inhibitors effect of a partially purified product from pineapple core with Palembang type pineapple (Ananas comosus [L.] Merr). The isolation process obtained crude enzymes with specific activity 42.60 Units/mg. Purification of crude enzymes using fractionation method with ammonium sulfate obtained bromelain fraction with the highest specific activity at 0-50 % saturation level of ammonium sulfate, which is 230.22 Units/mg with a purity level of 5.40 times from its crude extract. Further purification of 0-50 % ammonium sulfate fraction by gel filtration chromatography method using DEAE-Sephadex G-50 matrix obtained enzyme with specific activity 275.46 Units/mg with purity level 6.50 times from its crude extract. The optimum substrate concentration was at a concentration of 1.0 % (w/v). The purified bromelain has a Michaelis-Menten (Km) constant value and the maximum speed of the enzymatic reaction (Vmax) i.e. 0.52 % (w/v) and 0.51 Units/min. The proteolytic activity of bromelain is strongly inhibited by PCMB compound and activated by PMSF at 0.1 mM concentration of 67.27 %.

The aim of this study is to maintain the antiplatelet activity of pineapple core bromelain from degradation in gastric fluid by encapsulating the enzyme in alginate-guar gum crosslinked with glutaraldehyde. Bromelain isolation was followed by purification process such as ammonium sulfate precipitation and dialysis. The specific activity obtained showed an enhancement started for crude enzyme (20.11 U/mg), ammonium sulfate fraction (190.64 U/mg), and dialysis fraction (229.77 U/mg). Dialysis fraction was encapsulated by in situ loading method in alginate-guar gum hydrogel with 1.25% (v/v) glutaraldehyde concentration. The swelling ratio of the hydrogel is 234.37 % in acidity of 1.2; and 1375.91 % in acidity of 7.4; while the encapsulation efficiency amount was 60.42%. I n addition, maximum concentration of bromelain released in dissolution test was higher in artificial intestinal environment (1.79 mg/L) than in artificial gastric fluid (0.13 mg/L), with maximum proteolytic activity of 1.2 U/mL and 0.11 U/mL, respectively. In vitro study of antiplatelet activity showed a good inhibition for dialysis fraction (50.6 %) and dissolution fraction (34.86 %), indicating that the hydrogel is good enough to maintain the activity of bromelain.

Bromelain found in various tissues in the pineapple plant (Ananas Comusus [L] Merr. The bromelain will be taken from pineapple cores for kinetic study. The effect of addition organic compounds, such as phenylmethylsulfonyl fluoride (PMSF) and cysteine on its kinetics behaviour will be determined by proteolytic activities of the bromelain. The stages of this study were started from isolation of crude enzyme, fractionation with ammonium sulphate salt, dialysis, and ion-exchange column chromatography with CM sephadex C-50 for enzyme purification. The obtained enzyme fractions from crude enzyme, ammonium sulphate, dialysis, and CM-sephadex C-50 ion exchange chromatography have specific activities of 45.39, 238.31, 311.16, and 427.13 U/mg, respectively. The purification stages result in increasing of the bromelain specific activities by 9 times compared to the crude enzyme. The Michaelis-Menten constant (Km) value for the purified bromelain was 0.85 % casein and the Vmax value was 0.41 U/min. Addition of PMSF and cysteine compound effect on its Vmax and Km with various trends. The Vmax value will be decreased by addition of PMSF and increased by addition of cysteine. Thus, proteolytic activity of bromelain from pineapple cores can be significantly increased by the addition of cysteine compounds and inhibited by the addition of PMSF.

The aim of this study is to coat the bromelain from pineapple's core into chitosan microspheres crosslinked by glutaraldehyde to maintain its activity until it reaches the intestines. The crude enzyme was first purified by ammonium sulfate precipitation method, followed by the dialysis. The bromelain fraction of the dialysis has a specific activity of 294.44 U/mg with a purity level of 15.68 fold compared to the crude enzyme. Coating bromelain using glutaraldehyde crosslinked microsphere showed an efficiency of 87.14 %. The dissolution test results showed the release rate of bromelain in artificial gastric fluid is 9.35 % and in artificial intestinal environment is 79.92 %. The result of this study indicates that chitosan-coated microspheres are good enough as bromelain coatings for slow-release matrices.