IN VITRO STUDY OF BROMELAIN ACTIVITY IN ARTIFICIAL STOMACH JUICE AND BLOOD Poh Shiang Shiew, Yap Li Fang and Fadzilah Adibah Abdul Majid* Faculty of Chemical Engineering, Universiti Teknologi Malaysia (UTM) 3 rd International Conference on Biotechnology for the Wellness Industry 8-9 October 2010, PWTC KL
Overview Introduction Bromelain (general, pharmacological effects) Absorption of bromelain in body (stomach, blood) Objectives Methodology Measurement of clotting time and enzymatic activity. Pineapple juice processing. Analyze bromelain concentration/activity in artificial stomach juice. Analyze bromelain concentration/activity in artificial blood. Results and Discussion Activity of pineapple juice with different bromelain concentration after 4 hours reaction with artificial stomach juice. Bromelain concentration in pineapple juice within 8 hours reaction with artificial stomach juice. Bromelain concentration ti in pineapple juice within 8 hours reaction with artificial i blood. Bromelain concentration and activity before and after 4 hours exposure to artificial stomach juice and blood. Conclusion References
Introduction Bromelain Found in pineapple (Ananas comosus), Bromeliaceae family. Mixture of cysteine proteases and non proteases. Cysteine proteases: stem bromelain, fruit bromelain, ananain, comosain (Rowan et al., 1990). Catalytic activity- sulfhydryl group (SH) at the active site.
Introduction Bromelain Safe, therapeutic drugs for oral systemic treatment of inflammatory, blood-coagulationrelated and malignant diseases (Maurer, 2001). Rodents: LD50 of bromelain is >10g/kg (Kelly, 1996). Human:12g/day of bromelain without major side effects (Castell et al., 1997). Pharmacological actions: Antiedematous, antithrombotic, fibrinolytic (Moss et al, 1963 ; Pirotta and Giuli-Morghen 1978). Anti-inflammation inflammation, promotion of absorption of antibiotic drugs, inhibition of tumor cells growth (Desser et al., 1994; Tinozzi and Venegoni, 1978; Grabowska et al., 1997).
Introduction Absorption of bromelain Pineapple fruit provides health beneficial effects due to presence of bromelain. Is bromelain absorbed following oral application? Rabbits: increase plasmin serum level and prolong prothrombin and antithrombin times (Smyth et al., 1962). 40% of labelled bromelain is absorbed from intestine in high molecular form (Siefert et at., 1979). Bromelain is detected and retains its proteolytically activity in plasma (by immunoassay and proteolysis of model substrates) (Castell et al., 1997).
Introduction Absorption of bromelain
Introduction Absorption of bromelain In blood, bromelain is complexed with antiproteinases (α-2-macroglobulin and α-1-antichymotrypsin) (Castell et al., 1997). Clinical study: Oral administration of 400mg-800mg g tablets, 4 times daily up to 4days. Specific activity of bromelain in plasma is proportionally correlated with the respective dosage (Mai et al., 1996). Enzymes are absorbed from gastrointestinal tract in a functionally intact form. Relatively high doses is well tolerated. (Maurer, 2001)
Introduction Choose bromelain in pineapple juice rather that purified bromelain. Natural form, ease of processing, easy consume Consume sufficient i pineapple juice considerable amount of bromelain absorbed by body beneficial to health. However, bromelain are hydrolyzed by gastric acid and proteinases after oral administration. In this study: How doconditions of body fluid (t (stomach juice and blood) influence the bromelain concentration and activity after oral consumption of pineapple juice containing bromelain?
Objective To investigate the bromelain activity in pineapple juice after being exposed to artificial stomach juice and blood.
Methodology Measurement of clotting time and enzymatic activity Milk clot unit (MCU) assay (Balls et al., 1937) Enzymatic activity is related to time required to clot substrate in milk. Standard curve: clotting time (s) versus standard bromelain concentration (mg/ml) Sample activity it Milk Clotting Unit (U/ml) = Enzyme concentration (mg/ml)/ clotting time (s) Pineapple juice processing Collect cores of Gandul pineapple Juicing, centrifuge Measure clotting time of clarified juice
Analyze bromelain concentration in artificial stomach juice Artificial stomach juice preparation Contain pepsin (proteases), acidic solution (HCI, water). Measure clotting time (negative control). Pineapple juice with different bromelain concentration in artificial stomach juice Dilute pineapple juice into different concentration. Diluted juice + stomach juice, 37 ο C, 4 hours. Measure clotting time. Pineapple juice in artificial stomach juice for 8 hours Pineapple juice + stomach juice, 37 ο C, 8 hours. Measure clotting time every hour interval. Determine bromelain concentration for every hour sample.
Analyze bromelain concentration in artificial blood Artificial blood preparation Fetal bovine serum (FBS) Measure clotting time (negative control). Pineapple juice in artificial blood for 8 hours Pineapple juice + serum, 37 ο C, 8 hours Measure clotting time every hour interval. Determine bromelain concentration for every hour sample.
Result and Discussion Activity of pineapple juice with different bromelain concentration after 4hours reaction with artificial stomach juice 30.0000 25.00 g time (s) Clottin 20.0000 15.00 10.0000 5.00 y = -3.53x 353 + 37928 37.928 R² = 0.9866 000 0.00 0 2 4 6 8 10 12 Bromelain concentration in pinapple juice (mg/ml) Clotting time decreased with increasing bromelain concentration. High amount of bromelain could coagulate casein in milk at faster rate as compared to small concentration.
Result and Discussion Bromelain concentration in pineapple juice within 8 hours reaction with artificial stomach juice Bromelain Concentratio on (mg/ml) 10.0000 9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 y = -1.4278x + 8.9088 R² = 0.9396 y = -0.0227x + 3.7303 R² = 0.3333 0 1 2 3 4 5 6 7 8 Reaction duration (hour) Bromelain concentration decreased gradually from beginning (9.61mg/ml) to fourth hour (3.66mg/ml). Concentration remained constant after 4 hours reaction. 61.91% of bromelain being hydrolyzed in first 4 hours.
Result and Discussion Bromelain concentration in pineapple pp juice within 8hours reaction with artificial stomach juice Reduction in activity (from beginning to 4 th hour) due to inactivation and degradation of bromelain by gastric enzymes (pepsin) p and low ph (acidic phase solution). Remain constant in activity ii (from 4 th to 8 th hour) Bromelain of 3.66mg/ml could survive the reaction against gastric inactivation and became stable in artificial stomach juice condition. May due to pepsin-bromelain reaction in acidic phase solution reached saturation point. B l i f 3 66 / l ld b d bl d Bromelain of 3.66mg/ml could be passed to blood for further reaction.
Result and Discussion Bromelain concentration in pineapple juice within 8 hours reaction with artificial blood Br omelain Co oncentration n (mg/ml) 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 y = -0.306x + 3.642 R² = 0.9985 y = -0.001x + 2.452 R²=0.0313 0 1 2 3 4 5 6 7 8 Reaction duration (hour) Bromelain concentration decreased gradually from beginning (3.66mg/ml) to fourth hour (2.44mg/ml). Concentration remained constant after 4 hours reaction. 13% loss in bromelain concentration in first 4 hours.
Result and Discussion Bromelain concentration in pineapple juice within 8 hours reaction with artificial blood Reduction in activity (from beginning to 4th hour) due to degradation of bromelain by serum globulins or other components in blood. Bromelain of 2.44mg/ml is predicted to be absorbed by body for further reaction.
Result and Discussion Bromelain concentration and activity before and after 4 hours exposure to artificial stomach juice and blood. Condition Bromelain Bromelain % reduction in Residual concentration (mg/ml) (mg) in 250ml pineapple juice bromelain concentration bromelain activity (MCU) Starting 9.61 2402.50 0.00 600.63 After 4 hours exposure to artificial stomach juice After 4 hours exposure to artificial i blood 3.66 915.00 61.91 228.75 2.44 610.00 74.61 152.5
Conclusion Bromelain concentration and activity in artificial stomach juice and blood was determined. Obtain a better understanding of bromelain activity in human body. Bromelain concentration of 3.66mg/ml became stable after 4 hours reaction in artificial stomach juice. Bromelain concentration ti of 2.44mg/ml remained after 4 hours reaction in artificial blood. This concentration of bromelain is predicted to be absorbed by body for further reaction. If 250ml pineapple juice is consumed, the estimated bromelain activity absorbed by body remained 152.5MCU, about 74.61% activity loss as compared to that of original pineapple juice.
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