Poh Shiang Shiew, Yap Li Fang and Fadzilah Adibah Abdul Majid* Faculty of Chemical Engineering, Universiti Teknologi Malaysia (UTM)

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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

1 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

2 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

3 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.

4 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).

5 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).

6 Introduction Absorption of bromelain

7 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)

8 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?

9 Objective To investigate the bromelain activity in pineapple juice after being exposed to artificial stomach juice and blood.

10 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

11 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.

12 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.

13 Result and Discussion Activity of pineapple juice with different bromelain concentration after 4hours reaction with artificial stomach juice g time (s) Clottin y = -3.53x R² = 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.

14 Result and Discussion Bromelain concentration in pineapple juice within 8 hours reaction with artificial stomach juice Bromelain Concentratio on (mg/ml) y = x R² = y = x R² = 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 % of bromelain being hydrolyzed in first 4 hours.

15 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.

16 Result and Discussion Bromelain concentration in pineapple juice within 8 hours reaction with artificial blood Br omelain Co oncentration n (mg/ml) y = x R² = y = x R²= 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.

17 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.

18 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 After 4 hours exposure to artificial stomach juice After 4 hours exposure to artificial i blood

19 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.

20 References Rowan, A.D., Buttle, D.J. and Barrett, A.J. The cysteine proteinases of the pineapple plant. Biochem. J. 1990; 266: Maurer, H.R. Review: Bromelain: biochemistry, pharmacology and medical use. CMLS, Cell. Mol. Life Sci. 2001; 58: Kelly, G.S. Bromelain: A literature review and dermatological discussion of its therapeutic applications. Altern. Med. Rev. 1996; 1: Castell, J.V., Friedrich, G., Kuhn, C.S. and Poppe, G.E. Intestinal absorption of undegraded proteins in men: presence of bromelain in plasma after oral intake. Am. J. Physiol. 1997; 273: Moss, I.N., Frazier, C.V. and Martin, G.J. Bromelains-the pharmacology of the enzymes. Arch. Int. Pharmacodyn. 1963; 145: Pirotta, F. and de Giuli-Morghen, C. Bromelain: antiinflammatory and serum fibrinolytic activity after oral administration in the rat. Drug Exp. Clin. Res. 1978; 4:1-20. Desser, L., Rehberger, A., Paukovits, W. Proteolytic enzymes and amylase in human peripheral blood mononuclear cells in vitro. Cancer Biother. 1994; 9: Tinozzi, S. and Venegoni, A. Effect of bromelain on serum and tissue levels of amoxycillin. Drug Exp. Clin. Res. 1978; 1: Grabowska, E., Eckert, K., Fichtner, I., Schulze-Forster, K. and Maurer, H.R. Bromelain proteases suppress growth, invasion and lung metastasis of B16F10 mouse melanoma cells. Int. J. Oncol. 1997; 11: Smyth, RD R.D., Brennan, R., and Martin, G.J. GJ Systemic biochemical i changes following the oral administration of a proteolytic enzyme, bromelain. Arch. Int. Pharmacodyn. 1962; 136: Mai, I., Donath, F., Maurer, A., Bauer, S. and Roots, I. Oral bioavailability of bromelain and trypsin after repeated oral administration of a commercial polyenzyme preparation. Eur. J. Clin. Pharmacol. 1997; 50:548. Balls, A.K. and Hoover, S.R. The milk-clotting action of papain. J. Biol. Chem. 1937; 121:

European public MRL assessment report (EPMAR)

26 January 2018 EMA/CVMP/208391/2017 Committee for Medicinal Products for Veterinary Use European public MRL assessment report (EPMAR) Bromelain (porcine species) On 14 September 2017 the European Commission