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1 riginal Article Effect of heat processing of spices on the concentrations of their bioactive principles: Turmeric (Curcuma longa), red pepper (Capsicum annuum) and black pepper (Piper nigrum) D. Suresh, H. Manjunatha, Krishnapura Srinivasan Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore 57 2, India Abstract Studies were made to examine the loss of curcumin, capsaicin and piperine, the active principles of turmeric (Curcuma longa), red pepper (Capsicum annuum) and black pepper (Piper nigrum), respectively, as a result of subjecting the spices to domestic cooking processes. This involved heat treatment of each of these spices by: (i) boiling for 1 min, (ii) boiling for 2 min and (iii) pressure cooking for 1 min. Quantitation of the spice principles in the organic solvent extracts of the freeze-dried cooked spice samples was made with an appropriate HPLC method. Significant loss of spice active principles was observed when the spices were subjected to heat processing. Curcumin loss from heat processing of turmeric was 27 53%, with maximum loss in pressure cooking for 1 min. Curcumin loss from turmeric was similar even in the presence of red gram. In the presence of tamarind, the loss of Curcumin from turmeric was 12 3%. Capsaicin losses from red pepper ranged from 18% to 36%, with maximum loss observed in pressure cooking. Presence of either red gram or tamarind or both did not influence the loss of capsaicin. Piperine losses from black pepper ranged from 16% to 34%, with maximum loss observed in pressure cooking. The loss was somewhat lower in the presence of red gram. The results of this investigation indicated diminished availability of spice active principles from cooked foods when the food ingredients have been subjected to either boiling or pressure cooking for few minutes. Keywords: Capsaicin; Curcumin; Piperine; Loss during heat processing; Spice principles; Turmeric; Curcuma longa; Red pepper; Capsicum annuum; Black pepper; Piper nigrum 1. Introduction Spices are common food adjucts that impart flavour, aroma and colour to foods. Several common spices are now understood to exert many beneficial physiological effects (Srinivasan, 25a). Among these, their hypolipidemic and antioxidant properties have far-reaching health implications. Work in our laboratory on the physiological effects of spices has focused on their influence on lipid metabolism, their action as a digestive stimulant, the beneficial influence of hypocholesterolemic spices on cholesterol gallstone disease and diabetic nephropathy, Corresponding author. Tel.: ; fax: address: ksri@sancharnet.in (K. Srinivasan). and the beneficial influence of antioxidant spice principles on inflammatory disease (Srinivasan, 25b). Cooking or roasting alters the nature of many food constituents such as starches and proteins by changing their physical, chemical and nutritional characteristics (Belitz and Grosch, 1987). Heat processing also changes the bioavailability of proteins, carbohydrates, lipids and vitamins. Little information is available on the extent of destruction of bioactive principles of spices during food processing. Since the healthy, beneficial physiological effects of spices are attributable to their active principles (Srinivasan, 25a), there is a need to evaluate the availability of the spice active principles in their original form when spices are heat processed as in domestic cooking. Significant losses (if any) in the concentration of active principles would raise a question as to whether the

2 spices would retain beneficial health effects after conventional heat processing, as in domestic food preparation. In this context, three common spices, turmeric (Curcuma longa), red pepper (Capsicum annuum) and black pepper (Piper nigrum) were evaluated for the status of their respective active principles curcumin, capsaicin and piperine during domestic food processing with variables present such as those found in normal food ingredients of pulses (Cajanus cajan, or red gram, a protein-rich staple grain) and a food acidulant, tamarind. 2. Materials and methods 2.1. Materials Dry rhizomes of turmeric (Curcuma longa), dry red pepper (Capsicum annuum) and dry seeds of black pepper (Piper nigrum) were locally purchased and powdered to pass through a No. 5 mesh sieve. Tamarind (Tamarindus indica) dry pulp powder, decorticated red gram (Cajanus cajan) and common salt powder were obtained from a local market. Curcumin, capsaicin and piperine used as reference standard were procured from Fluka Chemie, Switzerland. Pepsin, pancreatin and bile extract of porcine origin were obtained from Sigma Chemical Co., USA. All other chemicals used were of analytical grade and the solvents were distiled before use Heat treatment of spice samples The test spices were subjected to heat treatment by (i) boiling for 1 min, (ii) boiling for 2 min and (iii) pressure cooking for 1 min at 15 p.s.i. to simulate what is normally encountered in domestic cooking processes. For each of the spices, the following were the sample variations: (1) spice alone, (2) spice+red gram (decorticated), (3) spice+red gram (decorticated)+tamarind, and (4) spice+tamarind. A precise quantity of the individual spice powder (1 g) was suspended in 1 ml water in parallel sets of 25 ml beakers. Tamarind powder (.5 g) was added in the 3rd and 4th set of samples. Addition of this acidulant brought down the ph by 1. unit. Portions of previously pressure-cooked (1 min) and mashed decorticated red gram equivalent to 5. g dry red gram were also added to the 2nd and 3rd set of samples. In the case of heat treatment by boiling, the contents of the beaker were boiled exactly 1 or 2 min while taking care to maintain the volume around 1 ml by intermittent addition of water. At the end of heat treatment (boiling or pressure cooking), the samples were cooled to room temperature. Appropriate controls were also included in which the samples did not undergo any treatment. Spice samples in the lyophilized food samples (5 g) were extracted with ethyl acetate in a Soxhlet apparatus for 4 h. The extracts were concentrated in a flash evaporator to a known volume (2 ml) and stored in the dark at 2 1C until further analysis. The individual spice principles were quantitated after separation by thin-layer chromatography (TLC) as described below, taking care to minimize the exposure to light during the extraction and TLC separation. Quantitation of the spice principles in the organic solvent extracts of the freeze-dried cooked spice samples was also made by using an appropriate HPLC method Quantitation of spice principles by TLC Aliquots of the ethyl acetate extracts (quadruplicate) of the lyophilized food material and reference curcumin (6 mg) were spotted on silica gel-g coated plates (2 2 cm). The plates were developed with the upper phase of the solvent system: benzene ethanol water acetic acid (1:27.5:7.5:.5 v/v/v/v) in a chamber pre-equilibrated with the above solvent system for 2 h. The yellow curcumin bands were collected, extracted with 4 ml acetone, and centrifuged at 2 rpm for 5 min. Curcumin was quantitated by rubrocurcumin reaction (Ravindranath et al., 1981) by mixing 2 ml of acetonic extract with 1 ml 7.5 mg% boric acid (in acetone) and 1 ml 5 mg% boric acid (in acetone). The contents of the tube were evaporated to dryness over a hot water bath. The residue was redissolved in 2 ml ethanol and absorption of the purple colour was measured at 55 nm using Shimadzu UV-visible Spectrophotometer. Aliquots of the ethyl acetate extracts of the food material were spotted on silica gel-g coated plates (2 2 cm) along with reference piperine (4 mg) and developed with petroleum ether (6 8 1C) acetone (65:35 v/v) in a chamber preequilibrated with the same solvent for 9 min (Bhat and Chandrasekhara, 1985). Thepiperinebandswerescannedin an automatic Camag TLC scanner (Model 2), mounted on a fluorimeter (Model 1, Turner Assoc.) attached to a W-W Recorder (Model III, Scientific Instruments Inc.) The individual lanes were scanned using a primary filter No and a neutral density filter No (1%) as a secondary filter, and lamp No (Emission nm). Piperine bands thus located were scraped and eluted with 2 ml chloroform. After centrifugation the piperine extracts were quantified by absorption measurement at 345 nm. For the determination of capsaicin, aliquots of ethyl acetate extracts (quadruplicate) were spotted on silica gel- G coated plates (2 2 cm) along with reference capsaicin (4 mg) and developed with petroleum ether (6 8 1C) acetone (65:35 v/v) in a chamber pre-equilibrated with the same solvent for 9 min (Srinivasan et al., 1981). The developed plates were air-dried and sprayed with Gibb s reagent (.1% 2,6-dichloroquinone-4-chlorimide in methanol). The plates were exposed to ammonia vapours in a closed chamber for exactly 1 min. The bluecoloured zones of capsaicin thus visualized were collected and extracted with 2 ml water and centrifuged. Absorption of the clear blue supernatants was read at 61 nm Quantitation of spice principles by HPLC Quantitation of spice principles in the extracts cleaned up by passing through 2 mm membrane filter was made by

3 HPLC in a Shimadzu HPLC LC-1AT system consisting of a photodiode array detector, binary pump and manual sample injector. Chromatographic separation was accomplished using SGE mm SSExcil C18 1-mm column. Curcumin was analysed by isocratic elution at 425 nm. Sample (1 ml) was injected on to a reverse-phase column and eluted with a mobile phase containing acetonitrile water acetic acid (5:49:1 v/v/v) at a flow rate of 1 ml/min (Asai and Miyazawa, 2). Quantitation of curcumin was made from peak area ratio, which was based on a calibration curve generated from standard curcumin. Piperine analysis was performed on reverse-phase column with an isocratic elution at 345 nm (Joe, 1996). A quantity of 1 ml of piperine sample was eluted with a mobile phase containing a mixture of acetonitrile water (5:5 v/v) at a flow rate of 1. ml/min. Quantitation of piperine was made from peak area ratio, which was based on a calibration curve generated from standard piperine. Capsaicin in the samples was separated and quantified by isocratic HPLC method using UV detection at a wavelength of 28 nm (Joe, 1996). An aliquot (1 ml) was injected onto a reversephase column and eluted with a mobile phase containing a mixture of methanol water (6:4 v/v) at a flow rate of 1. ml/min. Quantitation of capsaicin was made from peak area ratio, which was based on a calibration curve generated from standard capsaicin. HPLC elution profile of the three spice principles thus quantitated is given in Fig Simulated gastrointestinal digestion Since there was a general under-estimation of spice principles in samples containing red gram, a supplementary study was carried out to digest the protein matrix of red gram (Cajanus cajan) by a simulated gastrointestinal digestion procedure before extracting the spice principle (Miller et al., 1981) to verify if this would enhance the extractability of spice active principles. The specific samples containing the red gram were homogenized in additional amount of water; ph was adjusted to 2. with 6 M HCl. Fresh pepsin solution (1.6 g pepsin suspended in 1 ml.1 M HCl) was added (3 ml) and subjected to gastric digestion by incubation at 37 1C for 2 h with shaking. Intestinal digestion was simulated by incubating for 3 h with shaking after raising the ph to 7. with 1 M sodium hydroxide and addition of 5 ml freshly prepared pancreatin bile mixture (4 g pancreatin+25 g bile extract in 1 l.1 M sodium bicarbonate). Samples after simulated digestion as above were lyophilized and used for extraction of spice active principles. 3. Results and discussion In this investigation carried out to determine the extent to which the active principles of three spices curcumin, capsaicin and piperine survive the domestic cooking treatments, the spice ingredients were subjected to either mabs mabs mabs min Curcumin min Capsaicin min Piperine Fig. 1. HPLC separation profile of active principles of spices. boiling in water for 1 2 min or to pressure-cooking for 1 min along with other food ingredients. ph variation was brought about by the presence or absence of tamarind, which is a common acidulant in Indian homes. Inclusion at.5% level of tamarind brought down the ph of these samples by one unit from 6.2 to 5.2. Significant loss of spice active principles as quantitated by HPLC procedure was observed when these three commonly used spices were subjected to heat processing such as that found in domestic cooking (Tables 1 3). Curcumin loss from heat processing of turmeric was 27 53%, with maximum loss in pressure cooking for 1 min (Table 1). Curcumin loss from turmeric was similar even in the presence of red gram (42 5%) or red gram+tamarind (26 57%). In the presence of tamarind, the loss of curcumin from turmeric was comparatively lower (14 34%). Capsaicin losses from red pepper were in the range 18 36% with maximum loss being observed in pressure cooking (Table 2). Unlike for curcumin, the presence of the acidulant did not lower the loss of capsaicin during heat treatment. Piperine losses from black pepper were in the range 27 34% with maximum loss being observed in pressure cooking (Table 3). The loss was

4 Table 1 Concentration of spice principle (curcumin) in heat-processed turmeric Sample Curcumin concentration (mg/g spice) Raw (untreated) Heat treatment by boiling Pressure cooking (1 min) (2 min) (1 min) Turmeric (27.) (32.) (53.) Turmeric+red gram (42.3) (42.3) (5.2) Turmeric+red gram+tamarind (25.9) (37.) (57.2) Turmeric+tamarind (14.7) (21.7) (34.3) Values are mean7sem of 5 independent determinations. Figures in parenthesis denote % loss. Table 2 Concentration of spice principle (capsaicin) in heat-processed red pepper Sample Capsaicin concentration (mg/g spice) Raw (untreated) Heat treatment by boiling Pressure cooking (1 min) (2 min) (1 min) Red pepper (18.3) (21.9) (36.2) Red pepper+red gram (29.9) (34.9) (3.7) Red pepper+red gram+tamarind (29.) (29.) (28.8) Red pepper+tamarind (24.6) (24.2) (32.8) Values are mean7sem of 5 independent determinations. Figures in parenthesis denote % loss. Table 3 Concentration of spice principle (piperine) in heat-processed black pepper Sample Piperine concentration (mg/g spice) Raw (untreated) Heat treatment by boiling Pressure cooking (1 min) (2 min) (1 min) Black pepper (27.) (27.9) (33.9) Black pepper+red gram (16.) (17.) (2.) Black pepper+red gram+tamarind (26.4) (27.) (3.9) Black pepper+tamarind (18.) (21.) (32.7) Values are mean7sem of 5 independent determinations. Figures in parenthesis denote % loss. somewhat lower in the presence of red gram (16 2%) or tamarind (18 33%). The pattern of changes in the concentration of curcumin, capsaicin and piperine upon heat processing of the parent spices was found to be similar when these compounds were quantitated by appropriate TLC procedures (data not shown), although there were minor differences in the absolute values of spice principles between TLC procedure and HPLC procedure. Thus, loss of concentration of curcumin during domestic cooking procedure is maximum among the three spice principles, which have a somewhat similar structure with a common basic skeleton but different side chains (Fig. 2). A reduction of ph even by one unit produced a considerable reduction in the loss of this active principle. It has been reported that curcumin in aqueous media is highly stable at ph below 7. at ambient temperature, while at ph47., curcumin is extremely unstable even at ambient temperature (Tonnesen and Karlsen, 1985a). Alkaline degradation of curcumin has been reported to give ferulic acid and feruloyl methane, and that the feruloyl methane part of curcumin rapidly forms condensation products which are yellow to brownish yellow in colour (Tonnesen and Karlsen, 1985b). The degradation kinetics of curcumin under various ph conditions and the stability of curcumin in physiological matrices have been recently investigated (Wang et al., 1997). When curcumin was incubated in.1 M phosphate buffer and serum-free medium, ph 7.2 at 37 1C, about 9% decomposed within 3 min. A series of ph conditions ranging from 3 to 1 were tested and the result showed that decomposition was ph-dependent and

5 H 3 C H CH 3 H H Curcumin (Turmeric) H 3 C H NH CH 3 CH 3 Capsaicin (Red pepper) H 2 C N Piperine (Black pepper) Fig. 2. Structure of active principles of spices. occurred faster at neutral-basic conditions. It is more stable in cell culture medium containing 1% foetal calf serum and in human blood; less than 2% of curcumin decomposed within 1 h, and after incubation for 8 h, about 5% of curcumin still remained. Trans-6-(4 -hydroxy-3 - methoxyphenyl)-2,4-dioxo-5-hexenal was predicted as major degradation product and vanillin, ferulic acid, feruloyl methane were identified as minor degradation products. There is no information about the stability of curcumin in aqueous solutions at higher temperatures, except for our earlier report (Srinivasan et al., 1992). We have also observed a shift in the absorption maxima from 28 to 293 nm of capsaicin solution when the ph is changed from neutral to alkaline, the shift in absorption maxima could be attributable to deprotonation of hydroxyl group of capsacin in alkaline ph (our unpublished data). While we had earlier observed losses of active principles of turmeric, red pepper and black pepper based on TLC (Srinivasan et al., 1992), the present investigation has been an unambiguous study in that improved methodology, namely HPLC procedures have been employed for the quantitation of spice principles curcumin, capsaicin and piperine. Further both the presence and absence of red gram, a common pulse ingredient in Indian curry dishes has been examined in this context. The loss of curcumin which we have observed in the present study is contrary to the general belief that curcumin may be stable in aqueous solutions at ph either neutral or acidic. Generally, there was an under-estimation of all the three spice principles in the case of samples containing red gram. This could be due to reduced extractability of the spice principle due to high affinity binding to proteins present in Spice active principle (mg/g) Curcumin Capsaicin Piperine Spice Principle Before simulated gastro-intestinal digestion After simulated gastro-intestinal digestion Fig. 3. Improved extractability of spice principles in presence of red gram after simulated gastro-intestinal digestion. the pulse. Such a possibility was verified in a separate experiment by subjecting the food samples containing red gram to simulated gastrointestinal digestion following the heat treatment procedure and then extracting the respective spice principle with the organic solvent. As shown in Fig. 3, higher concentrations of each of these three spice principles was evidenced in samples subjected to simulated gastrointestinal digestion, which would have digested the protein present in the sample, thus facilitating extractability of the spice principle. The extractability of piperine was improved by 2.5-fold after simulated gastrointestinal digestion.

6 4. Conclusions The results of this investigation indicated diminished availability of spice active principles in cooked foods, when the food ingredients have been subjected to either boiling or pressure cooking for few minutes. Three common spices turmeric, red pepper and black pepper are considered to be nutraceuticals because of the multi-beneficial physiological effects they offer, attributable to their active principles (Srinivasan, 25a). In view of the significant extent of chemical alteration occurring to the spice active principles of turmeric and red pepper, it is desirable to determine the extent to which turmeric and red pepper retain their hypolipidemic and antioxidant potency upon thermal processing as in domestic cooking. Identification of the degradation products and/or altered products of spice principles of curcumin, capsaicin and piperine, arising out of heat processing of the respective parent spices, is in progress. References Asai, A., Miyazawa, T., 2. ccurrence of orally administered curcuminoid as glucuronide and glucuronide/sulfate conjugate in rat plasma. Life Science 67, Belitz, H.D., Grosch, W., Food Chemistry. Springer, Berlin. Bhat, B.G., Chandrasekhara, N., Determination of piperine in biological tissues by thin layer chromatography and ultraviolet absorption densitometry. Journal of Chromatography 338, Joe, B., Studies on the role of dietary lipids and antioxidants on the inflammatory mediators of macrophages. Ph.D. Thesis, University of Mysore. Miller, D.D., Schricker, B.R., Rasmussen, R.R., Vancanpen, D.R., An in vitro method for the estimation of iron availability from meals. American Journal of Clinical Nutrition 31, Ravindranath, V., Satyanarayana, M.N., Rao, M.V.L., Rubrocurcumin reaction and its use in micro-determination of certain organic acids. Indian Journal of Chemistry 2, Srinivasan, K., 25a. Role of spices beyond food flavouring: Nutraceuticals with multiple health effects. Food Reviews International 21, Srinivasan, K., 25b. Spices as influencers of body metabolism: an overview of three decades of research. Food Research International 38, Srinivasan, M.R., Satyanarayana, M.N., Rao, M.V.L., A thin layer chromatographic method for the estimation of capsaicin and related compounds. Journal of Research and Industry 26, Srinivasan, K., Sambaiah, K., Chandrasekhara, N., Loss of active principles of common spices during domestic cooking. Food Chemistry 43, Tonnesen, H.H., Karlsen, J., 1985a. Studies on curcumin and curcuminoids VI: kinetics of curcumin degradation in aqueous solution. Zeitshrung fur Lebensmittel und Unters Forschung 18, Tonnesen, H.H., Karlsen, J., 1985b. Studies on curcumin and curcuminoids V: alkaline degradation of curcumin. Zeitshrung fur Lebensmittel und Unters Forschung 18, Wang, Y.J., Pan, M.H., Cheng, A.L., Lin, L.I., Ho, Y.S., Hsieh, C.Y., Lin, J.K., Stability of curcumin in buffer solutions and characterization of its degradation products. Journal of Pharmacology and Biomedical Analysis 15,