Tissue distribution & elimination of capsaicin, piperine & curcumin following oral intake in rats

Indian J Med Res 131, May 2010, pp 682-691

682

Several of the common spices consumed as food adjuncts to impart flavour, aroma and colour to foods are also documented to exhibit several health beneficial physiological effects1. The beneficial physiological effects of the three common spices – turmeric (Curcuma

Tissue distribution & elimination of capsaicin, piperine & curcumin following oral intake in rats

D. Suresh & K. Srinivasan

Department of Biochemistry & Nutrition, Central Food Technological Research Institute (CSIR) Mysore, India

Received July 17, 2008

Background & objectives: Curcumin, capsaicin and piperine - the bioactive compounds present in spices-turmeric (Curcuma longa), red pepper (Capsicum annuum) and black pepper (Piper nigrum) respectively, have a considerable portion of structural homology. Tissue distribution and elimination of these three structurally similar bioactive compounds was examined following their oral intake in rats. Methods: Separate sets of animals (150 - 160 g) were orally administered the three spice principles at dosages of 30 mg (capsaicin), 170 mg (piperine) and 500 mg (curcumin) / kg body weight. The tissue concentrations of administered spice compounds were determined by HPLC.Results: Maximum distribution of 24.4 per cent of administered capsaicin was seen at 1 h, while no intact capsaicin was detectable after 4 days. Absorption of capsaicin was about 94 per cent and very rapid relative to other two compounds. A maximum of 10.8 per cent of administered piperine was seen in tissues at 6 h. Absorption of the administered piperine was about 96 per cent. Curcumin concentration was maximum in the intestine at 1 h; maximum in blood at 6 h and remained at significantly higher level even at 24 h. About 63.5 per cent of the curcumin dose was absorbed. Only a small portion of the administered dose of capsaicin (< 0.1%) and curcumin (0.173 %) was excreted in urine, whereas piperine was not detectable in urine. Enhanced bioavailability of curcumin was evidenced when the same was orally administered concomitant with piperine. Intestinal absorption of curcumin was relatively higher when administered concomitantly with piperine, and it stayed significantly longer in the body tissues. Intact curcumin was detected in brain at 24, 48 and 96 h with a maximum at 48 h. Conclusions: Considerable difference exists in the bioavailability of the three test compounds. Curcumin could be traced in the brain following its administration. Bioavailability of curcumin can be improved by co-administration with piperine.

Key words Bioavailability - capsaicin - curcumin - elimination - piperine - tissue distribution

longa), red pepper (Capsicum annuum) and black pepper (Piper nigrum) are incidentally attributable to their respective active principles - curcumin (yellow colouring compound of turmeric), capsaicin (pungent principle of red pepper) and piperine (the biting principle

of black pepper)1. These three spice compounds share a considerable amount of structural homology (Fig.1). Curcumin (Diferuloyl methane) contains two ferulic acid moieties joined by a methylene bridge. Capsaicin is 8-methyl-N-vanillyl-6-nonanamide. Piperine is the trans-trans isomer of 1-piperoyl piperidine.

Curcumin shows antioxidant, anti-inflammatory, anti-carcinogenic1 effective hypolipidemic agent, antilithogenic property2 and is an antidiabetic food adjunct3. Capsaicin is endowed with health attributes hypolipidemic, antilithogenic2, antioxidant4 and anti-inflammatory5,6 as also a pain reliever7,8. Piperine has been demonstrated to lower lipid peroxidation in vivo and beneficially influence antioxidant status9. The most far-reaching attribute of piperine has been its inhibitory influence on enzymatic drug biotransforming reactions in liver. Piperine enhances the bioavailability of a number of therapeutic drugs as well as phytochemicals9. Dietary capsaicin and piperine are known to enhance digestive capacity10.

In view of the diverse health beneficial potential of these three spice principles, it is most relevant to understand the bioavailability, tissue distribution profile, and the rate of elimination following their oral administration. Curcumin is known to possess low systemic bioavailability11, attributed to a generally poor absorption and faster metabolic alterations11,12. Information on the rate of intestinal absorption of capsaicin is limited. Considering the frequent

consumption of capsaicin as a food additive and its current medicinal use, assessment of absorption and tissue distribution of this compound is important. Mammalian metabolism of capsaicin has been also reported. Capsaicin appears to interact with xenobiotic metabolizing enzymes, particularly microsomal cytochrome P450-dependent monooxygenases which are involved in activation as well as detoxification of various chemical carcinogens and mutagens13. In vivo and in vitro metabolism of dihydrocapsaicin in rats has been reported by Kawada and Iwai14.

Recently, we have observed dissimilarities in the in vitro absorption of curcumin, capsaicin and piperine in everted rat intestines15. These three compounds of spices share a considerable similarity. Of the three spices active capsaicin is absorbed to least extent in everted rat intestines. The absolute amounts of piperine absorbed in this in vitro system exceeded the amounts of curcumin. Only a minimum portion of the absorbed curcumin was traced in serosal fluid at the end of 3 h incubation period, while most of it was still present in intestinal tissue. The relatively lesser recovery of curcumin in its native form suggested transformation of this compound in the intestine to a certain extent during its absorption. In the current investigation, studies the in vivo tissue distribution and subsequent elimination following oral administration of curcumin, capsaicin and piperine to rats are presented. Tissue distribution and elimination of curcumin orally administered concomitant with piperine was also examined.

Material & Methods

Chemicals and reagents: The three spice active principles - curcumin, capsaicin and piperine were procured from M/s Fluka Chemie, Switzerland. All other chemicals used were of analytical grade and the solvents were distilled before use.

Animal treatment: Experiments on animals were conducted with due approval of the procedures by the Institute’s Animal Ethics Committee with regard to the care and use of animals. In vivo tissue distribution and excretion studies with spice principles – curcumin, capsaicin and piperine were carried out in three separate experiments, on different sets (n = 42 per set) of Wistar male albino rats (120 - 125 g). The animals were maintained for 10 days on 18 per cent casein containing semi-synthetic diet with free access to food and water. They were housed individually in stainless steel metabolism cages, which permitted collection of urine and faeces. The animals weighed 150 - 160 g Fig. Structure of active principles of spices.

Piperine (Black pepper)

Capsaicin (Red pepper)

Curcumin (Turmeric)

SURESH & SRINIVASAN: PHARMACOKINETICS OF SPICE BIOACTIVE COMPOUNDS 683

on the day of administration of spice principles. The oral dosages (mg/kg body weight) of the three spice principles were: Capsaicin: 30 mg; Piperine: 170 mg and Curcumin: 500 mg. The doses of the three spice compounds selected here correspond to 5-times the average daily intake of the respective parent spices among Indian population16. These spice principles were administered by gavage to overnight fasted animals in the form of a suspension in 1.0 ml refined peanut oil. Six animals were sacrificed under ether anaesthesia at each of the time intervals 1 h, 3 h, 6 h, 1 d, 2 d, 4 d and 8 d following the oral administration of the spice compound. Blood was collected in plain tubes by heart puncture and serum was separated by centrifugation at 40C after 30 min. Liver, kidney and intestine were quickly excised. 24 h urine and faeces were collected for 8 days following the single oral administration of the test compound, in the set of animals which were retained for 8 days.

In a separate experiment, curcumin was orally administered (500 mg/kg body weight) concomitant with piperine (20 mg/kg body weight). Urine and faeces were collected. Groups of animals (6 per group) were sacrificed at time intervals: 3 h, 1 d, 2 d, 4 d and 8 d. Blood was collected by heart puncture and serum was separated by centrifugation at 40C after 30 min. Liver, kidney and brain were quickly excised.

All the tissues were rinsed immediately with ice-cold saline; intestines were flushed with ice-cold saline; tissues were stored at -20°C until extraction and analysis. Faeces samples were dried at 80ºC, and then powdered. Urine samples were concentrated to a convenient smaller volume by flash evaporation. Serum and urine samples were extracted for the spice principle with ethyl acetate by vortexing thoroughly and centrifuging; the supernatant was decanted and the extraction repeated thrice. Various other tissue samples and faeces samples were extracted for the spice principle by Folch procedure with chloroform-methanol (2:1 v/v)17. The extracts were analyzed for the concerned spice principle by an appropriate HPLC procedure described below. Tissues from rats not dosed with any spice principle served as controls. Control experiments were performed to assess the recovery of the spice active compounds and this involved spiking of tissue samples from untreated animals with appropriate spice principles. The recovery was > 90 - 95 per cent. Total blood volume (ml) in the rat was calculated using the formula: (Body weight in g X 0.0778 = Blood volume).

Quantitation of spice principles by HPLC : Quantitation of spice principles in the extracts cleaned up by passing through 0.22µ membrane filter was made by HPLC in a Shimadzu HPLC LC-10AT system using SGE 250 X 4.6 mm SS Excil C18 10-µm column. Assay performance and reproducibility were confirmed by using internal standards. Quantitation of spice compounds was made from peak area ratio, based on a calibration curve generated from the respective standard compound15.

Curcumin was analyzed by isocratic elution with a mobile phase containing acetonitrile-water-acetic acid (50:49:1 v/v/v) at a flow rate of 1 ml/min and monitoring at 425 nm after injecting 10 µl sample on to the reverse phase column18. Piperine analysis was performed on the reverse phase column with an isocratic elution with a mobile phase containing a mixture of 25 mM potassium dihydrogen phosphate (pH 4.5) and acetonitrile (35: 65 v/v) at a flow rate of 1 ml/min and monitoring at 345 nm19. Capsaicin in the samples was separated and quantified by isocratic elution with a mobile phase containing a mixture of methanol-water (60:40 v/v) at a flow rate of 1.0 ml/min and UV detection at 280 nm20.

Linearity was established by injecting the samples in triplicate, containing the test compound (curcumin, capsaicin and piperine) in the concentration range of 5–1000 ng/ml. Limits of detection and quantification were determined by calculation of signal-to-noise ratio. Signal-to-noise ratio of approximately 3:1 and 10:1 were used for estimating the detection and quantification limit, respectively. The lowest limit of detection was 5 ng/ml in the case of capsaicin, 10 ng/ml in the case of piperine and 5 ng/ml for curcumin. Intra-day precision was established by making six injections of lowest, middle and highest concentration in the above range in each case. The residual standard deviation (RSD) values for intra- and inter-day precision were <1.02 and <0.74 per cent, respectively, thereby indicating that the method was sufficiently precise. A similar qualitative separation of these compounds was obtained by analysis on a different chromatographic system on a different day, indicating that the method has good precision. These studies were also repeated on different days to determine inter-day precision. Accuracy was evaluated by spiking the samples with three known concentrations of the compound. Percentage recovery was calculated from the differences between the peak areas obtained for spiked and unspiked sample injections. Recoveries were made at each added concentration. The percentage recovery were between 88.3 and 92.6 per cent with <1.75 per cent RSD.

684 INDIAN J MED RES, MAy 2010

Pharmacokinetic parameters- Area under the curve (AUC) and half life (t½) for the administered spice compounds were calculated using standard formulae. Results are expressed as mean ± SEM and comparisons between groups were made by means of an unpaired Student’s t-test21. Differences were considered significant when P < 0.05.

Results & Discussion

The oral dosages of the three spice principles corresponded to about 5-times the concentrations normally encountered by our population16, are in fact the dosages previously well documented in our laboratory to effectively bring about several beneficial physiological effects22.

Tissue distribution and excretion of orally administered capsaicin: The distribution of capsaicin in the various tissues namely, blood, liver, kidney and intestine is given in Table I. Peak concentrations were observed in different tissues at different times following the single oral administration. Blood and intestine showed a highest concentration at 1 h, liver at 3 h, and kidney at 6 h. A total concentration of 1.24-24.4 per cent of the administered capsaicin was detected in blood, liver, kidney and intestine in 1-24 h. Whereas a maximum distribution of 24.4 per cent of administered capsaicin in these tissues was seen at 1 h, the same gradually reduced to 1.24 per cent in 24 h and to 0.057 per cent in 48 h. No capsaicin was detectable in these tissues beyond 96 h. Highest concentration of administered capsaicin in serum (1.90 µg/ml) was seen at 1 h, which decreased to less than half (0.83 µg/ml) in 6 h and further to about 2.5 per cent (0.05 µg/ml) by 24 h. A small amount of capsaicin was still seen in blood at 48 h (0.006 µg/ml serum) but not thereafter. Highest concentration of capsaicin was seen in liver at 3 h; the decline in concentration of capsaicin in liver was fast

thereafter. Its concentration in liver which was about 45 µg at 3 h was reduced to about 8.7 µg in 24 h. Absorption of orally administered capsaicin appears to be very rapid relative to the other two spice principles as indicated by its highest concentration in intestinal tissue at 1 h. Capsaicin content of intestine decreased from 1057 µg in 1 h to 249 µg in 6 h and further to 43.5 µg by 24 h. No capsaicin was detectable in kidney beyond 2 day and in intestine or liver beyond 4 day.

About 6.3 per cent of the administered capsaicin was excreted as such in the faeces over a period of 4 days, with the peak excretion occurring on the first day oral intake (Table II). Thus, nearly 94 per cent of orally administered capsaicin is absorbed. Only a small portion of capsaicin was also excreted intact in urine (0.095%). Unchanged dihydrocapsaicin (8.7% of the dose administered) and eight of its metabolites have been reported in the urine within 48 h of its oral administration (20 mg/kg) in rats by Kawada and Iwai23. Part of the unchanged dihydrocapsaicin (10% of total dose) was also excreted in the feces within 48 h. Thus, our current observation on the excretion of unchanged capsaicin in faeces and urine is in agreement with that reported by Kawada and Iwai23.

Kawada et al24 have reported from an in vivo study in rats that gastrointestinal absorption of capsaicin is rapid and about 85 per cent of the dose was absorbed within 3 h. And that the absorbed capsaicin is readily transported to the portal blood (about 85%) and partly metabolized during absorption to 8-methyl nonanoic acid. In anaesthetized rats intragastrically administered capsaicin was readily absorbed from the gastrointestinal tract; unchanged compound was present in portal blood, but was almost completely metabolized before reaching the general circulation after an effective metabolism in liver25. The metabolism of capsaicin

Table I. Tissue distribution of orally administered capsaicin in ratTime (h)

Serum(µg/ml)

Blood(µg/ total blood)

Liver(µg/ whole tissue)

Kidney(µg/whole tissue)

Intestine (µg/whole tissue)

1 1.90 ± 0.18 11.11 ± 1.05 24.7 ± 2.1 3.61 ± 0.32 1057.0 ± 157.03 1.47 ± 0.09 8.59 ± 0.53 44.7 ± 3.37 5.71 ± 0.33 700.2 ± 42.26 0.83 ± 0.10 4.85 ± 0.59 14.8 ± 1.50 6.73 ± 0.45 249.3 ± 24.024 0.05 ± 0.01 0.29 ± 0.06 8.71 ± 2.55 3.35 ± 0.45 43.5 ± 3.7548 0.006 ± 0.001 0.035 ± 0.006 0.60 ± 0.03 0.48 ± 0.09 1.14 ± 0.2196 0.00 0.00 0.045 ± 0.005 0.00 0.72 ± 0.01192 0.00 0.00 0.00 0.00 0.00

Values are mean ± SEM of 6 ratsCapsaicin was orally administered to rats at a dose of 30 mg/kg body weight


has been found to be similar in human, rat, and dog microsomes26. In these assays, three major metabolites were detected and identified as 16-hydroxycapsaicin, 17-hydroxycapsaicin, and 16,17-dehydrocapsaicin26. In addition to these three metabolites, rat microsomes also produced vanillylamine and vanillin. Biotransformation of capsaicin was slow in human skin in vitro, with the majority of the applied capsaicin remaining unchanged and a small fraction being metabolized to vanillylamine and vanillic acid. These data suggest that the metabolism of capsaicin by cytochrome P450 enzymes in skin is minimal, relative to hepatic metabolism.

Tissue distribution and excretion of orally administered piperine: The distribution of piperine in the various tissues namely, blood, liver, kidney and intestine is given in Table III. Upon oral administration of piperine to rats at a dose of 170 mg/kg by gavage, peak concentration of the same was observed in blood, intestine, liver, and kidney at 6 h. 3.1 – 10.8 per cent of the administered piperine was detected in blood,

liver, kidney and intestine from 1 - 24 h. A maximum of 10.8 per cent of administered piperine was seen in these tissues by 6 h after administration. The amount of piperine in serum which was 6.07, 9.75, and 11.1 µg/ml at 1, 3 and 6 h respectively, declined drastically to 0.93 µg/ml at the end of 24 h and was nil in the blood after 4 d. In rats administered piperine only 0.15 - 0.39 per cent could be detected in the liver during first 24 h, with a maximum of 0.39 per cent present at 6 h. The concentration of piperine in kidney was also maximum at 6 h (0.37%). Piperine concentration in the intestinal tissue gradually increased from 1.45 mg to a maximum of 2.45 mg by 6 h, and declined thereafter. The amount of piperine present in these tissues significantly reduced by 48 h (0.30%) and was not detectable beyond 96 h in blood, liver and intestine. The elimination pattern of piperine is presented in Table IV. Piperine was not detectable in urine at any time interval. On the other hand, 3.64 per cent of the administered piperine was excreted as such in the faeces over a period of 4 days, with the peak excretion occurring on the first day of oral intake. Thus, absorption of the administered piperine was about 96 per cent.

The tissue distribution of orally administered piperine in rats showed a similar maximum concentration of piperine at 6 h in blood, liver, kidney and spleen27. They however did not detect piperine in any of the tissue samples beyond 24 h, nor did they detect piperine in blood samples of 1 h and 24 h. Our data differs in that piperine could be detected in blood and other tissues even up to 4 days. It should be noted that the previous study had employed TLC - densitometry for the detection and estimation of piperine in tissue samples, while HPLC procedure was used in the present study.

Potential of piperine to increase the bioavailability of drugs in humans is of great clinical significance

Table II. Elimination of orally administered capsaicin in rat Day Faeces Urine 1 174.0 ± 11.3 4.05 ± 0.452 99.8 ± 5.03 0.225 ± 0.0353 11.3 ± 1.25 04 0.375 ± 0.032 05 0 06 0 07 0 08 0 0Total 285.5

(6.34% of administered dose)

4.275(0.095% of administered

dose)

Values expressed in µg are mean ± SEM of 6 rats.Capsaicin was orally administered to rats at the dose of 30mg/kg body weight

Table III. Tissue distribution of orally administered piperine in ratTime (h) Serum

(µg/ml)Blood

(µg/total blood)Liver

(µg/whole tissue)Kidney

(µg/whole tissue)Intestine

(µg/whole tissue)1 6.07 ± 0.82 35.53 ± 4.82 56.12 ± 1.01 12.51 ± 1.95 1450.0 ± 137.23 9.75 ± 1.07 57.03 ± 6.28 73.75 ± 2.05 22.0 ± 3.53 1725.2 ± 150.26 11.06 ± 0.80 64.71± 4.68 96.62 ± 5.71 91.75 ± 6.51 2450.3 ± 162.024 0.93 ± 0.16 5.48 ± 0.73 37.87 ± 3.81 28.75 ± 3.35 700.1 ± 112.548 0.37 ± 0.05 2.19 ± 0.36 4.77 ± 0.88 4.62 ± 0.47 62.3 ± 1.0896 0.078 ± 0.005 0.46 ± 0.03 0.33 ± 0.04 0.66 ± 0.13 4.47 ± 0.88192 0 0 0 0.63 ± 0.11 0

Values are mean ± SEM of 6 rats.Piperine was orally administered to rats at a dose of 170 mg/kg body weight


owing to its omnipresence in food. Differences in its metabolism in rats and humans have been reported. Increased excretion of conjugated glucuronides, sulfates and phenols in the urine for up to 8 days following oral administration of piperine in rats has been earlier evidenced which are presumably the transformed products of piperine27. The authors have suggested that piperine undergoes demethylenation of methylenedioxy group; glucuronidation and sulfation appear to be the major reactions in the metabolism of piperine in rats. They did not observe excretion of unmodified piperine in the urine of rats suggesting that most of the administered piperine is absorbed without any transformation during absorption by the intestine, and is later metabolized rapidly by other tissues. A new major urinary metabolite detected in rat urine and plasma has been characterized as 5-(3,4-methylenedioxy phenyl)-2E,4E-pentadienoic acid-N-(3-yl propionic acid)-amide28. The metabolite has a unique structure compared to the previously reported metabolites in that it retains methylenedioxy ring and

conjugated double bonds while the piperidine ring is modified to form propionic acid group. They suggest28 that urine is the major excretion route for piperine metabolites in rats as no metabolite could be detected in faeces.

Tissue distribution and excretion of orally administered curcumin: The distribution of curcumin in the various tissues namely, blood, liver, kidney and intestine is given in Table V. Upon oral administration of curcumin to rats at a dose of 500 mg/kg, peak concentration of the same was observed in the intestine at 1 h, while in blood, liver and kidney the peak concentrations were observed at 6 h. 3.88 – 48.3 per cent of the administered curcumin was detected in blood, liver, kidney and intestine from 1 - 24 h. Curcumin concentration was maximum in the intestine at the end of 1st h; the concentration of curcumin in the intestinal tissue gradually decreased from 36.2 mg at 1 h to 2.21 mg by 24 h. No curcumin was detectable in the intestine after 4 days. Concentration of curcumin in blood reached maximum (83.8 µg/ml) at 6th h and continued to remain at significantly higher level (52.6 µg/ml) even at 24 h. Curcumin concentration was maximum in liver and kidney at 6 h. Curcumin was not detectable in kidney after 24 h, but was present in liver tissue even up to 4 days.

After orally administration of curcumin in rats, could not detect intact curcumin in blood, liver and kidney up to 24 h29. Hence suggesting transformation as it is being absorbed from the intestine. Our current observation of the presence of intact curcumin in blood and other tissues thus differs from this and could be due to the more sensitive HPLC method employed by us for the detection and analysis of curcumin.

In an other study30, the major route of elimination of the labelled curcumin was the feces; the urinary

Table IV. Elimination of orally administered piperine in ratDay Faeces 1 737.0 ± 80.32 150.6 ± 15.83 20.0 ± 2.334 1.85 ± 0.355 06 07 08 0Total 909.5

(3.64 % of administered dose)

Values expressed in µg are mean ± SEM of 6 rats.Piperine was orally administered to rats at a dose of 170 mg/kg body weight; Piperine was not found in urine

Table V. Tissue distribution of orally administered curcumin in rat Time (h) Serum

(µg/ml)Blood



(µg/whole tissue)Intestine

(mg/whole tissue)1 2.94 ± 0.21 17.20 ± 1.23 16.46 ± 1.49 1.73 ± 0.33 36.19 ± 3.103 34.69 ± 2.21 203.0 ± 13.0 32.29 ± 4.25 4.39 ± 0.79 17.79 ± 1.686 83.80 ± 5.46 490.3 ± 32.0 135.2 ± 5.26 9.03 ± 1.11 11.83 ± 0.8324 52.56 ± 2.54 307.5 ± 14.9 86.46 ± 1.90 1.98 ± 0.76 2.21 ± 0.4348 9.57 ± 1.02 56.0 ± 6.00 20.00 ± 2.31 0 0.255 ± 0.02596 0.73 ± 0.08 4.27 ± 0.47 4.77 ± 0.69 0 0.027 ± 0.006192 0 0 0 0 0

Values are mean ± SEM of 6 rats.Curcumin was orally administered to rats at a dose of 500 mg/kg body weight


excretion of the label was very low regardless of the dose. At the dose of 80 mg of curcumin (which is comparable to the dose employed by us in the current study, most of the label was excreted within 72 h, while with 400 mg, considerable amounts of the label was present in the tissues 12 days after dosage. The percentage of curcumin absorbed (60-66% of the given dose) remained constant regardless of the dose administered. Pan et al31 have observed that about 2.25 µg/ml of curcumin appeared in the plasma in the first 15 min and declined thereafter following its i.p. administration (0.1 g/kg) to mice. One hour after administration, the levels of curcumin in the intestines, liver, and kidneys were 177.0, 26.9, and 7.51 µg/g, respectively, while only traces (0.41 µg/g) were observed in the brain at 1 h.

The elimination pattern of orally administered curcumin (Table VI) showed unchanged excretion mostly in the faeces (36.5%) of the single oral administration over a period of 8 days, Maximum faecal excretion (34 %) of curcumin occurred during first 24 h and gradually declined thereafter. Excretion

of intact curcumin in the urine was only minimal (0.173%). This figure for the absorption of curcumin is more than twice that reported32 and is about the same as after report29 . The reason for the higher absorption in the present investigation could be the lower dose (500 mg/kg body wt) used.

Tissue distribution and excretion of curcumin orally administered concomitant with piperine: The medicinal properties of curcumin are likely to be under-utilised because of poor bioavailability due to its rapid metabolism in the liver and intestinal wall. Piperine, the active principle of black pepper is known to be a strong inhibitor of hepatic and intestinal aryl hydrocarbon hydroxylation and glucuronidation. Piperine has been documented to enhance the bioavailability of a number of therapeutic drugs as well as phytochemicals by this very property9.

Upon oral administration of curcumin to rats along with piperine, curcumin could be found in blood even up to 8 days. (Table VII). For a similar concentration of administered curcumin, the concentration of intact curcumin present in blood at 3 h, 24 h, 96 h and 192 h was definitely higher (P< 0.01) than what is found when curcumin was administered alone. At 3 h and 24 h, the amount of curcumin in blood was 42 and 64 µg/ml which is about 20 per cent higher than that found when curcumin was administered alone. Similarly, the amounts of curcumin present in liver tissue at 3, 24, 48, 96 and 192 h was much higher (P< 0.01) than what is found when curcumin was administered alone. Curcumin concentrations in liver at 3, 24, 48 and 96 h were 52, 134, 43 and 13 µg respectively, which were 61, 55, 115 and 173 per cent higher (P< 0.01) that found when curcumin was administered alone. Curcumin concentration in kidney found at 3 and 24 h was higher (P< 0.01) than what is found when curcumin was administered alone; curcumin could be detected in kidney even at 2 and 4 day intervals.

Table VI. Elimination of orally administered curcumin in ratDay Faeces Urine 1 9.42 ± 0.66 0.105 ± 0.0052 7.06 ± 0.46 0.019 ± 0.0013 5.19 ± 0.43 0.006 ± 0.0014 2.85 ± 0.48 05 1.75 ± 0.28 06 0.75 ± 0.10 07 0.24 ± 0.03 08 0.12 ± 0.02 0Total 27.38

(36.5% of administered dose)

0.13(0.173% of administered

dose)Values expressed in mg are mean ± SEM of 6 rats.Curcumin was orally administered to rats at a dose of 500 mg/kg body weight

Table VII. Tissue distribution of curcumin in rat after oral administration concomitant with piperineTime (h) Serum

(µg/ml)Blood



(µg/whole tissue)Brain

(µg/whole tissue)3 42.05 ± 3.77 246.0 ± 22.1 52.07 ± 2.24 5.96 ± 1.14 ND24 64.29 ± 1.69 376.1 ± 9.90 134.3 ± 6.94 2.98 ± 1.03 1.84 ± 0.3348 8.04 ± 1.09 47.04 ± 6.38 43.09 ± 1.53 1.16 ± 0.37 5.87 ± 0.3896 2.53 ± 0.40 14.80 ± 2.34 13.00 ± 2.46 0.48 ± 0.10 0.43 ± 0.16192 0.64 ± 0.08 3.75 ± 0.47 1.86 ± 0.20 0.00 ND

Values are mean ± SEM of 6 rats.Curcumin was orally administered to rats at a dose of 500 mg/kg body weight concomitant with Piperine at a dose of 20 mg/kg body weight; ND, not determined


Thus, curcumin stayed significantly longer in the body tissues when administered concomitant with piperine. Curcumin was also detected in the brain tissue at 24, 48 and 96 h with a maximum at 48 h. The amount of curcumin present in brain at 48 h exceeded the amount present in kidney (5.87 µg vs.1.16 µg).

The elimination pattern of curcumin orally administered concomitant with piperine showed unchanged curcumin excretion mostly in the faeces extent of (22.1%) of the single oral administration over a period of 8 days (Table VIII). Maximum faecal excretion of curcumin occurred during first 24 h and gradually declined thereafter. Nearly 43 per cent of the eliminated curcumin was seen in the faeces of first 24 h. Excretion of intact curcumin in the urine was relatively higher (P<0.01) compared to what was seen when curcumin was administered alone and accounted for 1.43 per cent of the curcumin dose administered. Further, excretion of intact curcumin in the urine continued up to fifth day in this case. Thus, about 78 per cent of the oral curcumin dose was absorbed when administered concomitant with piperine. The extent of curcumin absorption was thus significantly higher than what is found when similar dose was administered alone.

Shoba et al33 have studied the effect of combining piperine, on the bioavailability of curcumin in rats and in human volunteers. When curcumin was given at a dose 2 g/kg to rats, moderate serum concentrations were achieved over a period of 4 h. Concomitant administration of piperine 20 mg/kg increased the serum concentration of curcumin for a short period of 1-2 h

post drug. Time to peak was significantly increased while elimination half life and clearance significantly decreased, and the bioavailability was increased by 154 per cent. On the other hand in humans after a dose of 2 g curcumin alone, serum levels were either undetectable or very low. Concomitant administration of piperine 20 mg/kg produced much higher concentrations from 0.25 to 1 h post drug; the increase in bioavailability was 2000 per cent. The authors33 infer that in the dosages used, piperine enhances the serum concentration, extent of absorption and bioavailability of curcumin in both rats and humans with no adverse effects. Thus, the results of the present investigation which has evidenced prolonged occurrence of curcumin in tissues when consumed concomitant with piperine, concurs with the earlier report33. The extent of absorption of curcumin when administered concomitant with piperine is 78 per cent compared to 63.5 per cent absorption observed when curcumin was administered alone. Thus, piperine has not only enhanced the bioavailability of curcumin so that it remains in the body tissues longer by reducing the rate of its metabolic breakdown, but also by enhancing the extent of intestinal absorption. Significantly higher excretion of unmetabolized curcumin in the urine when administered in presence of piperine is an additional evidence to this inference.

The pharmacokinetic parameters─ half life and Area under the curve (AUC) of the orally administered spice compounds calculated from the above four sets of data are presented in Table IX. The half life of orally administered curcumin is distinctly longer when co-administered with piperine. The same increased from 12.8 to 28.9 h in the presence of piperine. A comparison of the half life of the three orally administered spice compounds suggests that capsaicin has the least half life (7.9 h) while piperine stays longer (half life = 18.2 h) than either capsaicin or curcumin.

Table VIII. Elimination of curcumin in rat following oral administration concomitant with piperine Day Faeces Urine 1 7.13 ± 0.98 0.63 ± 0.092 4.51 ± 0.57 0.25 ± 0.033 2.78 ± 0.40 0.103 ± 0.0074 1.10 ± 0.24 0.075 ± 0.0055 0.49 ± 0.08 0.019 ± 0.0026 0.37 ± 0.06 07 0.15 ± 0.02 08 0.07 ± 0.01 0Total 16.60

(22.13 % of administered dose)

1.077(1.44 % of administered

dose)

Values expressed in mg are mean ± SEM of 6 rats.Curcumin was orally administered to rats at a dose of 500 mg/kg body weight concomitant with piperine at a dose of 20 mg/kg body weight

Table IX. Pharmacokinetic parameters of the orally administered spice compoundsSpice compound Half life (t½)

(h)Area under the curve (AUC)

(µg/ml.h)Capsaicin 7.88 15.6 Piperine 18.24 185.0 Curcumin (when administered alone)

12.83 2470.4

Curcumin (when co-administeredwith piperine)

28.88 2390.4

Values (calculated from data presented in Tables I,III,V and VII) are mean of 6 rats


The difference between the administered dose and the amount of parent compound found in the faeces (especially on the first day) represents the absorbed portion of the compound. The other possibility that the compound is not absorbed but metabolized by intestinal bacteria is less likely in view of the fact that faecal excretion of intact curcumin, capsaicin and piperine could be seen even after several days of its oral ingestion, which however tapered off from day 1 to day 8. Also, when the same amount of curcumin was administered concomitantly with piperine, the amount excreted in the faeces was lesser.

Epidemiological studies suggest that anti-inflammatory drugs reduce the risk of Alzheimer’s disease34. There is also substantial in vitro data indicating that curcumin has antioxidant, anti-inflammatory, and anti-amyloid activity35. In addition, studies in animal models of Alzheimer’s disease such as transgenic mice36 and aged female rats37 indicate a direct effect of curcumin in decreasing the amyloid pathology of Alzheimer’s disease. In this context, it is of interest to know if curcumin reaches the brain tissue crossing the blood brain barrier. The distribution of curcumin in brain tissue following its oral intake and its persistence in this tissue for a considerable duration is shown an study. This emphasizes the presence of curcumin in the brain tissue following its administration.

In summary, this animal study of the tissue distribution and elimination of the three structurally closer bioactive spice principles - curcumin, capsaicin and piperine following their oral intake indicated that intestinal absorption of capsaicin was relatively rapid. Absorption of the orally administered dose of capsaicin, piperine and curcumin was 94, 96 and 63.5 per cent respectively. Concentration of capsaicin and piperine in blood was maximum at 1 h and 6 h respectively, and decreased markedly thereafter. Curcumin concentration was maximum in blood at 6 h and remained at significantly higher level even at 24 h. Maximum tissue distribution of 24.4 per cent of administered capsaicin was seen at 1 h, while the same was not detectable after 4 days. Whereas piperine was not detectable in urine, only a small portion of the administered capsaicin (< 0.1%) and curcumin (0.173%) was excreted in urine. Bioavailability of curcumin was enhanced when the same was orally administered concomitant with piperine. Intestinal absorption of curcumin was relatively higher in this case, and it stayed for a longer duration in the body tissues. Curcumin was detected in brain with a maximum concentration at 48 h, and

its presence in brain tissue is of significance in the context of recent reports of beneficial role of the same in Alzheimer’s disease.

Acknowledgment

The first author (DS) is thankful to the Council of Scientific and Industrial Research, New Delhi for the award of Senior Research Fellowship.

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Reprint requests: Dr K. Srinivasan, Senior Scientist, Department of Biochemistry & Nutrition Central Food Technology Research Institute, Mysore 570 020, India e-mail: [email protected]


Tissue distribution & elimination of capsaicin, piperine & curcumin following oral intake in rats

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