critical publication review paper

Abstract: The medicinal properties of Curcumin obtained from Curcuma longa L. cannot be utilised because of poor bioavailability due to its rapid metabolism in the lover and intestinal wall. In this study, the effect of combining piperine, a known inhibitor of hepatic and intestinal glucuronidation, was evaluated on the bioavailability of Curcumin in rats and healthy human volunteers. When Curcumin was given alone, the dose 2g/kg to rats, moderate serum concentrations were achieved over a period of 4 h. Concomitant administration of piperine 20mg/kg increased (P < 0.02), and he bioavailability was increased by 154%. On the other hand in humans after a dose of 2g Curcumin alone, serum levels were either undetectable or very low. Concomitant administration of piperine 20mg produced much higher concentrations from 0.25 to 1h post drug (P < 0.01 at 0.25 and 0.5h; P < 0.001 at 1h), the increase in bioavailability was 2000%. The study shows that in the dosages used, piperine enhances the serum concentrations, extent of absorption and bioavailability of curcumin in both rats and humans with no adverse effects. Key words: Curcumin, piperine, pharmacokinetics, Curcuma longa, Zingiberaceae Introduction Curcumin in obtained from Curcuma longa L (Zingiberaceae), a perennial herb widely cultivated in tropical regions of Asian. Its rhizome is extensively used for imparting colour and flavour to food. Current traditional Indian medicine claims the use of its powder, turmeric, against a wide variety of diseases (1). Extensive scientific research (2) on curcumin had demonstrated a wide spectrum of therapeutic effects which range from anti- inflammatory, wound healing, antispasmodic, anticoagulant, antitumor activities (3) and recently, with potential utility in autoimmune deficiency syndrome (4). Pharmacokinetic properties of curcumin indicate that following oral administration, it is poorly absorbed (3) and only traces of the compound appear in the blood, while most of it is excreted in the faeces (5). The transformation of curcumin into an unidentified compound during absorption (6) and its glucuronidation in the liver (5, 7) are probably responsible for its low concentration in the blood.

Planta Medica 64 (1998) 353 – 356 ©Georg Thieme Veriag Stuttgart – New York

Black pepper (Piper nigrum L) and long pepper (Piper longum L) have been in use as spices from ancient time’s thoughout the world. A major component of the Piper species id the alkaloid piperine (l-piperoylpiprtidine), which had been reported to enhance the bioavailability of drugs by inhibition of glucuronidation in the liver (8) and small intestine (9). In view of the potential therapeutic utility of curcumin it appeared pertinent to examine the effect of piperine, a known hepatic and intestinal metabolic inhibitor, on the pharmacokinetic disposition of curcumin in animals and man, to provide a scientific rationale of assigning it a rightful place in the pharmacologist’s armamentarium. Materials and Methods Animal Studies Albina Wistar rats (n=96) of both sexes (150-200g) were chosen for the study. They were housed in well ventilated cages, fed on commercial rat pellets supplied by Hind Lever, Mumbai, with tap water as libitum. They were divided into two sex and weight matched groups (n=6/group/time cut), one group for administration of curcumin and the other for concomitant curcumin and piperine. Curcumin and piperine were supplied in pure powder for by Sami Chemicals and Extracts, Bangalore, India. Both the compounds were administered orally to fasted rats as an aqueous suspension. The in group that received both drugs, curcumin was administered first followed immediately by piperine. Control rats received water only. Curcumin was given in a dose of 2g/kg and piperine, 20mg/kg. Under ether anaesthesia, pre and post drug jugular vein blood samples were collected from both groups of rats into centrifuge tubes at the time intervals – 0, 0.25, 0.50, 0.75, 1, 2, 3, 4, 5 and 6h. The blood was allowed to clot at room temperature for about 1h and then centrifuged at 3000rpm for 10min. The serum was separated out carefully using Pasteur pipettes into storage tubes and frozen at -20°C prior to analysis.

Influence of Piperine on the Pharmacokinetics of Curcumin in Animals and Human Volunteers

Guido Shoba₁, David Joy₁, Thangam Joseph₁, M. Majeed₂. R. Rajendran₂, and P.S.S.R. Srinivas₂

₁Department of Pharmacology, St John’s Medical College, Bangalore, India ₂5AMI Chemicals & Extracts (P) Ltd., Banaglore, India

Received: August 1, 1997; Revision accepted: October 18, 1997

Human volunteer studies Ten healthy male volunteers, 20 to 26 years, weighing 50 – 75kg (mean 60 ± 1.93) participated in a randomized cross over trial, to determine the comparative bioavailability and pharmacokinetic profile of curcumin when given alone and with piperine. Complete physical examination and an electrocardiogram were done. Laboratory tests comprising complete blood counts and haemoglobin percentage, blood biochemistry consisting of blood urea nitrogen (BUN) serum creatinine, total and conjugated bilirubin, alkaline phosphatase, aspartate transaminase (ASAT), alanine transaminase (ALAT), urine albumin, and sugar were preformed to confirm that the subjects included in the study were normal. The study was formally approved by the Institutional Ethical Committee and informed consent was obtained for all subjects. Subjects abstained from food since 10pm of the previous evening and reported to the laboratory at 7am. Venepuncture was done using a 20g scalp vein set with heparin lock and left in situ. Blood samples (5ml) were collected (without anticoagulant) at 0, 0.25, 0.50, 0.75, 1, 2, 3, 4, 5 and 6h post drug. Blood was allowed to clot at room temperature for 1h. Serum separation and storage until analysis was as explained earlier. Following basal blood sample collection 2g of pure curcumin powder (4 capsules of 500mg curcumin each) or 2g of pure curcumin powder combined with 20mg of pure piperine powder (4 capsules of 500mg curcumin + 5mg piperine each; identical capsules prepared by Sami Chemicals and Extracts, Bangalore, India) was given with 150ml of water. Blood sampling after curcumin per se and curcumin with piperine was done two occasions, separated by a two week wash out period on the same volunteers. The following precautions were taken during the trial; subjects refrained from smoking, consuming alcohol, or beverages, and from taking drugs of any kind 24h prior to and during the trial. Standard meals were given to all the participants on the day of the test. Analytical methods Estimation of curcumin was done by reverse phase high pressure liquid chromatography (HPLC) using modification of the method described by Tannesen et al. (10). The modification was done by Sami Chemicals and Extracts, Bangalore, India, and is detailed below, the mobile phase used was ethanol : methanol (60 : 40) instead of only ethanol and the flow rate was changed from 1.2ml/min to 1ml/min. HPLC grade methanol and low actinic glassware protected from light were used for the entire procedure. Extraction and preparation of standard solution Curcumin (25mg) was dissolved and diluted to 25ml with methanol in a volumetric flask; 0.1ml (100 µm) of this was transferred to a volumetric flask and diluted with methanol up to 10ml making a 10ppm solution; 0.1ml of this 10ppm solution was transferred to another 10ml volumetric flask and the volume make up with methanol making a 0.1ppm solution.

Extraction of curcumin from serum and preparation of sample Serum samples stored t -20°C were equilibrated to room temperature before analysis. A portion of 1ml was transferred into a 10ml volumetric flask and about 5ml of methanol added. The mixture was shaken thoroughly and heated at 80°C on a water bath for half an hour. After cooling to room temperature, methanol was added to make up the volume to 10ml and mixed well. The turbid solution was transferred into a 15ml centrifuge tube and centrifuged at 4000 RPM for 10 minutes. The supernatant was collected by means of a 25ml syringe and 10cm needle (Luer lock) and the clear solution filtered through a 0.45µm, 13mm Millipore membrane filter, into a narrow end test tube. 20µl of the solution were injected into the chomatograph for carry out the HPLC analysis. Samples were read by UV absorbance of 254nM. The recovery rate experiments were carried out by adding a known amount of standard curcumin to the serum and the added curcumin extracted as per procedure and quantified. The recovery rate of curcumin from serum ranged from 87 – 89.9%. The minimum level of detection of curcumin was 0.001µg/ml. Calculation Content of curcumin in µg/ml in the test sample

Standard Reading x standard concentration Standard Reading x standard concentration

Treatment of pharmacokinetic data For calculation of pharmacokinetic parameters (PK), curve fitting was carried out by a model independent method with non-linear least-square regression analysis using a computer designed programme “PHARMKIT”. This programme uses an algorithm call “SIMPLEX” for calculating non-linear least-squares. The various PK parameters calculated were: absorption half-life (t½(a)), elimination half live (t½el), volume of distribution (Vd); and clearance (CI). Areas under the concentration time curve (AUCo-m) was calculated using the trapezoidal method. Maximum concentration (Cmax) and time to max (Tmax) are observed values. Relative bioavailability (F) was calculated using formula:

AUC Curcumin + piperine AUC Curcumin

Statistical analysis Serum concentration time curves and the PK parameter from animal data were analysed using the Students ‘t’ test while the paired ‘t’ test was used for comparing serum concentration curves in humans. PK parameters of curcumin when given alone in humans could not be calculated as curcumin could not be detected in most of the samples.

Planta Med. 64 (1998) Guido Shoba, David Joy, Thangam Joseph, M. Majeed. R. Rajendran, and P.S.S.R. Srinivas

=

F = x 100

Results Animal studies Curcumin alone at 2g/kg or when combined with piperine, 20mg/kg, was well tolerated by the rats as they showed no untoward effects for 48h. Yellow coloured faecal pellets appeared at 30h post drug and continued up to 48h. Perusal of Figure 1 indicates that when curcumin was given alone, peak serum concentrations of 1.00 ≠ 0.26 µg/ml were attained rapidly within 0.75h and plateaued till 1h. Thereafter, the levels declined gradually reaching zero at 5h. The plasma concentration time curve of curcumin in combination with piperine followed a similar pattern from 0 to 0.75h and 3 to 5h. However piperine produced higher serum concentrations of curcumin at 1 and 2 h (1.55 ± 0.21 and 1.50 ± 0.25 µg/ml) respectively, being significantly higher (P <0.02) at 2h. Thus piperine significantly enhanced the serum concentration of curcumin, albeit for a limited duration (although serum samples were collected up to 6h, values are depicted till 5h only, since the 6h value was also ‘0’ in all animals). Table 1 shows the values (mean ± SEM) of the pharmacokinetic parameters if curcumin per se and when combined with piperine. Cmax was increased from 1.35 ± 0.23 to 1.80 ± 0.16 µg/ml, but was not statistically significant, while Tmax was significantly increased from 0.83 ± 0.05 to 1.29 ± 0.23h (P <0.02). The t½(el) significantly decreased from 1.70 ± 0.58 to 1.05 ± 0.18h (P<0.002). Though t½(a) increased from 0.31 ± 0.07 to 0.47 ± 0.03h and AUC increased from 2.36 ± 0.28 to 3.64 ± 0.31 µg/h/ml, these increases were not statistically significant. CI significantly decreased from 713.00 ± 12.00 to 495.00 ± 37.00:/h (P<0.02) but the decrease in the Vd from 1366.00 ±248.70 to 782.60 ± 193.90L/kg was not significant. The relative bioavailability of curcumin when combined with

Piperine is 154%. Human volunteer studies Curcumin alone or when combined with piperine was well tolerated by all the subjects and there were no adverse or untoward reactions; 2 subjects dropped out of the study for non-medical reasons. Therefore all calculations presented here are based on the data obtained from 8 subjects. In figure 2 is shown the serum concentration if curcumin per se and when given with piperine. Although serum samples were collected up to 6h, we have depicted values till 3h, since the 4, 5 and 6h values were also ‘0’ in all subjects. Serum levels’ of curcumin when given alone were either very low or undetectable at mot time points in most subjects, explaining he almost flat serum concentration curve (Fig. 2). However, when piperine was added the serum concentrations of curcumin were significantly increased at the time points up to 0.75h; P <0.01

Planta Med. 64 (1998) Guido Shoba, David Joy, Thangam Joseph, M. Majeed. R. Rajendran, and P.S.S.R. Srinivas

Fig. 1 Serum concentrations µg/ml (mean ± SEM) of curcumin 20g/kg oral alone and with piperine 20mg/kg in

rats (n= 6/group/time cut). Significance as compared to

curcumin alone; *P <0.02.

Fig. 2 Serum concentrations µg/ml (mean ± SEM) of curcumin 2g oral alone and with piperine 20mg in humans

(n= 8). Significance as compared to curcumin alone;

*P <0.01 **P <0.001.

at 0.25h and 0.5h; P <0.001 and at 0.75h. Subsequently there was a rapid decline up to 1h and thereafter a gradual decline to zero by 3h. In Table 2 are depicted the PL parameters (Mean ± SEM) of curcumin when given alone and with piperine. Cmax (observed values) when curcumin was given alone was only 0.006 ± 0.005 µg/ml at 1h whereas when piperine was added the Cmax (observed value) was increased to 0.18 ± 0.16 µg/ml and was attained earlier, i.e. at 0.75h. Vd and CI could not be calculated with curcumin alone as serum levels were not detected at most time points in most subjects. The mean AUC(0-tn) however, was calculated using the trapezoidal method and was found to be 0.004 µg/ml, the relative bioavailability of curcumin when given with piperine was therefore 2000%.

Discussion The results obtained in the study demonstrate that piperine enhances the oral bioavailability of curcumin in both rats and humans at does that we devoid of adverse side effects. However, certain differences between rats and human with respect to curcumin were evident. Curcumin per se attained overall moderate serum concentrations over a 4h period in rats with peak levels occurring between 0.75h to 1h. On the other hand, in humans when curcumin was given alone only negligible serum concentrations of curcumin were detectable the serum concentration-time curve being almost flat. This difference may be due to high oral dose employed in the rat (2g/kg), whereas the human does was about 60 times less, approximately 33mg/kg. Curcumin serum concentrations reached zero at 5h in rats and 3h in humans. Further in rats with the addition of piperine, curcumin achieved high concentrations than in humans albeit for a short period, took s longer time to peak and declined slowly. Whereas in humans Tmax was attained earlier and then declined rapidly. This rapidity in decline is more apparent probably because of the high levels of curcumin achieved with piperine as compared to curcumin alone. There was an increase in the AUC though not significant as an increase in bioavailability of curcumin by about one and a half times as compared to curcumin given

alone in both rats and humans. In rats when piperine was added to curcumin both Vd and CI decreased which may have also contributed to the higher concentration, such a comparison was not possible in humans for season explained earlier. Our findings concerning absorption f curcumin in rats are in agreement with data obtained by Wahlstrom and Belnnow (11), who sowed that when Sprague Dawley rats were given curcumin 1g/kg p.o., measurement of blood plasma levels and biliary excretion indicated some absorption from the gut with no apparent toxic effect unto 5g/kg p.o. Likewise, Khanna et al. (12); found that after curcumin, 100mg/kg p.o., 74% was absorbed from the gastrointestinal tract within the first 5h, while complete elimination occurred within 48h. Our results are, however, in conflict with studies by Ravindranath and Chandrasekhara (6), who could not detect curcumin 400mg/kg p.o. They, however, did report 60% absorption of curcumin as determined by the amount excreted in the faeces. There is evidence that piperine is a potent inhibitor of drug metabolism, and glucuronidation altering the disposition and bioavailability of a large number of drugs (8). Further piperine at 20mg in humans has also been shown to produce earlier Tmax higher Cmax and AUC of drugs like propranolol and theophylline (13). This property of piperine suggests that it may be involved in inhibiting the metabolism of curcumin and enhancing bioavailability. In conclusion, the study shows that piperine enhances the serum concentration and bioavailability of curcumin in rats and man probably due to increased absorption and reduced metabolism. References

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2. Srimal, R.C., Dhawan, R.N. (1985) in: Development of Unuani drugs from herbal sources and the role of elements in their mechanism of action. Hamdard National Foundation Monograph. New Delhi.

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43. 86-92. 12. Khanna, M., Singh, S., Sarin, J.P.S. (1981) Indian Drugs 19. 31. 13. Bano, G., Raina, R.K., Zutshi, U., Bedi, K.L., Johri, R.K., Sharma,

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Planta Med. 64 (1998) Guido Shoba, David Joy, Thangam Joseph, M. Majeed. R. Rajendran, and P.S.S.R. Srinivas

Dr Guido Shoba Department of Pharmacology St. John’s Medical College Bangalore 560 034 India