Journal of Ethnopharmacology, 37 (1992) 85-91
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Elsevier Scientific Publishers Ireland Ltd.
Review Article
An Ayurvedic formulation 'Trikatu' and its constituents R . K . J o h r i a n d U. Z u t s h i Regional Research Laboratory, Jammu- Tawi (India)
'Trikatu' is an Ayurvedic preparation containing black pepper, long pepper and ginger, which is prescribed routinely for a variety of diseases as part of a multidrug prescription. These herbs along with piperine (alkaloid of peppers) have been shown to possess diverse biological activities in mammalian systems. A review is presented of these studies and it has been suggested that their use in the Indian system of medicine could be due to their bioavailability enhancing action on other medicaments.
Key words: Trikatu; peppers; piperine; bioavailability enhancer
Introduction
In Ayurveda, black pepper (Piper nigrum, Linn.) long pepper (Piper longum, Linn.) and ginger (Zingiber officinalis, Rosc.) in equal proportions are collectively termed Trikatu, a Sanskrit word meaning 'three acrids'. The ancient documented Ayurvedic Materia Medica which dates back to 6000 years B.C. mentions these three herbs as essential ingredients of numerous prescriptions and formulations used for a wide range of disorders (Charaka et al., 1941; Vagbhat, 1962; Kaviraj, 1963). Out of 370 compound formulations listed in Handbook of Domestic Medicines and Common .4yurvedic Remedies 210 contain either Trikatu or its individual ingredients (Anonymous, 1979). Use of the same herbs for different ailments was intriguing unless these possessed some unique activity of some use in the multidrug combinations (Raj and Nagarsheth, 1978). This continued to remain a scientific curiosity until recently when it was recognized that addition of these altered drug effects which could arise as a consequence of a change in bioavailability. Several studies have shown that Trikatu, its constituents and piperine, a major alkaloid from peppers possessed a bioavailability enhancing activity. Correspondence to: R.K. Johri, Regional Research Laboratory, Canal Road, Jammu-Tawi, 180001, India.
The objectives of this review are to illustrate the diverse pharmacological actions of Trikatu/ piperine followed by an evaluation of their biochemical effects which could be responsible for their potential role as a bioavailability enhancer. Tdkatu and other enhancer
Piper species as bioavailability
Bose (1929) while describing the anti-asthmatic property of vasaka leaves (Adhatoda vesica) affirmed that addition of long pepper to vasaka increased its efficacy. Experimental evidences show that the use of Trikatu and its constituents individually as well as collectively enhanced the bioavailability of a number of drugs like vasicine, spartein, sulphadiazine and tetracycline in experimental animals (Atal et al., 1981; Zutshi and Kaul, 1982). P. chaba, another Piper species was also found to be effective as a bioavailability enhancer with some anti-microbial drugs (Atal et al., 1980). Piperinc as bioavallability enhancer In subsequent studies carded out in animals as well as human volunteers it was noted that piperine (1-piperoyl piperidine, Fig. 1A) the active principle from the peppers (Atal et al., 1975; Lewis, 1977) was the major compound responsible for this effect. Enhancement of blood levels of the drugs such as rifampicin (Zutshi et al., 1984),
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86 phenytoin (Bano et al., 1987), pentobarbitone (Mujumdar et al., 1990), theophylline and propranolol (Bano et. al., 1991) was recorded when these drugs were co-administered with piperine. In the above reports which extended from laboratory animals to human volunteers piperine was used in the range of 1.0-30 mg as total dose. Medicinal properties of Trikatu/Piper species In Ayurveda Trikatu has been described as a major decoction useful in restoring the imbalance of kapha, rata and pitta which according to Ayurvedic concept represents the three humors of the body and the imbalance of which causes all types of diseases (Lakshmipati, 1946; Lele, 1986). Piper species have been used internally to treat various fevers, gastric and abdominal disorders and urinary difficulties and externally to treat rheumatism, neuralgia and boils; both P. Iongum and P. nigrum are reputed as folklore remedies for asthma, bronchitis, dysentry, pyrexia and insomnia (Youngken, 1950; Chopra and Chopra, 1959; Akamasu, 1970; Perry, 1980). In Chinese folklore medicine, P. nigrum is mentioned for treatment of epilepsy (Pei, 1983). The efficacy of the fruits of P. longum in reducing asthma in adult patients (Upadhyaya et al., 1982) and in children has been reported (Dahanukar et al., 1984). P. nigrum promoted the secretion of digestive juices (Shukla, 1984), increased appetite (Sumathikutty, 1979). P. longum was reported to be useful in patients with gastric disorders accompanied with clinical symptoms of achlorhydria and hypochlorhydria (Kishore et al., 1980).
and Sen, 1982). Experimental immunological response has been observed in rats and guinea pigs against antigen-induced bronchospasm under the influence of P. longum (Dahanukar and Karandikar, 1984). P. nigrum produced thyrogenic response in rats; it caused significant enhancement in tissue oxygen uptake, thyroid peroxidase activity and plasma thyroxine and triiodothyronine levels (Tripathi et al., 1989). In the above studies piper species were used as alcoholic extracts of either leaves or fruits and piperine was identified as the main active principle responsible for these effects. The subsequent sections have been divided into two major areas, one relating to the basic aspects of piperine pharmacology and other to its mode of action as bioavailability enhancer. Piperine Piperine isolated as the main alkaloid from peppers, is also present in the leaves of Rhododendron faurie (Ericaceae) (Kawaguchi et al., 1942; Kirtikar and Basu, 1975). The amount of piperine occurring in different parts of P. nigrum have been quantitated (Semler and Gross, 1990). In an earlier review various methods of isolation, estimation and chemical properties of piperine along with some of its biological activities have been discussed (Sumathikutti, 1981). The determination of piperine in biological tissue by thin layer chromatography and UV absorption densitometry has been described (Ganesh Bhat and Chandrasbekhara, 1985)
Pharmacology and pharmacokinetics of piperine Pharmacology of Piper species and ginger
Piper species exhibited diverse pharmacological activities. Both P. longum and P. nigrum increased pentobarbitone sleeping time, elevated blood pressure and produced conditioned avoidance response in dogs (Manavalan and Singh, 1979; Sridharan et al., 1979). P. longum exerted stimulatory effect on central nervous system (CNS) in rats (Kulshreshtha et al., 1969, 1971), while in mice the fruits of Piper retrofactum, another Piper species, were CNS depressant (Shin et al., 1979). P. longum and Z. officinalis registered marked anti-inflammatory activity in carageenininduced hind paw oedema in rats (Sharma and Singh, 1980). The antifertility activity of P. iongum (Kholkute et al., 1979; Garg, 1981) and anti-fungal activity of P. nigrum are documented (Chaudhary
Piperine showed CNS stimulant activity in rat (Singh et al., 1973). On the contrary Woo et al., (1979a, b) demonstrated that piperine possessed CNS depressant properties in laboratory animals and produced hypnotic effect. Piperine exhibited anti-pyretic activity against typhoid vaccination; analgesic activity against tail-clip pressure and writhing syndrome in mice and anti-inflammatory activity against carageenin-induced oedema in rats (Lee et al., 1984). Piperine interacted with serotonergic system of rat brain and it was suggested that this action of piperine could be responsible for its reported anti-epileptic activity (Liu et al., 1984). Piperine was found to deplete substance P in the rat spinal cord (Micevych et al., 1983). The neuromuscular transmission was affected by piperine; the activation of sensory terminals as
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well as a direct action on sensory receptors have been reported in rat (Patacchini et al., 1990). Piperine inhibited implantation, produced abortion and delayed labour in mice (Piyachaturawat et al., 1982). The anti-fungal activity of piperine was also documented (Madhyastha and Bhat, 1984) and it was suggested that it could be due to an effect of piperine on the bioenergetic functions of the cell like its inhibitory effect on mitochondrial electron transport in the rat liver (Reanmongkol et al., 1988). The pharmacokinetic profile of piperine has revealed 97% absorption irrespective of mode of dosing whereas 3% gets excreted in the faeces; but piperine was not detectable in urine. Maximum concentration of piperine were found in the stomach and small intestine by 6 h after oral or i.v. administration and by 24 h only traces of piperine remained in serum, kidney and spleen. The increased excretion of uronic acids, conjugated sulphates and phenols as a consequence of scission 0 II
o--v
(A) 0 II
of the methylenedeoxy groups of piperine, glucuronidation and sulfation appeared to be the major steps in its disposition (Ganesh Bhat and Chandrashekhara, 1986a). Figure 1 shows the metabolites of piperine which have been isolated in human urine: the compounds B, C detected in a majority of human beings whereas compound D was excreted in only 15% individuals. (Holzel and Spiteller, 1984).
Toxicology of piper/piperine Black pepper, its oleoresin or piperine fed to rats at 5-20 times the normal human uptake did not cause adverse effects on general growth pattern, food efficiency ratio and clinical pathology. Piperine (0.05%) in a synthetic diet caused increased food uptake in rats and an increase in liver weight mainly due to higher total and neutral lipid contents (Srinivasan and Satyanarayana, 1981). There was no adverse effect of black pepper and piperine in weanling rats (Ganesh Bhat and Chandrashekhara, 1986b). Acute, sub-acute and chronic toxicity studies have shown that at pharmacological effective doses piperine did not cause any abnormality in the general growth pattern, body to organ weight ratio and clinical pathology (Zutshi et al., 1989). LD50 values for piperine are depicted in Table 1 which shows a large margin of therapeutic index so far as it relates to its bioavailablilty enhancing effect.
How Trikatu/piperine increases the bioavailability some examples
(B) 0 II
H 0
A
0 II C--N~
It has been suggested earlier that the individual ingredients of Trikatu as well as piperine could in-crease bioavailability of drugs either by protecting the drug from being metabolized in its first passage through the liver after being absorbed or by promoting rapid absorption from the gastrointestinal tract (GIT) or by a combination of these two TABLE 1 LDs0 OF PIPERINE BY ORAL ROUTE
Fig. 1. Piperine (A) and its metabolites (B-D). (A) l-[5-(l,3benzodioxol-5-yl)- l-oxo-2,4-pentadienyl] piperidine. (B) 5-(3,4dihydroxyphenyl) valeric acid piperidide. (C) 5-(3,4dihydroxyphenyl) valeric acid-4-hydroxy piperidide. (D) 5-(3,4dihydroxyphenyi)-2,4-pentadienoic acid piperidide.
Animal
LDso (mg/kg)
authors
Mice Mice Rat weanling Rat Rat
1636.8 330.0 585.0 514.0 800.0
Woo et al., 1979a Piyachaturawat et al., 1983 Piyachaturaivat ¢t al., 1983 Piyachaturawat ¢t al., 1983 Zutshi et al., 1989
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mechanisms (Atal et al., 1981). The experimental evidence in favour of these hypotheses are presented in the following sections.
Effect on drug metabolizing enzymes The effect of long and black pepper on drug metabolizing enzymes have not been investigated in detail. In ginger-fed rats liver microsomal cytochrome P-450-dependent aryl hydrocarbon hydroxylase (AHH) activity and levels of tyrochrome P-450 and cytochrome b5 were enhanced whereas glucuronyl transferase activity remained unaffected (Sambaiah and Srinivasan, 1989). The effect of piperine on various drug metabolizing enzymes has been studied both in vivo and in vitro. Piperine administered orally inhibited hepatic AHH within 1 h and restored to normal by 6 h (Atal et al., 1985) but at higher doses, the activity of both AHH and aminopyrine N-demethylase was stimulated (Shin and Woo, 1985; Sambaiah and Srinivasan, 1989). In vitro studies demonstrated that piperine interacted with hepatic microsomal suspensions from rat and was a substrate for microsomal enzymes producing nonspecific inhibition of a variety of mixed function oxygenases. In addition piperine was found to potentiate hexobarbital-induced sleeping time and zoxazolamine paralysis time in mice (Atal et al., 1985; Shin and Woo, 1985; Woo et al., 1979b); it inhibited the rate of glucuronidation in rat liver (Singh et al., 1986). Potentiation of pentobarbitone sleeping time was also observed in rat which was suggested to be due to enhanced barbiturate uptake which was noted in the rat brain in presence of piperine or altered rate of pentobarbitone degradation observed in rat liver slices upto 1 h (Mujumdar et al., 1990). It is generally recognized that hexobarbital and barbiturates are useful reference drugs since their duration of action in the body is regulated largely by the levels of liver microsomal enzymes (Conney, 1967). Woo et al., (1978) screened the plant materials of 69 species belonging to 64 genera and 36 families and demonstrated that several plant extracts including family piperaceae possessed prolonging/inhibitory effects on barbiturate hypnosis which were suggestive of inhibitory and/or inductive activities of drug metabolizing enzyme systems in the liver. It is universally believed that most foreign compounds undergo a variety of metabolic changes by non-specific microsomal enzymes in mammals before they are excreted. However the response of microsomal reactions to different xenobiotics is not the same (Conney, 1967). The compounds
which induce/inhibit a microsomal enzyme system for metabolizing one substrate vary in their abilities to induce/inhibit those enzymes systems for metabolizing other substrates (Woo et al., 1978). This could possibly lead to altered pharmacokinetic profile of a drug in combination with the other. It seems likely that piperine might act by inhibiting primarily the monooxygenases thereby affecting drug biotransforming reactions in the liver (Singh et al., 1986). These events would in turn modulate the first passage and/or pattern of drug disposition. The inhibition of microsomal drug metabolizing enzymes may therefore be suggested as one of the mechanisms of potentiation of drug bioavailability by piperine.
Effects on gastrointestinal tract ( GIT) Peppers and ginger have been used as adjuncts to folklore medicines but there have been few attempts to elucidate the biochemical effects on GIT. Ginger was found to increase the bile acid secretion in rat (Sambaiah and Srinivasan, 1989), but the effect of ginger/peppers on the cholesterol and bile acid hydroxylation have not been studied. In view of the reported enhancement of bioavailability by Trikatu/piperine few investigations were carried out to explore the absorptive function of the intestine in this context. Annamalai and Manavalan (1990) demonstrated reduced acid secretion upto 1 h and enhanced blood flow to GIT up to 10 rain with enhancement of glucose absorption after oral administration of extracts or piperine to rabbits. It was suggested that prevention of degradation of active medicaments by the gastric acid and/or their accelerated transport could result in an enhanced bioavailability effect of drugs in presence of piperine. The extracts as well as piperine altered the motility of rabbit and guinea pig ileum (Neogi et al., 1971; Annamalai and Manavalan, 1990; Takaki et al., 1990) which could provide a longer contact of the drug with the absorptive surfaces of the GIT. When exerted sacs of rat intestines were incubated with piperine 47-64% of the added piperine disappeared from mucosal side. Only piperine could be detected in serosal fluid and the intestinal tissue indicating that piperine did not undergo any metabolic change during absorption (Ganesh Bhat and Chandrashekhara, 1986a). These findings raised the possibility that piperine could interact with and produce biochemical changes in intestinal epithelial cells which are the main site for the drugs and chemicals to enter the body. In vitro experiments showed that piperine stimulated ~-
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glutamyl transpeptidase (7-GT) activity, enhanced the uptake of amino acids and increased lipid peroxidation in isolated epithelial cells of rat jejunum. In the presence of benzyl alcohol, a membrane fluldizing agent, an enhanced 3,-GT activity due to piperine was maintained (Johri et al., 1992). These results suggested that piperine may interact with lipid environment to produce effect which lead to increased permeability of intestinal cells. These findings provided another clue to the mode of action of piperine which suggested that piperine mediated changes in the biochemical milieu of the epithelial ceils could result in the stimulation of the absorptive function of the intestine and thereby producing enhanced bioavallability effect of a drug when co-administered with piperine. Conclusion The evidence presented in the preceding sections reveal that the addition of Trikatu or its individual ingredients to a variety of Ayurvedic formulations was so that it could act as bioavailability enhancer, thus increasing the efficacy of the co-administered medicaments: this action results mainly due to the presence of piperine, the active principle of peppers. Further exploration in this area of combination therapy consisting of piperine with many modern drugs could prove advantageous in order to achieve an improved bioavailability and consequently an enhanced uptake, higher concentration and a longer stay of the drugs in the body leading to dose economy. The reduced dose of various highly toxic but essential drugs may help the body metabolize enzymes to handle smaller amounts in an effective manner. In addition many inherent kinetic problems of various drugs like poor absorption/rapid biotransformation could be overcome and this could help in designing orally effective formulations which are only parentally effective. Acimowledgemeat
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90 activity, amino acid uptake and lipid peroxidation. Biochemical Pharmacology 43, 1401-1407. Kaviraj, K.B. (1963) Sushruta Sanhita, vol. 3., 2nd edn., Chowkhamba Sanskrit Series, Varanasi, India. (In Sanskrit). Kawaguchi, R., Kim, K.G. and Kim, H.K. (1942) Components of the leaves of Rhododendron laurie var. refescens. Journal of Pharmacological Society, Japan. 62, 4-8. Kholkute, S.D., Kekare, M.B. and Manshi, S.R. (1979) Antifertifity effects of the fruits of Piper longum in female rats. Indian Journal of Experimental Biology 17, 289-290. Kirtikar, K.R. and Basu, B.D. (1975) Indian Medicinal Plants, vol. 3. 2nd edn. Lalit Mohan Basu, Allahabad, India, pp. 2128-2130. Kishore, P., Pandey, P.N., Pandey, S.N. and Dash, S. (1980) Preliminary trials of certain Ayurvedic drug formulation of 'Amalpitta'. Sachitra Ayurved 33, 40-45. Kulshreshtha, V.K., Singh, R.K., Srivastava, R.K. and Kohli, R.P. (1969) A study of central stimulant effect of Piper iongum. Indian Journal of Pharmacology 1, 8-11. Kulshreshtha, V.K., Singh, ~ , Srivastava, R.K., Rastogi, S.K. and Kohli, R.P. (1971) Analysis of central stimulant activity of Piper longum. Journal of Research in Indian Medicine 6, 17-23. Lakshmipati, A. (1946) Onehandred Useful Drugs, 3rd edn. Arogya Ashram Samithi, Madras, India. Lele, R.D. (1986) Ayurveda and Modern Medicine, Bhartiya Vidya Bhawan, Bombay. Lee, E.B., Shin, K.H. and Woo, W.S. (1984) Pharmacological study on piperine. Archives of Pharmacal Research 7, 127-132. Lewis, Y.S. (1977) Important spicesfrom south-east asia- their cultivation and technology. The Third Asian Symposium on Medicinal Plants and Spices, Columbo, pp. 139-151. Liu, G.Q., Algeri, S., Ceci, A., Gerattini, S., Gobi, M. and Murai, S. (1984) Stimulation of serotonin synthesis in rat brain after antiepilepserine, an antiepileptic piperine derivative. Biochemical Pharmacology 33, 3883-3886. Madhyastha, M.S. and Bhat, R.V. (1984) Aspergillus parasiticus growth and aflatoxin production on black and white pepper and the inhibitory action of their chemical constituents. Applied Environmental Microbiology 48, 376-379. Manavalan, R. and Singh, J. (1979) Chemical and some pharmacological studies on leaves of Piper Iongum Linn. Indian Journal of Pharmaceutical Sciences 41, 190-191. Micevych, P.E., Yaksh, T.L. and Szolesanyi, J. (1983) Effect of intrathecal capsaicin analogs on the immunofloreseence of peptides and serotonin in the dorsal horn in rats. Neuroscience 8, 123-128. Mujumdar, A.N., Dhuley, J.N., Deshmukh, V.K., Raman, P.H., Thorat, S.L. and Naik, S.R. (1990) Effect of piperine on pentobarbitone induced hypnosis in rat. Indian Journal of Experimental Biology 28, 486-487. Neogi, N.C., Haldar, R.K. and Rathor, R.S. (1971) Preliminary pharmacological studies on piperine. Journal of Research in Indian Medicine 6, 24-29. Patacchini, R., Maggai, C.A., Meli, A. (1990) Capsaicin-like activity of some natural punjent substances on peripheral endings of visceral primary affarents. Naanyn-Schmiedeberg's Archives of Pharmacology 342, 72-77. Pei, Y.Q. (1983) A review of pharmacology and clinical use of piperine and its derivatives. Epilepsia 24, 177-181. Perry, L.M. (1980) Medicinal Plants ofEast and Southeast Asia: attributed properties and uses, MIT Press, Cambridge. Piyachaturawat, P., Glinsukon, T. and Peugvicha, P. (1982)
Post-coital antifertility effect of piperine. Contraception 26, 625-633. Piyachaturawat, P., Giinsukon, T. and Tushkulkao, C. (1983) Acute and sub-acute toxicity of piperine in mice, rats and hamsters. Toxicology Letters 16, 351-359. Raj, K.P.S. and Nagarsheth, H.K. (1978) Pepper. Indian Drugs 16, 199-203. Reanmongkol, W., Janthasoot, W., Wattanatorn, W., Dhumma-Upakorn, P and Chudapongse, P. (1988) Effects of piperine on bioenergetic functions of isolated rat fiver mitochondria. Biochemical Pharmacology 37, 753-757. Sambaiah, K and Srinivasan, K. (1989) Influence of spices and spice principles on hepatic mixed function oxygenase system in rats. Indian Journal of Biochemistry and Biophysics 26, 254-258. Semler, U. and Gross, G.G. (1988) Distribution of piperine in vegetative parts of Piper nigrum. Phytochemistry 27, 1566-1567. Sfiarma, A.K. and Singh, R.H. (1980) Screening of antiinflammatory activity of certain indigenous drugs on carageenin-induced hind paw oedema in rats. Bulletin of Medico-Ethno-Botanical Research 1,262-271. Shin, K.H. and Woo, W.S. (1985) Effect of piperine on hepatic microsomal mixed function oxidase system. Korean Biochemical Journal 18, 9-15. Shin, K.H., Yun, H.S., Woo, W.S. and Lee, C. K. (1979) Pharmacologically active principle of Piper retrofactum. Korean Journal of Pharmacognosy 10, 69-71. Shulda, A.K. (1984) Endocrine response of certain spices on albino rats. Ph.D. Thesis, Banaras Hindu University, Varanasi, India. Singh J., Dubey, R.K. and Atal, C.K. (1986) Piperine-mediated mediated inhibition of glucuronidation activity in intestine: Evidence that piperine lowers the endogenous UDPglucuronic acid content. The Journal of Pharmacology and Experimental Therapeutics 2236, 488-493. Singh, N., Kulshreshtha, V.K., Srivastava, R.K. and Kohfi, R.P. (1973) Studies on the analeptic activity of some Piper Iongnm alkaloids. Journal of Research in Indian Medicine 8, 1-4.
Sridharan, K., Kalla, A.K. and Singh, J. (1979) Chemical and pharmacological screening of Piper nigrum Linn. leaves. Journal of Research in Indian Medicine, Yoga and Homoeopathy 13, 107-108. Srinivasan, M.R. and Satyanarayana, M.N. (1981) Effect of black pepper (Piper nigrum) Einn. and piperine on growth, blood constituents and organ weights in rats. Nutrition Reports International 23, 871-877. Sumathikutty, M.A., Rajaraman, K., Sankarikutty, B. and Mathew, A.G. (1979) Chemical composition of pepper grades and products. Journal of Food Science and Technology 16, 249-254. Sumathikutty, M.A., Rajaraman, K., Sankarikutty, B., Narayana, C.S. and Matthew, A.G. (1981) Piperine. Lebensm-Weiss Technology 14, 225-228. Takaki, M., Jin, J-G., Ui, Y-F. and Nakayama, S. (1990) Effects of piperine on motility of isolated guinea-pig ileum and its comparison with capsaicin. European Journal of Pharmacology 186, 71-75. Tripathi, P., Tripathi, G.S. and Tripathi, Y.B. (1989) Thyrogenic response of Piper nigrum. Fitoterupia LX, 539-541. Upadhyaya, S.D., Kansal, C.M. and Pandey, N.N. (1982) Clinical evaluation of (Piper longum ) on patients of bron-
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Elsevier Scientific Publishers Ireland Ltd.
Review Article
An Ayurvedic formulation 'Trikatu' and its constituents R . K . J o h r i a n d U. Z u t s h i Regional Research Laboratory, Jammu- Tawi (India)
'Trikatu' is an Ayurvedic preparation containing black pepper, long pepper and ginger, which is prescribed routinely for a variety of diseases as part of a multidrug prescription. These herbs along with piperine (alkaloid of peppers) have been shown to possess diverse biological activities in mammalian systems. A review is presented of these studies and it has been suggested that their use in the Indian system of medicine could be due to their bioavailability enhancing action on other medicaments.
Key words: Trikatu; peppers; piperine; bioavailability enhancer
Introduction
In Ayurveda, black pepper (Piper nigrum, Linn.) long pepper (Piper longum, Linn.) and ginger (Zingiber officinalis, Rosc.) in equal proportions are collectively termed Trikatu, a Sanskrit word meaning 'three acrids'. The ancient documented Ayurvedic Materia Medica which dates back to 6000 years B.C. mentions these three herbs as essential ingredients of numerous prescriptions and formulations used for a wide range of disorders (Charaka et al., 1941; Vagbhat, 1962; Kaviraj, 1963). Out of 370 compound formulations listed in Handbook of Domestic Medicines and Common .4yurvedic Remedies 210 contain either Trikatu or its individual ingredients (Anonymous, 1979). Use of the same herbs for different ailments was intriguing unless these possessed some unique activity of some use in the multidrug combinations (Raj and Nagarsheth, 1978). This continued to remain a scientific curiosity until recently when it was recognized that addition of these altered drug effects which could arise as a consequence of a change in bioavailability. Several studies have shown that Trikatu, its constituents and piperine, a major alkaloid from peppers possessed a bioavailability enhancing activity. Correspondence to: R.K. Johri, Regional Research Laboratory, Canal Road, Jammu-Tawi, 180001, India.
The objectives of this review are to illustrate the diverse pharmacological actions of Trikatu/ piperine followed by an evaluation of their biochemical effects which could be responsible for their potential role as a bioavailability enhancer. Tdkatu and other enhancer
Piper species as bioavailability
Bose (1929) while describing the anti-asthmatic property of vasaka leaves (Adhatoda vesica) affirmed that addition of long pepper to vasaka increased its efficacy. Experimental evidences show that the use of Trikatu and its constituents individually as well as collectively enhanced the bioavailability of a number of drugs like vasicine, spartein, sulphadiazine and tetracycline in experimental animals (Atal et al., 1981; Zutshi and Kaul, 1982). P. chaba, another Piper species was also found to be effective as a bioavailability enhancer with some anti-microbial drugs (Atal et al., 1980). Piperinc as bioavallability enhancer In subsequent studies carded out in animals as well as human volunteers it was noted that piperine (1-piperoyl piperidine, Fig. 1A) the active principle from the peppers (Atal et al., 1975; Lewis, 1977) was the major compound responsible for this effect. Enhancement of blood levels of the drugs such as rifampicin (Zutshi et al., 1984),
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86 phenytoin (Bano et al., 1987), pentobarbitone (Mujumdar et al., 1990), theophylline and propranolol (Bano et. al., 1991) was recorded when these drugs were co-administered with piperine. In the above reports which extended from laboratory animals to human volunteers piperine was used in the range of 1.0-30 mg as total dose. Medicinal properties of Trikatu/Piper species In Ayurveda Trikatu has been described as a major decoction useful in restoring the imbalance of kapha, rata and pitta which according to Ayurvedic concept represents the three humors of the body and the imbalance of which causes all types of diseases (Lakshmipati, 1946; Lele, 1986). Piper species have been used internally to treat various fevers, gastric and abdominal disorders and urinary difficulties and externally to treat rheumatism, neuralgia and boils; both P. Iongum and P. nigrum are reputed as folklore remedies for asthma, bronchitis, dysentry, pyrexia and insomnia (Youngken, 1950; Chopra and Chopra, 1959; Akamasu, 1970; Perry, 1980). In Chinese folklore medicine, P. nigrum is mentioned for treatment of epilepsy (Pei, 1983). The efficacy of the fruits of P. longum in reducing asthma in adult patients (Upadhyaya et al., 1982) and in children has been reported (Dahanukar et al., 1984). P. nigrum promoted the secretion of digestive juices (Shukla, 1984), increased appetite (Sumathikutty, 1979). P. longum was reported to be useful in patients with gastric disorders accompanied with clinical symptoms of achlorhydria and hypochlorhydria (Kishore et al., 1980).
and Sen, 1982). Experimental immunological response has been observed in rats and guinea pigs against antigen-induced bronchospasm under the influence of P. longum (Dahanukar and Karandikar, 1984). P. nigrum produced thyrogenic response in rats; it caused significant enhancement in tissue oxygen uptake, thyroid peroxidase activity and plasma thyroxine and triiodothyronine levels (Tripathi et al., 1989). In the above studies piper species were used as alcoholic extracts of either leaves or fruits and piperine was identified as the main active principle responsible for these effects. The subsequent sections have been divided into two major areas, one relating to the basic aspects of piperine pharmacology and other to its mode of action as bioavailability enhancer. Piperine Piperine isolated as the main alkaloid from peppers, is also present in the leaves of Rhododendron faurie (Ericaceae) (Kawaguchi et al., 1942; Kirtikar and Basu, 1975). The amount of piperine occurring in different parts of P. nigrum have been quantitated (Semler and Gross, 1990). In an earlier review various methods of isolation, estimation and chemical properties of piperine along with some of its biological activities have been discussed (Sumathikutti, 1981). The determination of piperine in biological tissue by thin layer chromatography and UV absorption densitometry has been described (Ganesh Bhat and Chandrasbekhara, 1985)
Pharmacology and pharmacokinetics of piperine Pharmacology of Piper species and ginger
Piper species exhibited diverse pharmacological activities. Both P. longum and P. nigrum increased pentobarbitone sleeping time, elevated blood pressure and produced conditioned avoidance response in dogs (Manavalan and Singh, 1979; Sridharan et al., 1979). P. longum exerted stimulatory effect on central nervous system (CNS) in rats (Kulshreshtha et al., 1969, 1971), while in mice the fruits of Piper retrofactum, another Piper species, were CNS depressant (Shin et al., 1979). P. longum and Z. officinalis registered marked anti-inflammatory activity in carageenininduced hind paw oedema in rats (Sharma and Singh, 1980). The antifertility activity of P. iongum (Kholkute et al., 1979; Garg, 1981) and anti-fungal activity of P. nigrum are documented (Chaudhary
Piperine showed CNS stimulant activity in rat (Singh et al., 1973). On the contrary Woo et al., (1979a, b) demonstrated that piperine possessed CNS depressant properties in laboratory animals and produced hypnotic effect. Piperine exhibited anti-pyretic activity against typhoid vaccination; analgesic activity against tail-clip pressure and writhing syndrome in mice and anti-inflammatory activity against carageenin-induced oedema in rats (Lee et al., 1984). Piperine interacted with serotonergic system of rat brain and it was suggested that this action of piperine could be responsible for its reported anti-epileptic activity (Liu et al., 1984). Piperine was found to deplete substance P in the rat spinal cord (Micevych et al., 1983). The neuromuscular transmission was affected by piperine; the activation of sensory terminals as
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well as a direct action on sensory receptors have been reported in rat (Patacchini et al., 1990). Piperine inhibited implantation, produced abortion and delayed labour in mice (Piyachaturawat et al., 1982). The anti-fungal activity of piperine was also documented (Madhyastha and Bhat, 1984) and it was suggested that it could be due to an effect of piperine on the bioenergetic functions of the cell like its inhibitory effect on mitochondrial electron transport in the rat liver (Reanmongkol et al., 1988). The pharmacokinetic profile of piperine has revealed 97% absorption irrespective of mode of dosing whereas 3% gets excreted in the faeces; but piperine was not detectable in urine. Maximum concentration of piperine were found in the stomach and small intestine by 6 h after oral or i.v. administration and by 24 h only traces of piperine remained in serum, kidney and spleen. The increased excretion of uronic acids, conjugated sulphates and phenols as a consequence of scission 0 II
o--v
(A) 0 II
of the methylenedeoxy groups of piperine, glucuronidation and sulfation appeared to be the major steps in its disposition (Ganesh Bhat and Chandrashekhara, 1986a). Figure 1 shows the metabolites of piperine which have been isolated in human urine: the compounds B, C detected in a majority of human beings whereas compound D was excreted in only 15% individuals. (Holzel and Spiteller, 1984).
Toxicology of piper/piperine Black pepper, its oleoresin or piperine fed to rats at 5-20 times the normal human uptake did not cause adverse effects on general growth pattern, food efficiency ratio and clinical pathology. Piperine (0.05%) in a synthetic diet caused increased food uptake in rats and an increase in liver weight mainly due to higher total and neutral lipid contents (Srinivasan and Satyanarayana, 1981). There was no adverse effect of black pepper and piperine in weanling rats (Ganesh Bhat and Chandrashekhara, 1986b). Acute, sub-acute and chronic toxicity studies have shown that at pharmacological effective doses piperine did not cause any abnormality in the general growth pattern, body to organ weight ratio and clinical pathology (Zutshi et al., 1989). LD50 values for piperine are depicted in Table 1 which shows a large margin of therapeutic index so far as it relates to its bioavailablilty enhancing effect.
How Trikatu/piperine increases the bioavailability some examples
(B) 0 II
H 0
A
0 II C--N~
It has been suggested earlier that the individual ingredients of Trikatu as well as piperine could in-crease bioavailability of drugs either by protecting the drug from being metabolized in its first passage through the liver after being absorbed or by promoting rapid absorption from the gastrointestinal tract (GIT) or by a combination of these two TABLE 1 LDs0 OF PIPERINE BY ORAL ROUTE
Fig. 1. Piperine (A) and its metabolites (B-D). (A) l-[5-(l,3benzodioxol-5-yl)- l-oxo-2,4-pentadienyl] piperidine. (B) 5-(3,4dihydroxyphenyl) valeric acid piperidide. (C) 5-(3,4dihydroxyphenyl) valeric acid-4-hydroxy piperidide. (D) 5-(3,4dihydroxyphenyi)-2,4-pentadienoic acid piperidide.
Animal
LDso (mg/kg)
authors
Mice Mice Rat weanling Rat Rat
1636.8 330.0 585.0 514.0 800.0
Woo et al., 1979a Piyachaturawat et al., 1983 Piyachaturaivat ¢t al., 1983 Piyachaturawat ¢t al., 1983 Zutshi et al., 1989
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mechanisms (Atal et al., 1981). The experimental evidence in favour of these hypotheses are presented in the following sections.
Effect on drug metabolizing enzymes The effect of long and black pepper on drug metabolizing enzymes have not been investigated in detail. In ginger-fed rats liver microsomal cytochrome P-450-dependent aryl hydrocarbon hydroxylase (AHH) activity and levels of tyrochrome P-450 and cytochrome b5 were enhanced whereas glucuronyl transferase activity remained unaffected (Sambaiah and Srinivasan, 1989). The effect of piperine on various drug metabolizing enzymes has been studied both in vivo and in vitro. Piperine administered orally inhibited hepatic AHH within 1 h and restored to normal by 6 h (Atal et al., 1985) but at higher doses, the activity of both AHH and aminopyrine N-demethylase was stimulated (Shin and Woo, 1985; Sambaiah and Srinivasan, 1989). In vitro studies demonstrated that piperine interacted with hepatic microsomal suspensions from rat and was a substrate for microsomal enzymes producing nonspecific inhibition of a variety of mixed function oxygenases. In addition piperine was found to potentiate hexobarbital-induced sleeping time and zoxazolamine paralysis time in mice (Atal et al., 1985; Shin and Woo, 1985; Woo et al., 1979b); it inhibited the rate of glucuronidation in rat liver (Singh et al., 1986). Potentiation of pentobarbitone sleeping time was also observed in rat which was suggested to be due to enhanced barbiturate uptake which was noted in the rat brain in presence of piperine or altered rate of pentobarbitone degradation observed in rat liver slices upto 1 h (Mujumdar et al., 1990). It is generally recognized that hexobarbital and barbiturates are useful reference drugs since their duration of action in the body is regulated largely by the levels of liver microsomal enzymes (Conney, 1967). Woo et al., (1978) screened the plant materials of 69 species belonging to 64 genera and 36 families and demonstrated that several plant extracts including family piperaceae possessed prolonging/inhibitory effects on barbiturate hypnosis which were suggestive of inhibitory and/or inductive activities of drug metabolizing enzyme systems in the liver. It is universally believed that most foreign compounds undergo a variety of metabolic changes by non-specific microsomal enzymes in mammals before they are excreted. However the response of microsomal reactions to different xenobiotics is not the same (Conney, 1967). The compounds
which induce/inhibit a microsomal enzyme system for metabolizing one substrate vary in their abilities to induce/inhibit those enzymes systems for metabolizing other substrates (Woo et al., 1978). This could possibly lead to altered pharmacokinetic profile of a drug in combination with the other. It seems likely that piperine might act by inhibiting primarily the monooxygenases thereby affecting drug biotransforming reactions in the liver (Singh et al., 1986). These events would in turn modulate the first passage and/or pattern of drug disposition. The inhibition of microsomal drug metabolizing enzymes may therefore be suggested as one of the mechanisms of potentiation of drug bioavailability by piperine.
Effects on gastrointestinal tract ( GIT) Peppers and ginger have been used as adjuncts to folklore medicines but there have been few attempts to elucidate the biochemical effects on GIT. Ginger was found to increase the bile acid secretion in rat (Sambaiah and Srinivasan, 1989), but the effect of ginger/peppers on the cholesterol and bile acid hydroxylation have not been studied. In view of the reported enhancement of bioavailability by Trikatu/piperine few investigations were carried out to explore the absorptive function of the intestine in this context. Annamalai and Manavalan (1990) demonstrated reduced acid secretion upto 1 h and enhanced blood flow to GIT up to 10 rain with enhancement of glucose absorption after oral administration of extracts or piperine to rabbits. It was suggested that prevention of degradation of active medicaments by the gastric acid and/or their accelerated transport could result in an enhanced bioavailability effect of drugs in presence of piperine. The extracts as well as piperine altered the motility of rabbit and guinea pig ileum (Neogi et al., 1971; Annamalai and Manavalan, 1990; Takaki et al., 1990) which could provide a longer contact of the drug with the absorptive surfaces of the GIT. When exerted sacs of rat intestines were incubated with piperine 47-64% of the added piperine disappeared from mucosal side. Only piperine could be detected in serosal fluid and the intestinal tissue indicating that piperine did not undergo any metabolic change during absorption (Ganesh Bhat and Chandrashekhara, 1986a). These findings raised the possibility that piperine could interact with and produce biochemical changes in intestinal epithelial cells which are the main site for the drugs and chemicals to enter the body. In vitro experiments showed that piperine stimulated ~-
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glutamyl transpeptidase (7-GT) activity, enhanced the uptake of amino acids and increased lipid peroxidation in isolated epithelial cells of rat jejunum. In the presence of benzyl alcohol, a membrane fluldizing agent, an enhanced 3,-GT activity due to piperine was maintained (Johri et al., 1992). These results suggested that piperine may interact with lipid environment to produce effect which lead to increased permeability of intestinal cells. These findings provided another clue to the mode of action of piperine which suggested that piperine mediated changes in the biochemical milieu of the epithelial ceils could result in the stimulation of the absorptive function of the intestine and thereby producing enhanced bioavallability effect of a drug when co-administered with piperine. Conclusion The evidence presented in the preceding sections reveal that the addition of Trikatu or its individual ingredients to a variety of Ayurvedic formulations was so that it could act as bioavailability enhancer, thus increasing the efficacy of the co-administered medicaments: this action results mainly due to the presence of piperine, the active principle of peppers. Further exploration in this area of combination therapy consisting of piperine with many modern drugs could prove advantageous in order to achieve an improved bioavailability and consequently an enhanced uptake, higher concentration and a longer stay of the drugs in the body leading to dose economy. The reduced dose of various highly toxic but essential drugs may help the body metabolize enzymes to handle smaller amounts in an effective manner. In addition many inherent kinetic problems of various drugs like poor absorption/rapid biotransformation could be overcome and this could help in designing orally effective formulations which are only parentally effective. Acimowledgemeat
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