Piperine is an interesting compound because it is a good biomarker together with other pure bioactive components or their derivatives or with drug molecules and hence proves to be potential drug compound. Since from the ancient time, piperine the bioactive natural compound belongs to the Piperaceae family, detected in piper species such as Piper nigrum L. (Black Pepper) and Piper longum L. (Long Pepper) [1] and has been investigated its utilization in various applications. Apart from piper species, it also exists in the leaves of Rhdodendron faurie (Ericaceae) [2], Vicoaindica (Asteraceae), seeds of Anethumsowa (Apiaceae), Fructus piperis Longi [3] and bark of Careya arborea (Lecythidaceae) [4]. Piperine is an alkaloid with a formula C₁₇H₁₉NO₃ and molecular weight of 285.34 daltons. It belongs to the largest class of plant secondary metabolite termed as alkaloids. It is a solid substance, insoluble in water, and has weak basicity character, tasteless at first, but leaves a burning taste after a while. The alkaloid piperine is rationally used for the pungency of the plant [1] which belongs to the vanilloid family of compounds; i.e., capsaicin, the pungent substance found commonly in hot chilli peppers. However, plants containing the alkaloid, piperine are commonly found to be used in various traditional systems of medicines for curing an array of disorders and as spices globally [1, 5]. This review presents all available information on the biosynthesis, extraction methodologies, chemical synthesis, analytical data reports and biological properties of piperine and is summarized to create an overview of piperine content.

Piperine belongs to the alkaloidal class of secondary metabolite whose biosynthesis is probably initiated by sequence of reactions i.e. condensation, decarboxylation, oxidative deamination and cyclization. The various investigation suggests that biosynthetic pathway enroute to piperine would involve the amino acid precursor L-lysine. The whole biosynthetic process involves two steps. Step 1 primarily occurs through condensation reaction involving N-heterocycle piperidine and thioester piperoyl- CoA, which provides the group-transfer potential required in the reaction (Fig. 1b) [6]. The N-heterocycle piperidine generates from the decarboxylation of amino acid L-lysine in the presence of pyridoxal phosphate (PLP) to cadaverine, which go through oxidative deamination directly by the enzyme diamine oxidase to yield an amino aldehyde. Then this amino aldehyde further put up cyclization to give the imine, Δ1- piperideine, which further reduces to piperidine (Fig. 1a) and then this generated piperidine reacts with piperoyl-CoA (step 2) to give piperine [7]. However, this propose mechanistic study has not been proven yet; therefore, various enzymatic studies based on the biosynthesis of analogous compounds such as coumaroylagmatine [8, 9] and feruloyltyramine [10, 11] were carried out, which presents clear evidence that the formation of such type of acid amide linkages can be met via intermediate acyl-CoA derivatives.

Fig. (1). (a) Biosynthesis of Piperidine from L-Lysine (b) Reaction catalysed by piperidine piperoyltransferase from Piper nigrum.

In case of concerning the biosynthesis of natural alkaloid piperine, the two major requirements are successfully fulfilled by the preparation of piperoyl-CoA [12] and by this discovery it was demonstrated that a new acyltransferase obtained from the shoots of Piper nigrum is capable to synthesize appreciable amount of piperine in the presence of piperoyl-coenzyme A and 2 piperidine and thus represent a potential enzymatic source [13]. Various studies provides information regarding the reaction rate while using different substrates and minor reaction rates are observed with bases like pyrrolidine and 3-pyrroline where as other related heterocyclic nucleus like pyridine, pyrrole, tetrahydropyran etc. are not accepted as good substrates.

Piperine is a pungent natural alkaloidal product and secondary metabolite extracted from the plant of the genus Piper. It is an obtained or isolated from the oleoresin of P. nigrum or P. longum. Various extraction methodologies were employed to isolate the piperine which depend upon the scale and as well as the purpose of the product and operation. Maceration or Soxhlet extraction of fruit powder with polar organic solvents obtains dark-brown oily substance, which on fractionation by column chromatography using hexane and diethylether (1:2) as mobile phase gives piperine and related amides [14] (Figs. 2 and 3). Pure piperine can also be obtained by supercritical fluid extraction, followed by crystallization from ethanol [3, 15]. However, Supercritical fluid extraction of Pepper generally requires high rate of pressures and temperatures and helpful in consuming the reduce amount of solvent. In addition, the molecule gains an important position in today’s discovery programme and it is being tempted by the people to be developed or isolated in its purest form by employing newer green technologies: using microwave [16] and by hydrotropic solubilisation [17], so that it can be directly used medicinal preparations without any further processing. Furthermore, piperine like compounds from Piper species have been extensively isolated, fractionated and identified by employing High performance liquid chromatography [18, 19]. It is also reported in literature that previously developed methods measures the plasma as well as tissue levels of piperine in animals [20, 21]. In recent years, piperine content can be detected upto nanogram/pictogram level using HPLC as well as HPTLC methods [22-24].

Fig. (2). Brief outline of obtained piperine.

4. CHEMICAL SYNTHESIS OF PIPERINE AND RELATED COMPOUNDS

Due to the most effective bioactive constituent, it received a potential greater interest in their synthesis so they can be employed as potential pharmaceuticals. Piperine exits as trans-trans isomer of 1-piperoyl piperidine. The above structure of piperine (Fig. 4) has been unambiguously confirmed by number of synthetic routes [25-32]. Piperine can be stereo selectively synthesize by Horner- Wadsworth-Emmons Reaction (Fig. 5). Preparation of piperine involves reaction of phosphonate ester of methyl-4-bromo-2-butenoate and piperonal in the presence of sodium methoxide. This generates the phosphate carbanion which further undergoes a Wittig type reaction to form the trans alkene, methylpiperate {(E, E)-5-(3, 4-methylenedioxyphenyl)-2,4- pentadienoate}. Thus, reaction of methyl piperate with piperidine in the presence of sodium methoxide and methanol gives piperine [33]. A more recent preparation of piperine analogues is given by Schobert which utilizes intermolecular three component reaction between aldehydes (alkyl or aryl), amines (1° or 2°) and ketenylidenetriphenylphospharane (Fig. 6) [34]. Apart from this, various amide analogues of piperine were synthesized by using substituted aromatic aldehydes [35] which generally undergoes alkaline hydrolysis in presence of ethanol and provides the basic units such as piperidine, pyrollidine or isobutyl amine and their corresponding acids such as piperic acid or piperylinic acid or guineensic acid, depending on the nature of the carbon chain of the respective amide [36, 37].

Fig. (3). Piperine-type amides from the genus Piper.

Fig. (4). Structure of Piperine.

5. ANALYTICAL METHODS AND TECHNIQUES FOR DETERMINATION OF PIPERINE

From the commencement of any official pharmaceuticals analytical assay methods were included in compendia monographs with aim to characterize the drug molecule. In recent years, the assay methods in monographs include titrimetry, spectrometry, chromatography, electrophoresis and other analytical methods can be seen in literature. An analytical method plays an important role from the stage of drug development to marketing. Thus, in the field of pharmaceutical research, the analytical investigation of any active constituent, intermediates, drug products, drug formulations, degradation product and biological samples is very important. Considering these analytical principles, chemical degradation, transformation, analyses by spectroscopic methods have been extensively useful in the characterization of piperine analogues. Thus, in case of amides which possess full unsaturation in their hydrocarbon chain or partly saturated hydrocarbon chain which possess an α, β- unsaturated amide carbonyl chromophore. It was observed that the IR spectrum displays absorption bands at 1650 (α,β-unsaturated amide carbonyl), 1605 (conjugated trans CH=CH), 1000 (styryl bond) and 925 (methylenedioxy group), while the perhydro- and the Δ β, γ -dihydropiperine and related amides lack the band at about 1605 [38]. The typical ¹H spectroscopic analysis of piperine analogs by NMR, UV, MS fragmentation had been extensively reported and discussed by a number of workers in the past few decades. The ¹H NMR signals are reported in low and as well as in high field region. Both the regions shows significant peaks at chemical shift value of 6.25, 6.65-7.65 for ethylenic and aromatic protons and at chemical shift value at 5.96 for methylenedioxy protons, as recorded for piperlonguminine, piperine, wisanine, trichostachine and sylvatine [37, 39, 40]. The UV absorption peaks for piperine type chromophore is at 250, 304, 378 nm irrespective of the aliphatic amino moiety such as piperine and piperlonguminine which display identical UV characteristics. However, tetrahydropiperine and its analogues have absorption peak at 232 and 285 nm which are very close to 3, 4-methylenedioxybenzenes [38, 40].

Fig. (5). Synthesis of Piperine from Methyl-4-bromo-2-butenoate.

Fig. (6). Three-component reaction between aldehydes, amines and ketenylidenetriphenylphosphorane.

The mass spectra of piperine analogs, including piperine, sylvatine, trichostachine, piperlonguminine were shows M⁺ at 201 (C-N bond), 135 (formation of methylene dioxy tropylium ion) and the base peak at 173 and 172 representing loss of -CO (28) from m/e 201 which is followed by the loss of a hydrogen atom, respectively (Fig. 7) [36, 41]. Thus, to differentiate the conjugation and non-conjugation of the aromatic ring with the amide carbonyl function present in piperine and related amides is characterized by IR and UV absorptions and in the MS fragmentation patterns (Table 1). This is exemplified by the reported spectral data for guineensine and sylvatine that the peak at m/e 161 is for guineensine and related Piper amides, but does not for sylvatine and piperine (Table 1), (Fig. 7) [40, 42].

Fig. (7). MS fragmentations of sylvatine (a) and a guineensine-type amide (b).

Piperine content was also estimated and quantified by UV spectrophotometry, TLC –UV Densitometry, HPTLC and HPLC. Mukherjee et al., observed the presence of piperine content by HPLC in fruits of P. nigrum and P. longum was 3-6% and 0.6-1.6%. In the year 2005, Santosh et al. reports that content of piperine in fruit and root of P. longum was 0.879% and 0.31% and in fruit of P. nigrum was 4.5% by RP-HPLC. Hue et al. showed that the total yield of piperine was 0.79% by RP-HPLC technique 0.99% in thin root and 0.14% in the thick root ; 0.44% in the cortex and 0.29% cm in stele of root. The average count batches of pepper roots were in the range of 6.67 -6.77 mg-g. Hamrapurkar et al. reports that this alkaloid can be quantified with excellent accuracy with in short time period through HPTLC method. The accuracy values obtained to 98.57% in P. nigrum and 96.50% to 97.50% in P. longum [43].

Piperine structure consists of: i) Methylenedioxyphenyl (MDP) ring. (ii) Side chain with conjugated double bonds. (iii) Basic piperidine moiety attached through carbonyl amide linkage to side chain and has possible number of conformers. Numerous researchers reports that the piperine helps in inhibiting both constitutive and inducible Cytochrome P450 (CYP)-dependent drug metabolizing enzymes. Total thirty eight analogs of piperine were prepared by carrying out modification into the phenyl nucleus, side chain and the basic moiety to study the effect of different types of moieties in analyzing the structure activity relationship. The prepared analogs were subjected to mono-oxygenase reactions namely AHH and MOCD, which results in inhibition of cytochrome P450 in rat microsomal fraction which was prepared from untreated, 3- ethylcholanthrene and phenobarbital treated rat liver in vitro.

As a result, it was also observed that inhibitory potential of the parent molecule is mostly lost with modification in any of the three components of piperine. The study concludes that if saturation is carried out in the side chain it results in significant enhanced inhibition of Cytochrome P450 while modifications in phenyl and basic moieties offered selective inhibition either inconstitutive or inducible CYP [35]. In another study, a new series of piperine analogs were evaluated against human transient receptor potential cation channel subfamily V member 1 (TrpV1) expressed in HEK293 cells. The compound piperine founds to be good and strong activator for non- selective cation channel transient receptor potential vanilloid 1 (TRPV1), which in 1997 was identified and proved as the receptor for capsaicin. The receptor TRPV1 functions as to detect and regulate the body temperature. In addition, it provides sensation of scalding heat as well as pain. This study also concludes that the length of carbon chain in piperine amides effects the agonistic potential of the same where as structure changes in double bond and in stereochemistry of aliphatic chain of the prepared amides didn’t effect or change the potency or efficacy which indicates the presence of high level of rigidity or planarity in the piperine molecule. It was also observed that the opening of methylenedioxy ring opening or change in the heterocyclic nucleus of the parent molecule, piperine reduces or abolishes the biological response. Furthermore, piperine amides which are inactive or doesn’t possess any agonistic potential, which were further investigated for antagonist activity; did not display any activity [44].

Quantitative structure activity relationship (QSAR) analysis of new chemical entities obtained from natural and synthetic one has been performed in order to generate highly accurate model as inhibitors of effluxpump NorA for Staphylococcus aureus. To generate the model variables are selected by genetic function approximation method in Cerius 2.

Among them, various types of descriptors were considered; out of which partial negative surface area of the compounds, area of the molecular shadow in the XZ plane and heat of formation are important descriptors responsible for describing the activity of S. aureus NorA efflux pump inhibitors.

This theoretical approach indicates that on increasing the exposed partial negative surface area, the inhibitory activity of the compound also increases against NorA whereas on increasing the area of the molecular shadow in the XZ plane decreases the inhibitory activity. However, the model relates and explains the relationship between heat of formation and inhibitory potential but the generated model doesn’t able to predict the activity of new compounds it only explains the important regions of the molecules quantitatively [45].

Piperine has a broad spectrum of biological properties, many of them have been confirmed by in vivo and in vitro studies. Piperine contains three major moieties or sites in their structure which are found to be responsible for various bioactivities. Thus, a brief description of some pharmacological activities is provided as best.

Recently the piperine action as bioenhancer has been found by Indian Institute of Science, Bangalore using Mycobacterium smegmatis as the test organism [73]. The study provides an interesting observation that piper alone even at high concentrations does’nt inhibits the growth of mycobacteria but it shows a remarkable growth reduction /inhibition on the microorganism and this inhibition is higher than that of rifampicin alone. As rifampicin acts on RNA polymerase where as piperine abrogates the non specific transcription which is catalysed by M. smegmatis RNA polymerase. When RNA polymerase was purified from a rifampicin resistant strain of M. smegmatis, the enzymatic activity, otherwise resistant to rifampicin, found 12 to be significantly decreased in presence of piperine along with Rifampicin.

The study carried out by Reen’s observes and concludes that methylenedioxyphenyl ring founds to be responsible in inhibiting the drug metabolizing enzymes [119]. Another study by Atal in 1985 concludes that we can enhance the in-vivo drug bioavailability by inhibiting the hepatic and non hepatic drug metabolizing enzymes [59]. The property of enhancing the bioavailability of drugs is partly due to the increased blood supply to the intrinsic vessels as a result of local vasodilation. In case of enzymatic activity, Glucoronyl transferase inhibits the endogenous UDP glucoronic acid content and by decreasing transferase activity [119]. One more mechanism of action of piperine was also explains by IIIM, Jammu, which states that Piperine act as a cell membrane modulator and thus helps in transportation of the drugs molecules across the barriers. It was speculated that piperine may also called as thermonutrient as it increases the GIT absorption of certain nutritional substances and thus helpful in production of local thermogenic action. To enhance the mechanism for drug bioavailability, following possible explanations’ are:

1. Increased blood supply to GIT, b) Increased enzymatic activities like gamma- glutamyl transpeptidase c) Non-specific mechanisms inhibiting enzymes involving in biotransformation of drugs, preventing their inactivation and elimination [70, 120].

A study proves that Piperine interacts and interferes both in vitro and in vivo with the metabolism and degradation related enzymes and thus act as a nonspecific inhibitor of drug metabolism. Piperine inhibits a series of enzymes mainly related to P-gp and cytochrome P 450 family other ezymes are: Aryl hydrocarbon hydroxylase (Microsomal enzyme system), Ethyl morphine-N demethylatse, 7-Ethoxycoumarin-O-de-ethylase, 3- Hydroxy-benzo(a)pyrene glucou-ronidase, Uridine di phosphate glucose dehydrogenase (UDP-GDH), Uridine di phosphate glucuronyl transferase (UDP-GT), 5-Lipoxegenase (5-LOX) and Cyclo-oxegenase-I (COX-I) [121].

The study concludes that a supplementation of piperine to fat, carbohydrate, fructose and cornstarch fed rats normalizes the blood pressure, improve glucose tolerance and reduce plasma parameters of oxidative stress and inflammation. In addition to this, it also improves the liver function [122].

To study the pharmacokinetic parameters and tissue distribution of piperine, lipid nanospheres of piperine (LN-P), pegylated lipid nanospheres LN-P-PEG and positively charged stearylamine LN-P- SA were prepared by process of homogenization which was further followed by ultrasonication and evaluated on male albino mice. The pharmacokinetic parameters of LN-P- PEG and LN-P- SA were: AUC(0-24): 372.1 +/- 71.6 and 162.2 +/- 36.4 microgram/ hour/ ml, clearance 13 +/- 2.5 and 32 +/- 7.5 ml/ hour, Cmax: 24.7 +/- 1.5 and 22.3 +/- 1.0 microgram/ ml, Vd: 0.45 +/- 0.02 and 0.66 +/- 0.06 liter/ Kg. Pharmacokinetics of lipid nanospheres of piperine shows a biexponential decline with significantly high AUC, lower rate of clearance with a smaller volume of distribution than piperine [123].

Ayurvedic formulation: ChitrakadiVati [124], Trikatu Churna [125, 126], Pippli Churna [127], Sitopaladichurna (STPLC) [128], Ajmodadichurna (AJC) [129, 130], Triphalaguggulu [131], Eladi Gutika [132], LasunadiVati, Marichyadivati and Kaphaketu rasa [133]. Unani formulation: Hab-e-Azarakhi [134], Jawarish-e-Bisbasa [135], Habb-e- Khardel [136]. Dosage forms 1) Vista Nutrition Curcumin with Piperine (Capsule) Description: Vista Nutritions Curcumin with Piperine capsules has anti-oxidant, anti- inflammatory effects. It contains curcumin and piperine that restrains the enzyme called CYP3A4 to increase the oral bioavailability of compounds. Vista Nutritions Curcumin with Piperine helps in maintaining the healthy liver and protecting the brain cells. 2) Zenith Nutrition Curcumin with Piperine (Capsules) Description: Zenith Nutrition Curcumin with Piperine contains curcumin and piperine. The active component of turmeric (curcumin) is responsible for its rich color and potential health benefits. The powerful antioxidant helps in protection of cells from free radical damage, in maintaining a healthy inflammatory response, protection of brain cells, in maintaining a healthy liver even under challenging circumstances. Standardization of preparation also ensures the consistent levels of active ingredients in every dose.

The clinical studies of piperine shows that it may enhance the bioavailability of resveratrol a substance commonly found in red grapes, peanuts and chocolate which help in translating the potential benefit of the same to humans. Resveratrol (3, 5, 4′-trihydroxystilbene) is chemically a phytoalexin possess numerous health-promoting properties in pre-clinical studies. On comparing the resveratrol alone, combination with piperine increases the degree of exposure of resveratrol to 229%, while exposure to the compound principal metabolite decreased by about 80%. Piperine enhances the reservatol pharmacokinetic parameters via inhibiting its glucuronidation, and hence thereby slows the process of elimination [137]. Recent studies, in collaboration with Oregon Health and Science University (OHSU) dermatologists, the combination piperine with narrow band ultra violet B (NBUVB) was applied to National Institutes of Health/ National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH/NIAMS) to fund a Phase I clinical trial for vitiligo disease. Moreover, the reviewers expressed enthusiasm for piperine as a novel treatment for vitiligo, and for the design of the clinical trial, a major concern was piperine unknown effect on melanoma development, particularly if used in conjunction with UVB. This concern is a major hurdle to the future clinical investigation of piperine in vitiligo, and clearly must be addressed. Therefore the effect of piperine, alone or with UV radiation, on melanoma development using the HGF-BL6 mouse model of melanoma was investigated. However, the result shows, despite its stimulatory effects on normal melanocyte proliferation, PIP will not promote, and may even reduce, melanoma formation in a murine model of UV-induced melanoma [138, 139].

Piperine is used as spice and nutrient enhancer. The study conclude that, consecutive administration of piperine (2.25 and 4.5 mg/kg, p.o.) for five days causes a significant reduction in total leukocyte counts, increases the percentage of neutrophils and suppresses the mitogenic response of B-lymphocyte to lipopolysaccharide in swiss male mice. Moreover, the treatment at higher dose results into significant decrease in the weight of spleen, thymus and mesenteric lymph nodes.

However, at lower dose (1.12 mg/kg, p.o.) it may be considered as immunologically safe [140]. The previous research concludes and suggests that piperine should be co-administered with drugs i.e. P-gp substrates, particularly for patients whose diet heavily relies on Pepper.

Piperine concentration ranging from 10 to 100 μM, inhibits P-gp mediated efflux transport of [3H ]-digoxin across LMDR1 and Caco-2 cell monolayers. This acute inhibitory effect depends on concentration of piperine with abolishment of [3H]-digoxin polarized transport which was attained at 50 μM of piperine. In contrast, the prolonged (48 and 72 h) co- incubation of Caco-2 cell monolayers with piperine (50 and 100 μM), increases P-gp activity through an up-regulation of cellular P-gp protein and MDR1 mRNA levels. The up-regulated protein was functionally active, as it is demonstrated with a higher degree of [3H]-digoxin efflux across the cell monolayers, but the induction was totally readily reversed by the removal of piperine as spice from the culture medium. Oral administration of piperine (112 μg/kg) for 14 consecutive days increases intestinal P-gp levels in male wistar rats. Thus, it was observed that concomitant reduction in the rodent liver P-gp whereas kidney P-gp level remains unaffected [141].

It was well that Cadmium (Cd) is an industrial and environmental ubiquitous pollutant, and it is toxic to several tissues, most notably hepatic and renal acute administration results its chronic exposure thus, a study by Khandelwal evidences that piperine at 2.5 mg/kg/day significantly decreases the hepatic and renal Cadium levels in mice [142].

In the present review, an attempt has been made to congregate the erudition of versatile molecule, PIPERINE. Although it have medicinal applications from time immemorial, but todays need is to develop modern drugs with effective extensive investigation for its bioactivity, mechanism of action, pharmacotherapeutics, and toxicity and after proper standardization and clinical trials. Reviewed interest among the researchers all around the world in the structural modification and synthesis of novel analogues of the privileged molecule piperine is attributed to the wide array of biological activities it possesses. It appears to top in the list of bioenhancers as it has been used as bioenhancer for Allopathic, Ayurvedic and Unani drugs. Several therapeutically as well as industrially useful preparations have been marketed which generates encouragement among the scientists in exploring this medicinal important moiety. As it is very well evident from the literature that piperine has got tremendous potential, thus the appropriate modification in the molecule and synthesis of its analogues to attenuate the toxicity with better economic investment and with good therapeutic utilization presents greater benefit particularly in various treatments and therapies.