Introduction

Spices are widely used in human food due to their taste and burning sensations caused by their pungent principles. Black pepper (family Piperaceae) alone accounts for about 35% of the world's total spice trade. Piperine is the active principle of black pepper (Piper nigrum L.) and long pepper (Piper longum L.). It is also the principal alkaloid of these plants.(₁) The piperine content is 3-9% and 3-5% (on dry weight basis) in P. nigrum and P. longum respectively. The average daily consumption of black pepper in food in the U.S. is 446 mg, while the average human consumption of chemical piperine is 21.0 mg. (₂) Apart from being used as for its food flavoring properties, the pungent piperine has important pharmacological actions. Capsaicin as well as piperine act as legends for the vanilloid receptor (TRPV1, formerly VR1)(₃), a member of the transient receptor potential (TRP) family of ion channels, a large group of proteins involved in the detection and integration of sensory stimuli.(₄) TRPV1 is a cation channel that is highly expressed in peripheral and central terminals of a subset of primary afferent neurons (with unmyelinated C fiber or thinly myelinated A δ fibers) and is directly activated by a wide range of stimuli including noxious heat, protons, endogenous lipoxygenase products and fatty acid amides as well as capsaicin, gingerols, eugenol, resiniferatoxin, piperine and camphor.(₅,₆,₇,₈) Piperine in addition, exhibits a number of pharmacological effects. It has been shown to possess antioxidant properties(₉), protect against AFB1-induced cytotoxicity(₁₀) and to inhibit enzymatic drug biotransforming reactions in the liver(₁₁). In the digestive tract, piperine exerted gastric protective effects(₁₂) (Szolcsányi , 1990) and delayed gastrointestinal motility(₁₃).

Piperine also exerts antidepressant like effects in mice.(₁₄) Contradictory data, however, has been reported as regards the effect of piperine in models of hepatic injury. Piperine pretreatment has been reported to inhibit(₁₅) or potentiate(₁₆) the hepatotoxicity of CCl4 in rodents. The present study was therefore designed to examine the effect of piperine treatment on liver damage caused in rats by ligation of the common bile duct. This model closely mimics the situation of surgical obstruction of the common bile duct in man such as that occurring due to stricture or malignant growth etc... The effect of piperine was compared with that of silymarin, a standardized extract, derived from the milk thistle plant and is used as a hepatoprotective agent worldwide(₁₇). We also aimed to bone changes and the possible effect of piperine in this model of obstructive jaundice.

Materials and methods

Drugs

Animals

Surgery

Design of experiments

Biochemical assessment

Histological studies

Statistical analysis

Results

Biochemical changes

Effect of piperine on serum transaminases, alkaline phosphatase and biliruin

Results are presented in table 1. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and bilirubin levels were significantly higher in bile duct ligated rats compared with their Sham-treated counterparts. Serum ALT increased by 78.1%(31.0 ± 2.7 vs 56.2 ± 4.7 U/l), serum AST by 403.6% (78.2 ± 6.4 vs 393.8 ± 31.3 U/l), serum ALP by 850% (326.0 ± 26.3 vs 3098.8 ± 121.6 U/l), respectively. Serum total bilirubin increased markedly in BDL compared to sham operated rats (6.7 ± 0.5 mg/dl vs 0.2 ± 0.1 mg/dl, p < 0.05, n = 6 per group).

The administration of piperine at 20, 40 or 80 mg/kg to bile duct ligated rats reduced elevated plasma ALT by 16.2, 23.3, 37.5%, AST by 15.4, 19,26.8%, ALP by 60.5, 72.4, 72.7% and bilirubin by 28.4, 44.8,46.3%, respectively. In comparison, the elevated serum ALT, AST, ALP and bilirubin levels decreased to 28.3%, 20%, 62.8 and 31.3% of controls, respectively by 22 mg kg of silymarin. ALT by 16.2, 23.3, 37.5%, respectively.

Figure 1

Table 1: Serum total bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in bile duct-ligated (BDL) rats treated with piperine or silymarin

Effect of piperine on serum calcium

Calcium levels in plasma of BDL rats were not significantly altered by piperine, however calcium level values in BDL-rats plus 40 or 80 mg/kg piperine were significantly less than normal (Sham) rats (table 2).

Figure 2

Table 2: Thickness of bone tissue at the diaphysis of right tibia in bile duct-ligated rats treated with piperine

Histological results

Effect of piperine on liver injury

Examination of sections of liver tissue from a BDL-control rats showed severe fibrosis accompanied with obvious lymphocytic infiltration at the portal areas (Fig. 1A). Sections of liver tissue from rats treated with piperine 20 mg/kg still showed fibrosis at the portal areas with slight lymphocytic infiltration (Fig. 1B). Fibrosis was markedly reduced in sections of liver tissue from rats treated with piperine 40 mg/kg (Fig. 1C), while sections of liver tissue from rats treated with piperine 80 mg/kg showed nearly normal architecture of liver tissue (Fig. 1D).

Figure 3

Figure 1: A photomicrograph of a section of liver tissue from; (A) a control bile duct ligated (BDL) rat showing severe fibrosis accompanied with obvious lymphocytic infiltration at the portal areas extending from it outwards surrounding the hepatic lobule. The lymphocytic infiltration take the shape of a lymphatic nodule (arrow). The normal architecture of liver tissue is still preserved, inspite of the dilated blood sinusoids denoting edema. Many of the hepatocytes show small dense nuclei; (B) BDL rat treated with piperine 20 mg/kg showing fibrosis at the portal areas with slight lymphocytic infiltration and spots of hemorrhage in between (arrow). Strands of fibrous tissue extend from the portal areas in between the hepatocytes. The central vein is slightly dilated and congested with blood (C). Signs of edema is still present and many hepatocytes show small dense nuclei specially near the fibrotic areas denoting second degree of degeneration; (C) BDL rat treated with piperine 40 mg/kg showing a lesser amount of fibrous tissue – if compared with the previous sections- is present at the corners of the hepatic lobule ( at the portal areas) accompanied with slight lymphocytic infiltration. Strands of fibrous tissue extend from these areas in between the hepatocytes in the direction of central vein ( arrows). The portal vein in these areas show congestion with very slight dilatation ( P). Also the central vein (C) shows slight dilatation and congestion. Dilatation of blood sinusoids is still present and only a few of the hepatocytes suffer from pyknosis; (D) BDL rat treated with piperine 80 mg/kg showing nearly normal architecture of liver tissue. Neither fibrosis nor edema is seen in this section. Only a few lymphocytes and collagenous fibers are observed in the portal area (arrow), around the portal vein that shows slight dilatation and congestion (P). Most of the hepatocytes appear normal in shape and color ( Hx. & E. X 100).

Effect of piperine on bone changes

Section of bone tissue from a BDL-control rats showed some small gaps in the matrix of the tissue with widening of the canals normaly present. The osteocytes appeared small in size with dense flattened nuclei. The internal surface of the bone showed irregularities and invasion at some points (Fig. 2A). In rats treated with piperine 20 mg/kg, there were increased number and size of the gaps in the matrix of bone (Fig. 2B), but with the higher dose of 40 mg/kg, decrease in number of the gaps in the bone matrix was seen together with an increase in the amount of collagenous fibers in the matrix of bone tissue. Areas of basophilic cartilaginous tissue appear within the bone tissue representing areas of regeneration (Fig. 2C). Sections of bone tissue from rats treated piperine 80 mg/kg showed increased density of the bone matrix as a result of increased amount of collagenous fibers that become more regularly arranged. No gaps appeared in the bone tissue matrix, but areas of basophilic cartilaginous tissue are still present (Fig. 2D).

Figure 4

Figure 2: A photomicrograph of a section of bone tissue from a control bile duct ligated (BDL) rat showing some small gaps in the matrix of the tissue with widening of the canals normaly present. The osteocytes appear small in size with dense flattened nuclei. The internal surface of the bone (facing the bone marrow cavity) show irregularities and invasion at some points ( arrow); (B) BDL rat treated with piperine 20 mg/kg showing increased number and size of the gaps in the matrix of bone that are localized in the middle of the thickness of bone tissue. Larger gaps are formed from the invasion by osteoclasts from the endosteum and closure after that by bone tissue (arrow). These large gaps carry with them bone marrow tissue. Irregularities begin to appear at the outer surface of the bone (R); (C) BDL rat treated with piperine 40 mg/kg showing much decrease in number of the gaps in the bone matrix in comparison with the previous section and increase in the amount of collagenous fibers in the matrix of bone tissue. Areas of basophilic cartilaginous tissue appear within the bone tissue representing areas of regeneration (arrow). The osteocytes become larger is size (younger and more active cells) and greater in number; (D) BDL rat treated with piperine 80 mg/kg showing increased density of the bone matrix as a result of increased amount of collagenous fibers that become more regularly arranged. The osteocytes are greater in number in comparison with the first 2 sections and smaller in size. Both the external and the internal surfaces of the bone (facing the periosteum and the endosteum respectively) are smooth & regular. No gaps appear in the bone tissue matrix, but areas of basophilic cartilaginous tissue are still present (Hx. & E. X 100).

Discussion

In the present study, we have shown that piperine, the active principle of black pepper and a common dietary additive, is able to protect in a dose-dependent manner against hepatic damage caused by bile duct ligation in rats. Other researchers have shown that piperine protected against tert-butyl hydroperoxide and carbon tetrachloride hepatotoxicity(₁₅). In a previous study, it was shown that capsaicin, the active principle of hot red and green pepper and a legend for the vanilloid receptor (TRPV1) reduced hepatic injury caused by the hepatotoxin CCl₄ in rats. Liver enzymes (alanine transaminase, aspartate transaminase) in the serum and liver necrosis evaluated histologically were significantly reduced in the capsaicin-treated rats. The CCl₄-related disturbances of cellular dysfunction indicated by reduced glycogen and protein content and DNA alterations were ameliorated by capsaicin(₂₁). In the digestive tract, capsaicin-sensitive afferent innvervation participates in nocioception, gastroprotection and intestino-intestinal activation of inhibitory refexes. Sensory neuron stimulation by intragastric administration of capsaicin protects the gastric mucosa of experimental animals against a variety of injurious factors, whereas ablation of extrinsic afferent neurons with a neurotoxic dose of capsaicin impaired gastric mucosal defense with the resultant exacerbation of the chemical-induced injury.(₂₂,₂₃,₂₄) Similar protective effects for sensory nerve stimulation has been demonstrated in the colon(₂₅). In the stomach, sensory neuron-mediated protection of the mucosa involves the protective rise of gastric mucosal blood flow caused by the release of CGRP.(₂₆,₂₇,₂₈) The latter is a potent vasodilator and has been shown to prevent experimental gastric and colonic injury.(₂₉,₃₀,₃₁) Piperine also activates vanilloid receptors (₃₂,₃₃) and although piperine was a less potent agonist than capsaicin , it demonstrated a much greater efficacy (approximately two-fold) at TRPV1.(₇) Piperine also showed gastric mucosal protective properties(₁₂,₃₄), but unlike capsaicin, the mechanism of which is not yet elucidated.

Piperine is well absorbed from the gastrointestinal tract of rat. After oral or intraperitoneal administration, about 97% was absorbed irrespective of the mode of dosing. About 1-2.5% of the intraperitoneally administered piperine was detected in the liver during 0.5-6 h after administration as contrasted with 0.1-0.25% of the orally administered dose(₃₅). When sacs of rat intestines were incubated with piperine, about 44-63% of piperine disappeared from the mucosal side with 7-12% of the absorbed piperine being found in the serosal fluid(₃₆).

It in this way that piperine which activates vanilloid receptors (₃₂,₃₃), can reach hepatobiliary tract and excite sensory nerve endings. Piperine was shown to evoke the release of substance P and CGRP, respectively, from sensory nerves (₃₇,₃₈). The potency ratio between piperine and capsaicin in releasing substance P, however, is 1/50 (₃₈).

The rat hepatobiliary tract is densely innervated by CGRP-containing fibers form dense networks in the fibromuscular layer of the biliary tree and surrounding the portal vein. This suggests the involvement of these peptidergic visceral afferents in regulating hepatobiliary activities, including hemodynamic functions of the hepatic vasculature (₃₉).

Piperine also can reduce hepatic tissue injury by virtue of its antioxidant properties. In this context, it has been shown that piperine exerted a significant protection against tert-butyl hydroperoxide and carbon tetrachloride hepatotoxicity by reducing both in vitro and in vivo lipid peroxidation, and by preventing the depletion of glutathione and total thiols in the intoxicated mice (₁₅). In rats fed a high-fat diet Simultaneous supplementation with black pepper or piperine maintained superoxide dismutase, catalase and reduced glutathione in the liver (₉).

Metabolic bone disease is common among patients with chronic liver disease with osteoporosis accounting for the majority of cases (₄₀) (Rouillard and Lane, 2001). Osteopenia is a classic complication of chronic cholestasis related to primary biliary cirrhosis or primary sclerosing cholangitis (₄₁,₄₂) (Heathcote, 2000; Le Gars, 2002). Both decreased osteoblastic activity and increased osteoclastic activity contribute to the development of osteoporosis in PBC patients (₄₃) (Hodgson et al., 1985).

The present study has in addition demonstrated that bile duct ligation for one month is associated with marked bone changes in the form of gaps in the matrix of the tissue with widening of the canals normally present. The internal surface of the bone showed irregularities and invasion at some points. These bone alterations appear to be decreased by piperine treatment with a decrease in number of the gaps in the bone matrix together with an increase in the amount of collagenous fibers in the matrix of bone tissue and increased density of the bone matrix. The mechanism by which piperine affect bone is not clear and the clinical significance of these findings is yet to be elucidated in view of the fact that piperine is a dietary constituent in humans.