Bioactivities of Iridoids

Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 2007, 6, 307-314 307 1871-5230/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd.Bioactivities of IridoidsIrene M. Villaseñor *Institute of Chemistry, University of the Philippines, Diliman, Quezon City 1101 PhilippinesAbstract: Iridoids discussed in this review were chosen based on their anti-inflammatory activity and their having variousdifferent bioactivities. Harpagoside is the major iridoid glycoside (0.5-1.6%) in Harpagophytum procumbens , an herbalmedicine for rheumatologic conditions. The stem bark of C. ovata is used as an anti-inflammatory drug and catalposide isthe main constituent. Geniposide is a main iridoid glucoside of Gardenia jasmoides , 56.2 mg / 500 mg extract. Gardeniafruits are used for the treatment of anti-inflammatory, hepatic and gall bladder diseases. Catalpol, known for its neuropro-tective activity, is the major constituent of the roots of Rehmannia glutinosa , which are traditionally used for the treatmentof auditory diseases such as tinnitus. Aucubin, which is frequently found as a natural constituent of many traditional me-dicinal plants, is used in the alleviation of chronic allergic inflammatory disease. PicroLiv, an anti-inflammagen, is a stan-dardized fraction from the root and rhizome of Picrorhiza kurroa containing picrosides I and II and kutkoside. Both aucu-bin and PicroLiv have potent hepatoprotective activity.BIOSYNTHESIS OF IRIDOIDSThe iridoids are a group of metabolites characterized byskeletons in which a six-membered ring containing an oxy-gen atom is fused to a cyclopentane ring. They are a widelydistributed class of natural substances, not terpenoid com-pounds in the usual meaning of the term, but of mevalonatein origin and isoprenoid in carbon skeleton.Tracer experiments have shown that iridoids are derivedfrom mevalonic acid. Geranylpyrophosphate [1] is the parentcompound for the monoterpenes. A slightly different foldingof geraniol gives the cyclopentane skeleton of the iridoids.Both hydroxygeraniol and hydroxynerol are efficient precur-sors, as are the corresponding dialdehydes. Cyclization of thedialdehyde into a cyclopentane ring gives iridodial. Furtheroxidation yields an iridotrial which, in its hemiacetal form,yields deoxyloganic acid.The use of labeled iridoids established the steps in theirbiosynthesis with several proposed biogenetic pathways [2].Majority of iridoid compounds are characterized by a cis ringfusion, a cis -2-oxabicyclo-[4,3,0]-nonane, and an 8- -configuration. Deoxyloganic acid was incorporated intotrans -fused iridoid glycosides [3], which indicated that theymay have been formed late in the biosynthesis.The four major groups of iridoids are iridoid glycosides,simple or non-glycosylated iridoids, secoiridoids, and bisiri-doids [1]. Simple iridoids have an iridane skeleton with amethyl at C-8 and another carbon bonded to C-4. Glycosidiclinkages are usually formed at the aglycone C-1 or C-11 hy-droxyl. Cleavage of the 7,8-bond of the cyclopentane ringforms secoiridoids while bisiridoids result from the dimeriza-tion of both iridoids and secoiridoids.Introduction of additional carbons, functional groups anddouble bonds into the iridane skeleton give structural diver-*Address correspondence to this author at the Institute of Chemistry, Uni-versity of the Philippines, Diliman, Quezon City 1101 Philippines; E-mail:irene.villasenor@.phsity to iridoids. Some structural variations are the oxidation of C-6, C-7, C-8 and C-10-methyl, epoxidation of the cy-clopentane ring, introduction of unsaturation, acetoxy and carboxyl groups. IRIDOIDS AS DEFENSE COMPOUNDS The term iridoid is derived from a species of ants, Irido-myrmex , which utilize iridomyrmecin and iridodial in defen-sive secretions. Iridoids have been isolated from the secre-tion of several insects where they play a role in chemical defense and communication. They are irritants and deter most predators. Iridoid glycosides, aucubin and catalpol, present in Plantago lanceolata (Plantaginaceae) deter feed-ing by the beet armyworm herbivore Spodoptera exigua and the biotropic fungal pathogen Diaporthe adunca [4]. A cya-nogenic glycoside esterified with an iridoid glycoside, 2R-[(2-methoxybenzoylgenoposidyl)-5-O-beta-D-apiofuranosyl-(1 6)-beta-glucopyranosyloxy]-2-phenyl acetonitrile, sho-wed weak retarding activity towards neonate larvae of Spodoptera littoralis , commonly known as Egyptian cotton leafworm [5]. Induction of allelochemicals is another way that plants efficiently counteract injury as shown by increased levels of aucubin and catalpol in P. lanceolata following damage by D. adunca [6] and by caterpillars of the specialist Junonia coenia , the Common Buckeye butterfly [7]. Iridoids also affect the development of herbivores and their parasitoids [8] and some sequester these iridoids for their own defense [9]. The larvae of Ceratomia catalpae (Sphingidae) (Catalpa sphinx or Catawba worm), which are the major defoliators of Catalpa species, and the hemolymph in which its parasitoid Cotesia congregate wasp larvae grew contained high amounts of catalpol [10]. J. coenia larvae also sequestered aucubin and catalpol and both are retained in the pupae [11]. Oviposition of Melitaea cinxia , a very common butterfly, was enhanced by aucubin and catalpol and both acted as feeding stimulants to the larvae [12] and as feeding deter-rents to Cotesia melitaearum , its parasitoid wasp [13] .308 Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 2007, Vol. 6, No. 4 Irene M. VillaseñorBIOACTIVITIES OF IRIDOIDSAlthough iridoids have a wide range of biological activi-ties, they are most commonly known for their anti-inflammatory activity. Harpagoside, catalposide, geniposide and derivatives genipin, geniposidic acid and pentaacetyl-geniposide, catalpol and aucubin, and PicroLiv, a mixture of picroside I, picroside II and kutkoside (Fig. 1) are representa-tive of anti-inflammatory iridoids that are multifunctional.Fig. (1). Structures of IridoidsHARPAGOSIDEHarpagoside is the major substituent (0.5-1.6%) of Har-pagophytum procumbens (Pedaliaceae, subfamily Scrophu-lariaceae), an herbal medicine for rheumatologic conditions [14]. H. procumbens is used in the treatment of rheumatoid arthritis, osteoarthritis and low back pain [15-17], glomerular inflammatory disease [18] and arthrosis of the hip and knee [19, 20]. The crude methanolic extract of H. procumbens secondary roots caused a reduction of arterial blood pressure, heart rate and coronary flow and showed a protective action against arrhythmias [21, 22]. Its dried aqueous [23] and ethanolic [24] extracts showed anti-inflammatory and anal-gesic effects.The anti-inflammatory actions of H. procumbens may be due to its action on eicosanoid biosynthesis [14] and / or the migration of interleukins and leucocytes to the inflamed joint area [24]. It decreased ionophore A23187-stimulated cyste-inyl-leukotriene (cys-LT) levels [25]. Its aqueous extract suppressed prostaglandin E2 (PGE2) synthesis and cyclooxy-genase-2 (COX-2) and inducible nitric oxide synthase (i-NOS) expressions in fibroblast cell line L929 [26]. Extracts interfere with the transcriptional activation of iNOS [18]. iNOS generates high amounts of nitric oxide (NO), which mediates and regulates inflammatory responses, in activated inflammatory cells.H. procumbens prevented lipopolysaccharides (LPS)-induced synthesis of tumour necrosis factor alpha (TNF-alpha) [27].TNF-alpha is a cytokine involved in systemic inflammation and it causes apoptotic cell death, cellular pro-liferation, differentiation, inflammation, tumorigenesis and viral replication. It regulates immune cells and its overpro-duction leads to a variety of human diseases.H. procumbens contains 0.5-3% iridoid glycosides. The iridoid glycosides harpagoside, 6’-O- -D-galactopyranosyl-harpagoside, harprocumbide A (6”-O- -D-galactopyranosyl-harpagoside), harpagide, 8-O-feruloylharpagide, 8-O-(p-coumaroyl)harpagide, 8-O-(cis-p-coumaroyl)harpagide, 8-cinnamoylmyoporoside, procumbide, 6”-O-(p-coumaroyl) procumbide, harprocumbide B (6”-O-(cis-p-coumaroyl) pro-cumbide) [28], 6’-O-(p-coumaroyl)harpagide and 6’-O-(cis-p-coumaroyl)harpagide [29] were isolated from the tubers of H. procumbens.Harpagophytum extracts and its pharmaceutical prepara-tions are standardized [30, 31] with at least 50 mg harpa-goside [32-36]. Harpagoside is effective in the treatment of degenerative rheumatoid arthritis, osteoarthritis, tendonitis, kidney inflammation and heart disease [37]. It showed anal-gesic activity [23] but did not possess anti-inflammatory properties [23, 38] although harpagoside-enriched extracts showed high anti-inflammatory activity [39]. It suppressed NF-kappaB activation and inhibited the expression of cy-clooxygenase-2 and iNOS in human HepG2 hepatocarci-noma cells [40]. NF-kappaB is a transcription factor present in immune cells that plays an important role in the inflamma-tory and immune response by regulating pro-inflammatory cytokines, interleukin-1, -2, -6, -8, and pro-inflammatory mediators, ICAM-1 (Intercellular Adhesion Molecule – 1), iNOS and COX-2.Pharmacokinetic studies showed maximum levels of plasma harpagoside were reached after 1.3 to 2.5 hours and serum harpagoside levels inhibited leukotriene biosynthesis [25]. It had no effect on LPS-induced TNF-alpha release [27] but it was able to inhibit thromboxane- synthase enzyme, a cytochrome P450 enzyme that catalyzes the conversion of the prostaglandin endoperoxide into thromboxane A2, a po-Bioactivities of Iridoids Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 2007, Vol. 6, No. 4 309tent vasoconstrictor and inducer of platelet aggregation. [41].It also interfered with the mechanisms that regulate the in-flux of calcium in smooth muscle cells [42]. CATALPOSIDEThe iridoids 6-O-trans- and 6-O-cis-p-coumaroyl-7-deoxyrehmaglutin A, 6-O-trans- and 6-O-cis-p-coumaroyl-3--O-methyl-7-deoxyrehmaglutin A, 6-O-trans- and 6-O-cis-p-coumaroyl-3- -O-methyl-7-deoxyrehmaglutin A, 6-O-trans- and 6-O-cis-p-coumaroyl-1 -O-methyllovatofuranicacid methyl esters were isolated from Catalpa ovata G. Don(Bignoniaceae) [43] with catalposide as the main constituent. The stem bark of C. ovata is used as an anti-inflammatory drug in traditional Korean medicine [44]. It controls inflam-mation by inhibiting the production of TNF-alpha and NO on RAW 264.7 macrophages with significant decreases in mRNA levels of TNF-alpha and iNOS. The leaves have also been used for the treatment of burns and athlete’s foot and the extract showed antibacterial activity [43].Catalposide possessed antinociceptive and anti-inflammatory activities [45] and may be used for the treat-ment of diseases characterized by mucosal inflammation[46]. It suppressed the expression of TNF-alpha, interleukin-1beta (IL-1beta), intercellular adhesion molecule-1 and the nuclear translocation of NF-kappa B p65 in trinitrobenzene sulfonic acid (TNBS)-induced inflammatory colitis [46] as well as IL-6 in LPS-activated RAW 264.7 macrophages [47]. It also decreased IL-8 secretion, increased TNF-alpha-mediated p38 and extracellular signal-regulated kinase (ERK) phosphorylation and decreased NF-kappaB-mediated transcriptional activation and Ikappa-Balpha degradation in human intestinal epithelial HT-29 cells [46]. It significantly inhibited NO production in LPS-stimulated RAW 264.7 macrophages, cytokine-stimulated human DLD-1 and rat vascular smooth muscle (VSM) cells [48].Catalposide also have antimicrobial, especially on gram-negative bacterial infections [47], and antitumour activities.It is also a potent inducer of heme-oxygenase-1 (HO-1),which explains the protective role of catalposide against oxi-dative injury [49].GENIPOSIDE, GENIPIN, GENIPOSIDIC ACID Geniposide is a main iridoid glucoside of Gardenia jas-moides Ellis (Rubiaceae), 56.2 mg / 500 mg extract [50]. The fruits of Gardenia are used as a food colorant and as a tradi-tional Chinese medicinal for the treatment of anti-inflammatory, hepatic [51] and gall bladder [52] diseases. It has angiogenic [53], anxiolytic [54] and analgesic [55] ac-tivities.Geniposide lowered serum IL-1beta and TNF-alpha lev-els in rheumatoid arthritic rats. It showed angiogenic activityin the chick embryo chorioallantoic membrane (CAM) assay and inhibited the growth of transformed NIH3T3 [53]. It exhibited potential anti-asthma activity in guinea pig trachea [56]. It suppressed collagen-induced mouse platelet aggrega-tion and thus has anti-thrombotic activity [57].Geniposide is a potential chemopreventive agent against tumor promotion [58] and early acute aflatoxin B1 (AFB1) hepatotoxicity [59]. Its topical application decreased 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted mouse skin tumors, TPA-caused induction of epidermal ornithine decarboxylase (ODC) activity, and TPA-induced edema of mouse ears. Pre-treatment with geniposide inhibited TPA-induced hyperplasia in ear skin and formation of hydrogen peroxide and myeloperoxidase in mouse skin. Geniposide pre-treatment of AFB1 treated rats decreased AFB1-DNA adduct formation and lowered the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma-glutamyltranspeptidase (gamma-GT). AST, ALT and gamma-GT are marker enzymes of hepatic dam-age.The chemopreventive activity of geniposide may be due to its ability to enhance the activity of glutathione-S-transferase (GST), a major group of detoxification enzymes, and glutathione-peroxidase (GSH-Px) [59, 60] by activating and increasing expression of Ras/Raf/MEK-1 signaling me-diators [61]. Another anti-tumour mechanism is its ability to inhibit topoisomerase I [62], but not topoisomerase II. Its hepatoprotective activity is due to its ability to decrease liver P-450 dependent monooxygenases and increase total hepatic glutathione content and hepatic cytosolic glutathione-S-transferase activity [51].Geniposide also exhibited immunosuppressive activity [63] as shown by its inhibition of IL-2 secretion by phorbol myristate acetate and anti-CD28 monoclonal antibody co-stimulated activation of human peripheral blood T cells. It protected neuronal cells from damage [64] and induced neu-ronal differentiation of paraneurons rat pheochromocytoma (PC12) cells with resulting neuritis outgrowth [65] through activation of intracellular signal transduction pathway [66].When geniposide is orally administered, it is metabolized to genipin, its aglycone, in the intestine [67]. This may ex-plain the apparent hepatotoxicity of geniposide when admin-istered orally [68] as evidenced by increased serum ALT and AST activities. The hepatotoxicity of geniposide is compara-ble to the hepatotoxic effects of intraperitoneal (ip) admini-stration of genipin. The hepatotoxicity of geniposide upon oral administration was suppressed by ip administration of geniposide or by pretreatment with chloramphenicol.Genipin is also naturally present in the ethanol extract of Gardenia fruit [55] and mimics the bioactivities of geniposide. It is a stronger anti-inflammagen than geniposide in carrageenan-induced rat paw edema and inhibited more the production of exudates and NO in the carrageenan-induced rat air pouch assay. It also exhibited anti-inflammatory effect when applied topically to croton oil-induced edema in mice [69]. Its anti-inflammatory activity is due to the decrease in NO production and iNOS expression through the inhibition of NF-kappaB activation. It is a radi-cal scavenger as it markedly decreased lipid peroxidation and is a potent angiogenic in the chick embryo CAM assay.Genipin induced apoptotic cell death in FaO rat hepatoma cells and human hepatocarcinoma Hep3B cells [70] mediated by NADPH oxidase-dependent generation of reactive oxy-gen radicals (ROS). Ip administration of genipin showed bile acid-independent choleretic activity [71]. Infusion of genipin in rat liver increased bile flow and biliary secretion of biliru-bin conjugates but did not increase bile acid secretion [72].310 Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 2007, Vol. 6, No. 4 Irene M. VillaseñorIts choleretic activity may be due to the stimulation of multidrug resistance-associated protein 2 (Mrp2-Abcc2) in the canalicular membrane vesicles. Genipin has prominent neuritogenic activity [66]. It also protected the hippocampal neurons from amyloid beta protein (Abeta) toxicity, which is the main constituent of amyloid plaques in the brains of Alz-heimer’s disease patients [73]. Geniposide is less effective in preventing Abeta toxicity.Geniposide is metabolized to geniposidic acid by es-terases. However, in vitro enzymatic studies showed that geniposide is first hydrolyzed to genipin by beta-D-glucosidases and then to the aglycone of geniposidic acid by esterases [67]. Geniposidic acid is a more potent mouse tu-mor growth inhibitor than geniposide and decreased undesir-able radiation damage after high dose, whole body X-irradiation. [74]. Geniposidic acid may also be effective against aging as it improved the turnover rate in the stratum of false aged model rats [75] and stimulated collagen synthe-sis [76]. Both geniposide and geniposidic acid showed activ-ity against osteoporosis [77]. Both increased the proliferation of osteoblasts, to facilitate osteogenesis, and inhibited prolif-eration of osteoclast, to inhibit osteolysis. Geniposidic acid significantly decreased heart rate, coronary perfusion rate (CPR) and the strength of the contractility of the heart mus-cle in rat hearts [78].Another structurally related iridoid is pentaacetyl geniposide, which was obtained from a modified extract of Gardenia fructus [79,80]. It inhibited the growth and devel-opment of C-6 glioma cells inoculated in rats [81] resulting to cell death, chromatin condensation, and internucleosomal DNA ladder [82]. It reduced plating efficiency and DNA synthesis in cultured C-6 glioma cells but had little effect on RNA and protein synthesis [79]. Cell death is mediated by apoptosis-related Bcl-2 family proteins and cell cycle arrest at G(0)/G(1) induced by p53 and c-Myc [82]. Correlated with increased p53 levels is the increased expression of p21 protein, a cyclin-dependent kinase (CDK) inhibitor, that di-rectly inhibited the activity of cyclin D1/ CDK 4 complex [83]. Furthermore, pentaacetyl geniposide activated neutral spingomyelinase (N-SMase) [84], enhanced the binding of nerve growth factor (NGF) p75, which activated protein kinase C delta (PKC delta) [85] and signaled apoptosis by c-Jun N-terminal phosphorylation (JNP) involving the stress kinase JNK (Jun N-terminal kinase) [86] thus expressing Fas-L and caspase 3 activation. Moreover, it decreased DNA damage and AFB1-induced hepatocarcinogenesis by activat-ing GST and GSH peroxidase [52]. In vivo experiments showed that pentaacetyl geniposide is not harmful to liver, heart and kidney.CATALPOLCatalpol is the major constituent of the roots of Rehman-nia glutinosa Libosch.Steamed roots have been traditionally used for the treatment of auditory diseases such as tinnitus and hearing loss [87,88], dementia [89], inflammations [90], and age-related diseases [91]. Its ethanol extract showed anti-oxidant activity [87, 88] on HEI-OC1 auditory cells. It increased the scavenging activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), glutathione (GSH) [87], ascor-bate peroxidase, non-specific peroxidase [92] and reduced lipid peroxidation [88]. Acteoside, a phenolic compound, was identified as the antioxidant component [93].Oral administration of stachyose extract from R. gluti-nosa lowered plasma glucose in normal, glucose- and adrenaline-induced hyperglycemic and alloxan-induced dia-betic rats [94]. When tested using the same protocols, R. glutinosa oligosaccharide was inactive against adrenalec-tomized rats, which indicates that its mechanism on glucose metabolism may be affected by the neuroendocrine system [95].The aqueous extract of R. glutinosa steamed root has anti-inflammatory activity [96]. It decreased TNF-alpha se-cretion by inhibiting IL-1 secretion in substance P and LPS-stimulated mouse astrocytes. It also selectively inhibited cyclooxygenase activity [90]. Its administration in rats with ischemia-reperfusion induced acute renal failure repaired renal defects and improved creatinine clearance, urinary so-dium excretion, urinary osmolality and solute-free reabsorp-tion [97].Its hot water extract inhibited the proliferation of rat (H-4-II-E) and human (HA22T/VGH) hepatocellular carcinoma cell lines and stimulated p53-mediated apoptosis [98]. R. glubosa extracts also enhanced bone metabolism in osteopo-rosis [99] in both in vitro and in vivo, using ovariectomy-induced osteoporotic rats, tests. It increased cell proliferation and alkaline phosphatase activity, mRNA expression of bone-related genes, and osteoprotegenin secretion of os-teoblasts and prevented osteoporotic bone loss.The neuroprotective activity of catalpol is well-studied. Its neuroprotective activity in gerbils subjected to transient global cerebral ischemia is apparent when applied immedi-ately and up to 3 hours after ischemia and in both short (12 days) and long (35 days) post-ischemic periods (100, 101). Catalpol significantly reduced lipid peroxide content in the cortex and hippocampus, increased the activity of GSH-Px and decreased the activity of iNOS (102), which indicates that neuroprotection is partly due to its anti-oxidant activity and the reduction of NO formation. Catalpol inhibited apop-tosis by modulating the expressions of anti-apoptotic Bcl-2 and pro-apoptotic Bax genes (103).Catalpol also increased hippocampal levels of synapto-physin, which enhances neuroplasticity and neuronal hyper-trophy; PKC, which phosphorylates growth associated pro-tein (GAP) 53; brain derived neurotrophic factor (BDNF), loss of which leads to brain deterioration; and GAP 53, which, in phosphorylated form, enhances learning and mem-ory (91). The neuroprotection of catalpol extends to dopa-minergic neurons (104) and may thus be effective against inflammation-related neurodegenerative diseases such as Parkinson’s disease.Catalpol inhibitedH2O2-induced apoptosis in PC12 cells [105] by up-regulating Bcl-2 gene, down-regulating Bax gene, suppressing the release of mitochondrial cytochrome c to the cytosol and thus weakening caspase- and caspase-3 activation. Catalpol is a Taq DNA polymerase inhibitor and a possible mechanism is its competition with dNTPs at the binding site of the enzyme (106).Bioactivities of Iridoids Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 2007, Vol. 6, No. 4 311AUCUBINAucubin is frequently found as a natural constituent of many traditional medicinal plants. It exhibited significant anti-inflammatory activity in carrageenan-induced hind paw edema assay and antinociceptive activity in p-benzoquinone-induced writhings in mice [107] but it did not affect the pro-duction of TNF-alpha in RAW 264.7 cells [108]. Hydrolysis of aucubin by beta-glucosidase yielded a product that blocked TNF-alpha formation, I-kappa B alpha degradation and NF-kappa B translocation. The anti-inflammatory activ-ity of aucubin in antigen (Ag)-induced rat basophilic leuke-mia (RBL)-2H3 mast cells justified its use in the treatment of chronic allergic inflammatory disease [109]. It inhibited Ag-induced TNF-alpha and IL-6 production and expression, NF-kappa B nuclear translocation and I-kappa B alpha degrada-tion.Aucubin showed potential as an anti-aging agent. It in-hibited the formation of matrix metalloproteinase-1 (MMP-1), a matrix degrading protease involved in skin photodam-age, and expression of MMP-1 mRNA in ultraviolet B (UVB)-irradiated human fibroblasts HS68 cell line [110], which was induced by ROS [111]. Pretreatment with aucubin also decreased senescence-associated-beta-galactosidase, an enzyme expressed by cells no longer dividing but still me-tabolically active. Aucubin also induced collagen synthesis in aged model rats [112].Other biological activities ascribed to aucubin are its try-panocidal activity against Trypanosoma brucei rhodesiense [113]; its potential activity against osteoporosis by enhancing osteogenesis and suppressing osteolysis [77]; its anti-tumour activity by inhibiting topoisomerase I [62]; its immunostimu-lating activity as a result of enhanced human lymphocyte proliferation and secretion of interferon gamma (IFN-gamma) [114]; its antispasmodic activity in uterine, through non-competitive antagonism against acetylcholine and com-petitive antagonism of calcium, and vas deferens muscles [115]; and its weak anti-leukemic [116] activity against hu-man leukemia and lymphoma lines.The hepatoprotective activity of aucubin is well-studied [117]. It protected mice from liver damage induced by CCl4, as shown by decreased serum glutamic-oxaloacetic (GOT) and glutamic pyruvic transaminases (GPT) levels [118], which are high in acute and chronic hepatitis, obstructive jaundice, carcinoma of liver, myocardial infraction and mus-cular dystrophy. It also protected mice from alpha-amanitin poisoning [119]. Upon ingestion, alpha-amanitin binds to RNA polymerase II causing cytolysis of hepatocytes leading to renal and liver failure. It was, however, aucubigenin, the aglucone of aucubin, which inhibited both DNA and RNA polymerase in Hep G2 cells [120]. Aucubigenin can form imine bonds with proteins [121] through Schiff reactions [122] mainly due to its structural similarity to glutaralde-hyde.PICROSIDE I, KUTKOSIDE, PICROSIDE IIPicroLiv is a standardized fraction from the root and rhi-zome of Picrorhiza kurroa Royle ex Benth (Scrophu-lariaceae) or kutki, which is found in the North-Western Hi-malayan region from Kashmir to Kumaun and Garhwal re-gions in India and Nepal. Powdered roots of P. kurroa, its alcoholic extract and active constituents, kutkin, picroside I and kutkoside showed anti-inflammatory activity in adju-vant-induced arthritis, a model of chronic inflammation that exhibits several pathological changes similar to those occur-ring in rheumatoid arthritis [123], formaldehyde- and car-rageenan-induced edema [124].The effect of PicroLiv against hepatotoxicity and hepato-carcinogenesis is well-documented. It prevented aflatoxin B1- , a potent hepatotoxic and hepatocarcinogenic myco-toxin, [125-127], D-galactosamine (GalN)- [128], N-nitrosodiethylamine (NDEA)-, a hepatocarcinogen, [129], ethanol- [130], monocrotaline-, a pyrrolizidine alkaloid, [131], CCl4- [132], paracetamol- [133, 134], rifampicin- [135], thioacetamide- [136] and A. phalloides- [137, 138] induced liver damage. In all cases, PicroLiv significantly normalized the liver histology and biochemical changes in liver and serum of rats. Its hepatoprotective effect was ap-parent when administered simultaneously or as a pre-treatment. PicroLiv may affect the biotransformation of these toxic substances and its protective effect may be due to the decreased formation of reactive metabolites [139].Pretreatment with PicroLiv also protected rats against hepatic ischemia-reperfusion injury [140] by decreasing apoptosis, oxidant induced cellular damage, neutrophil infil-tration and tissue levels of IL-1 alpha and IL-I beta; and in-creasing the levels of intracellular superoxide dismutase and proliferative cell nuclear antigen (PCNA) immunoreactivity. In patients with acute viral hepatitis, PicroLiv-treated pa-tients showed significant difference in bilirubin, serum GOT and GPT values compared to placebo [141]. In vitro studies showed that PicroLiv has anti-hepatitis virus surface antigen (HBsAg) like activity and inhibited purified hepatitis B virus (HBV) [142]. The HBsAg positive serum was obtained from patients with HBV associated acute and chronic liver dis-eases and healthy HBsAg carriers.PicroLiv is a potent antioxidant and its hepatoprotective activity is partially due to its ability to suppress lipid peroxi-dation. If effectively reversed the marked increase in lipid peroxide levels and decreased antioxidant levels in aflatoxin B1-induced lipid peroxidation in rat liver and kidney [143]. It also inhibited the lipid peroxidation of serum beta-lipoproteins of M. coucha infected with P. berghei, rodent malarial parasites [144]. Pretreatment of rats with renal ischemia-reperfusion injury reduced lipid peroxidation, im-proved antioxidant levels and reduced apoptosis [145]. PicroLiv mimics inhibitors of xanthine oxidase, superoxide dismutase, and metal ion chelators because it effectively quenched the enzymatic, in xanthine –xanthine oxidase sys-tem, and non-enzymatic, in phenazine methosulphate NADH system, generation of superoxide anions [146]. It also sup-pressed malonaldehyde generation in rat liver microsomes stimulated by ascorbate-Fe+2 and NADPH-ADP-Fe+2 sys-tems. It improved the activities of various microsomal en-zymes related to drug metabolism, such as aniline hydroxy-lase, aminopyrine-N-methylase, benzopyrene hydroxylase, NADH/NADPH cytochrome C reductase, glutathione-S-transferase, cytochrome P-450, and cytochrome b5, in CCl4-intoxicated rats [147]. Even after partial hepatectomy, rats pretreated [148] or treated [149] with PicroLiv recovered。