Galactosamine-induced hepatotoxic effect and hepatoprotective role of a protein isolated from the herb Cajanus indicus L in vivo

dd(+)-Galactosamine is a well-known experimental hepatotoxin. The present study was conducted to determine the protective role of a 43-kD protein isolated from the leaves of the herb Cajanus indicus L against D(+)-galactosamine (GalN) induced liver damage in mice. Both preventive and curative effects of the protein have been investigated in the study. The protein was administered intraperitoneally at a dose of 2 mg/kg body weight for 4 days before and after GalN intoxication at a dose of 800 mg/kg body weight for 3 days. The increased activities of serum marker enzymes, alanine aminotransferase, and alkaline phosphatase because of GalN administration, were significantly reduced by the protein treatment. The protein also normalized the altered activities of antioxidant enzymes superoxide dismutase, catalase, glutathione reductase, and glutathione-S-transferase as well as the levels of cellular metabolites, reduced glutathione, glutathione disulfide, and total thiols. In addition, the enhanced hepatic lipid peroxidation because of GalN intoxication was also effectively inhibited by the protein treatment. Results suggest that GalN caused hepatic damages via oxidative insult and that the protein provided protection through its antioxidant mechanism.     

Protective effect of a 43 kD protein from the leaves of the herb, Cajanus indicus L on chloroform induced hepatic-disorder

Cajanus indicus is a herb with medicinal properties and is traditionally used to treat various forms of liver disorders. Present study aimed to evaluate the effect of a 43 kD protein isolated from the leaves of this herb against chloroform induced hepatotoxicity. Male albino mice were intraperitoneally treated with 2 mg/kg body weight of the protein for 5 days followed by oral application of chloroform (0.75 ml/kg body weight) for 2 days. Different biochemical parameters related to physiology and pathophysiology of liver, such as, serum glutamate pyruvate transaminase and alkaline phosphatase were determined in the murine sera under various experimental conditions. Direct antioxidant role of the protein was also determined from its reaction with Diphenyl picryl hydroxyl radical, superoxide radical and hydrogen peroxide. To find out the mode of action of this protein against chloroform induced liver damage, levels of antioxidant enzymes catalase, superoxide dismutase and glutathione-S-transferase were measured from liver homogenates. Peroxidation of membrane lipids both in vivo and in vitro were also measured as malonaldialdehyde. Finally, histopathological analyses were done from liver sections of control, toxin treated and protein pre- and post-treated (along with the toxin) mice. Levels of serum glutamate pyruvate transaminase and alkaline phosphatase, which showed an elevation in chloroform induced hepatic damage, were brought down near to the normal levels with the protein pretreatment. On the contrary, the levels of antioxidant enzymes such as catalase, superoxide dismutase and glutathione-S-transferase that had gone down in mice orally fed with chloroform were significantly elevated in protein pretreated ones. Besides, chloroform induced lipid peroxidation was effectively reduced by protein treatment both in vivo and in vitro. In cell free system the protein effectively quenched diphenyl picryl hydroxyl radical and superoxide radical, though it could not catalyse the breakdown of hydrogen peroxide. Post treatment with the protein for 3 days after 2 days of chloroform administration showed similar results. Histopathological studies indicated that chloroform induced extensive tissue damage was less severe in the mice livers treated with the 43 kD protein prior and post to the toxin administration. Results from all these data suggest that the protein possesses both preventive and curative role against chloroform induced hepatotoxicity and probably acts by an anti-oxidative defense mechanism.     

A 43 kD protein isolated from the herb Cajanus indicus L attenuates sodium fluoride-induced hepatic and renal disorders in vivo

The herb, Cajanus indicus L, is well known for its hepatoprotective action. A 43 kD protein has been isolated, purified and partially sequenced from the leaves of this herb. A number of in vivo and in vitro studies carried out in our laboratory suggest that this protein might be a major component responsible for the hepatoprotective action of the herb. Our successive studies have been designed to evaluate the potential efficacy of this protein in protecting the hepatic as well as renal tissues from the sodium fluoride (NaF) induced oxidative stress. The experimental groups of mice were exposed to NaF at a dose of 600 ppm through drinking water for one week. This exposure significantly altered the activities of the antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione reductase (GR) and the cellular metabolites such as reduced glutathione (GSH), oxidized glutathione (GSSG), total thiols, lipid peroxidation end products in liver and kidney compared to the normal mice. Intraperitoneal administration of the protein at a dose of 2 mg/kg body weight for seven days followed by NaF treatment (600 ppm for next seven days) normalized the activities of the hepato-renal antioxidant enzymes, the level of cellular metabolites and lipid peroxidation end products. Post treatment with the protein for four days showed that it could help recovering the damages after NaF administration. Time-course study suggests that the protein could stimulate the recovery of both the organs faster than natural process. Effects of a known antioxidant, vitamin E, and a non-relevant protein, bovine serum albumin (BSA) have been included in the study to validate the experimental data. Combining all, result suggests that NaF could induce severe oxidative stress both in the liver and kidney tissues in mice and the protein possessed the ability to attenuate that hepato-renal toxic effect of NaF probably via its antioxidant activity.     

Effect of crude sulphated polysaccharide from brown algae against acetaminophen-induced toxicity in rats

This study was conducted to examine the protective role of crude polysaccharide from brown seaweed Sargassum polycystum against acetaminophen-induced abnormality in blood glucose, serum albumin/globulin ratio, and liver glycogen, lactate, and pyruvate. Liver and renal tissue histology was performed to confirm the efficacy of Sargassum polysaccharide. A toxic dose of acetaminophen (800 mg/kg body weight intraperitoneally) induced severe abnormality in all basic parameters with apparent toxicity in liver (enlargement of hepatocytes, loss of cytoplasmic content with disruption in the hepatic plates and sinusoidal dilation) and renal tissue (glomerular damage with congestion of tubules). The isolated liver cells were stained with acridine orange and examined under fluorescence microscope, which revealed that the acetaminophen induced significant damage. In contrast, the rats pretreated with Sargassum polysaccharide (200 mg/kg body weight) daily for 3 weeks did not show liver and renal tissue with these severe aberrations induced by acetaminophen. Histology results were also consistent with analyzed basic biochemical parameters, which confirmed the effectiveness of the crude polysaccharide against acetaminophen-induced abnormality in rats.     

Protective effects of ginkgo biloba against acetaminophen-induced toxicity in mice

Background: The analgesic acetaminophen (AAP) causes a potentially fatal, hepatic centrilobular necrosis when taken in overdose. It was reported that these toxic effects of AAP are due to oxidative reactions that take place during its metabolism. Objective: In this study, we aimed to investigate the possible beneficial effect of Ginkgo biloba (EGb), an antioxidant agent, against AAP toxicity in mice. Methods: Balb/c mice were injected i.p. with: (1) vehicle, control (C) group; (2) a single dose of 50 mg/kg Ginkgo biloba extract, EGb group; (3) a single dose of 900 mg/kg i.p. acetaminophen, AAP group, and (4) EGb, in a dose of 50 mg/kg after AAP injection, AAP + EGb group. Serum ALT, AST, and tumor necrosis factor-alpha (TNF-α) levels in blood and glutathione (GSH), malondialdehyde (MDA) levels, myeloperoxidase (MPO) activity, and collagen contents in liver tissues were measured. Formation of reactive oxygen species in hepatic tissue samples was monitored by using chemiluminescence (CL) technique with luminol and lusigenin probe. Tissues were also examined microscopically. Results: ALT, AST levels, and TNF-α were increased significantly (p < 0.001) after AAP treatment, and reduced with EGb. Acetaminophen caused a significant (p < 0.05–0.001) decrease in GSH levels while MDA levels and MPO activity were increased (p < 0.001) in liver tissues. These changes were reversed by EGb treatment. Furthermore, luminol and lusigenin CL levels in the AAP group increased dramatically compared to control and reduced by EGb treatment (p < 0.01). Conclusion: Our results implicate that AAP causes oxidative damage in hepatic tissues and Ginkgo biloba extract, by its antioxidant effects protects the tissues. Therefore, its therapeutic role as a “tissue injury-limiting agent” must be further elucidated in drug-induced oxidative damage.     

Characterization of a proteinase inhibitor from Cajanus cajan (L.)

A protein proteinase inhibitor (PI) has been purified from pigeonpea Cajanus cajan (L.) PUSA 33 variety by acetic-acid precipitation, salt fractionation and chromatography on a DEAE-Cellulose column. The content of inhibitor was found to be 15 mg/20 g dry weight of pulse. The molecular weight of the inhibitor as determined by SDS-PAGE under reducing conditions was found to be about 14,000. It showed inhibitory activity toward proteolytic enzymes belonging to the serine protease group, namely trypsin and alpha-chymotrypsin. The inhibitory activity was stable over a wide range of pH and temperatures. Estimation of sulfhydryl groups yielded one free cysteine and at least two disulfide linkages. N-terminal sequence homology suggests that it belongs to the Kunitz inhibitor family. Structural analysis by circular dichroism shows that the inhibitor possesses a largely disordered structure.     

Protective effect of diallyl sulfone against acetaminophen-induced hepatotoxicity in mice

Diallyl sulfone (DASO₂) is a metabolite of diallyl sulfide, a compound derived from garlic. The present study investigated the effect of DASO₂ as a protective agent against acetaminophen (APAP)-induced hepatotoxicity in mice. Oral administration of DASO₂ protected mice against the APAP-induced hepatotoxicity in a dose- and time-dependent manner. When administrated 1 hour prior to, immediately after, or 20 minutes after a toxic dose of APAP, DASO₂ at a dose of 25 mg/kg completely protected mice from development of hepatotoxicity, as indicated by liver histopathology and serum lactate dehydrogenase levels. Protective effect was observed when DASO₂ at a dose as low as 5 mg/kg was given to mice 1 hour prior to APAP administration. Oral administration of DASO₂ to mice 1 hour prior to a toxic dose of APAP significantly inhibited the APAP-induced glutathione depletion in the liver. DASO₂ treatment also decreased the levels of oxidative APAP metabolites in the plasma without affecting the concentrations of nonoxidative APAP metabolites. In liver microsomes, 0.1 mM of DASO₂ caused a 60% decrease in the rate of APAP oxidation to N-acetyl-p-benzoquinone imine, which was determined as glutathione conjugate. This inhibitory effect is mainly due to its inhibition of cytochrome P450 2E1 activity; with an IC₅₀ value equal to 0.11 mM. DASO₂ also slightly inhibited the activities of P450s 3A and 1A, with IC₅₀ values >5 mM. Furthermore, a single oral dose of DASO₂ inactivated P450 2E1- and P450 1A-dependent activities in liver microsomes. The results suggest that the protective effect of DASO₂ against APAP-induced hepatotoxicity is due to its ability to block acetaminophen bioactivation mainly by the inactivation and inhibition of P450 2E1. © 1996 John Wiley & Sons, Inc.     

Rescue effect of curcumin against copper toxicity

Turmeric has long been used not only as an indispensable part of Asian cuisine but as a medicinal herb for dressing wounds, bites, burns, treating eye infections and acne. Curcuminoids are the active substances and their synthetic derivatives (i.e. diacetylcurcumin (DAC) and metal-curcumin complexes) possess an incredibly wide range of medicinal properties that encompass chelation capacity for multiple heavy metals, antioxidant activity, anti-inflammatory properties, cytotoxicity against cancerous cells, antiviral and antibacterial effects, antihypertensive and insulin sensitizing role, and regulatory role on apoptosis. The aforementioned properties have put curcumin on spotlight as a potential treatment for ailments such as, hepatic diseases, neurodegenerative diseases, metabolic syndrome, dyslipidemia, cardiovascular disease, auto-immune diseases, malignancies and conditions associated with metal overload. Copper is essential for major biological functions, however, an excess causes chronic ailments including neurodegenerative disorders. The fascinating approach of curcumin could alleviate such effect by forming a complex. Thus, this review aims to present available data on the effect of copper-curcumin interaction in various in vitro, ex-vivo in vivo, and clinical studies.     

Direct Differentiation of Shoot Buds from Leaf Explants of Cajanus Cajan L.

A protocol was developed for direct differentiation of multiple shoot buds from leaf explants of Cajanus cajan. In a modified Murashige and Skoog's medium supplemented with 2.22 µM benzyladenine (BA), 0.57 µM indole-3-acetic acid (IAA) and 41 µM adenine sulphate (AdS), the segments of basal halves of the first two leaves of a young seedling incubated on filter paper bridges in liquid medium took 20 – 25 d to differentiate shoot buds. The explants after transfer to solidified medium, with lower concentration of BA (0.22 M) resulted in fast growing healthy shoots. The developed shoots (measuring ca. 3 cm) were rooted in a medium supplemented with 1.42 µM IAA. They were subsequently grown in pots with soil with more than 80 % transplantation success.     

Role of interleukin-6 in hepatic heat shock protein expression and protection against acetaminophen-induced liver disease

Recent experimental data suggest that the idiosyncratic nature of drug-induced liver disease (DILD) may be due in part to a deficiency of one or more hepatoprotective factors. In this study we have investigated whether interleukin (IL)-6 may also be one of these factors. Following the induction of liver injury with acetaminophen (APAP), a time-dependent increase in liver mRNA expression of IL-6 and its family members IL-11, leukemia inhibitory factor, and oncostatin M was observed in wild type (WT) mice, suggesting a possible hepatoprotective role played by this cytokine family. Indeed, mice lacking IL-6 (IL-6-/-) were more susceptible than were WT mice to APAP-induced liver injury. The increased susceptibility of the IL-6-/- mice was associated with a deficiency in the expression of hepatic heat shock protein (HSP)25, 32, and 40 as well as inducible HSP70 following APAP treatment. These results suggest that IL-6 and possibly other family members may protect the liver from injury, at least in part, by up-regulating the hepatic expression of several cytoprotective HSPs.     

Organogenesis and embryogenesis from diverse explants in pigeonpea (Cajanus cajan L.)

Seed and seedling expiants of pigeonpea were evaluated for organogenesis and somatic embryogenesis. De novo plant regeneration through organogenesis was obtained from mature cotyledons, primary leaves and roots of seedlings. Production of multiple shoots from the cotyledonary node was observed in cultures of whole seeds on 6-benzylaminopurine enriched medium. Somatic embryos were induced from immature cotyledons and embryonal axes, however, well-developed plants could not be derived from these embryos. The regenerants obtained through organogenesis were transferred to the field and grown to maturity.     

大豆肽体外抗氧化活性研究

研究大豆肽的体外抗氧化的作用。采用邻二氮菲-Fe^2＋检测大豆肽对羟自由基（·OH）的清除作用,邻苯三酚检测大豆肽对超氧阴离子（·O2^-）的清除作用,用硫代巴比妥酸法测定小鼠肝匀浆丙二醛（MDA）的含量,用比色法测定小鼠红细胞溶血度,来研究大豆肽的抗氧化效果。结果表明：大豆肽可以清除·OH和·O2^-,抑制·OH所致的丙二醛的产生,减少H2O2所致的红细胞溶血,在2～15g／L内均具有明显的量效关系。表明大豆肽在体外具有明显的抗氧化效果。     

Metallothionein: a multifunctional protein from toxicity to cancer

The metallothionein (MT) family is a class of low molecular weight, intracellular and cysteine-rich proteins presenting high affinity for metal ions. Although the members of this family were discovered nearly 40 years ago, their functional significance remains obscure. Four major MT isoforms, MT-1, MT-2, MT-3 and MT-4, have been identified in mammals. MTs are involved in many pathophysiological processes such as metal ion homeostasis and detoxification, protection against oxidative damage, cell proliferation and apoptosis, chemoresistance and radiotherapy resistance. MT isoforms have been shown to be involved in several aspects of the carcinogenic process, cancer development and progression. MT expression has been implicated as a transient response to any form of stress or injury providing cytoprotective action. Although MT participates in the carcinogenic process, its use as a potential marker of tumor differentiation or cell proliferation, or as a predictor of poor prognosis remains unclear. In the present review the involvement of MT in defense mechanisms to toxicity and in carcinogenicity is discussed.     

Purification and characterization of urease from dehusked pigeonpea (Cajanus cajan L) seeds

Urease has been purified from the dehusked seeds of pigeonpea (Cajanus cajan L.) to apparent electrophoretic homogeneity with approximately 200 fold purification, with a specific activity of 6.24 x10(3) U mg(-1) protein. The enzyme was purified by the sequence of steps, namely, first acetone fractionation, acid step, a second acetone fractionation followed by gel filtration and anion-exchange chromatographies. Single band was observed in both native- and SDS-PAGE. The molecular mass estimated for the native enzyme was 540 kDa whereas subunit values of 90 kDa were determined. Hence, urease is a hexamer of identical subunits. Nickel was observed in the purified enzyme from atomic absorption spectroscopy with approximately 2 nickel ions per enzyme subunit. Both jack bean and soybean ureases are serologically related to pigeonpea urease. The amino acid composition of pigeonpea urease shows high acidic amino acid content. The N-terminal sequence of pigeonpea urease, determined up to the 20th residue, was homologous to that of jack bean and soybean seed ureases. The optimum pH was 7.3 in the pH range 5.0-8.5. Pigeonpea urease shows K(m) for urea of 3.0+/-0.2 mM in 0.05 M Tris-acetate buffer, pH 7.3, at 37 degrees C. The turnover number, k(cat), was observed to be 6.2 x 10(4) s(-1) and k(cat)/K(m) was 2.1 x 10(7) M(-1) s(-1). Pigeonpea urease shows high specificity for its primary substrate urea.