Methyl Brevifolincarboxylate Attenuates Free Fatty Acid-Induced Lipid Metabolism and Inflammation in Hepatocytes through AMPK/NF-κB Signaling Pathway.

The prevalence of non-alcoholic fatty liver disease (NAFLD) is one of the leading causes of chronic liver diseases worldwide. This study examined the potential protective effects of a naturally occurring polyphenolic compound, methyl brevifolincarboxylate (MBC) on fatty liver injury in vitro. The results showed that MBC at its non-cytotoxic concentrations, reduced lipid droplet accumulation and triglyceride (TG) levels in the oleic acid (OA)-treated human hepatocarcinoma cell line, SK-HEP-1 and murine primary hepatocytes. In OA-treated SK-HEP-1 cells and primary murine hepatocytes, MBC attenuated the mRNA expression levels of the de novo lipogenesis molecules, acetyl-coenzyme A carboxylase (Acc1), fatty acid synthase (Fasn) and sterol regulatory element binding protein 1c (Srebp1c). MBC promoted the lipid oxidation factor peroxisome proliferator activated receptor-α (Pparα), and its target genes, carnitine palmitoyl transferase 1 (Cpt1) and acyl-coenzyme A oxidase 1 (Acox1) in both the SK-HEP-1 cells and primary murine hepatocytes. The mRNA results were further supported by the attenuated protein expression of lipogenesis and lipid oxidation molecules in OA-treated SK-HEP-1 cells. The MBC increased the expression of AMP activated protein kinase (AMPK) phosphorylation. On the other hand, MBC treatment dampened the inflammatory mediator's, tumor necrosis factor (TNF)-α, interleukin-6 (IL-6), IL-8, and IL-1ß secretion, and nuclear factor (NF)-κB expression (mRNA and protein) through reduced reactive oxygen species production in OA-treated SK-HEP-1 cells. Taken together, our results demonstrated that MBC possessed potential protective effects against NAFLD in vitro by amelioration of lipid metabolism and inflammatory markers through the AMPK/NF-κB signaling pathway.

Lead accumulation, growth responses and biochemical changes of three plant species exposed to soil amended with different concentrations of lead nitrate.

Lead (Pb) contamination of soil is a serious environmental problem, adversely affecting ecosystems, globally. Phytoremediation is an alternative to conventional methods of soil remediation. The success of phytoremediation depends on the identification of suitable native plant species with high biomass to deal with metal contamination. In the present experiment, response of Eclipta prostrata (L.) L., Scoparia dulcis L. and Phyllanthus niruri L. to increase in concentrations of PbNO3·5H2O in the soil for a period of 30 days was tested to assess their suitability in phytoremediation. Pb accumulation in all the three plants was in a concentration-dependent manner. Although S. dulcis survived the soil metal concentrations, it exhibited a stunted growth; P. niruri was found susceptible to Pb toxicity; E. prostrata recorded a maximum uptake of 12484 µg/g dry weight in its root and 7229 µg/g dry weight in its shoot, without any adverse impact on growth traits. Bioconcentration factor and translocation factor of the three plants were also calculated, which revealed that E. prostrata has Pb accumulation potential. Therefore, enzymatic antioxidant activities and transmission electron microscopic analysis were carried out to determine the physiological adaptation and tolerance of E. prostrata to Pb stress. Overall, E. prostrata is identified as a tolerant plant showing Pb hyperaccumulation tendencies with essential features for phytoextraction.

Chitosan mediated enhancement of hydrolysable tannin in Phyllanthus debilis Klein ex Willd via plant cell suspension culture.

Phyllanthus debilis Klein ex Willd. is wild medicinal plant used in the traditional system of medicine. This plant has been actively used for hepatoprotection and to cure many diseases including jaundice and so on; which leads to complete extinction of this particular species. Therefore, the chitosan mediated cost effective cell suspension method has been developed for the production of hydrolysable tannin. The hydrolysable tannins are the main therapeutically active constituents with antioxidant, anticancer, and antimicrobial properties. An in vitro cell suspension culture was optimized by adding chitosan for production of hydrolysable tannin. According to the growth kinetics, a maximum biomass of 4.46±0.06g fresh cell weight and 1.33±0.04g dry cell weight were obtained from the optimal suspension medium consisted of MS medium+0.5mgL-1 BAP+1.5mgL-1 NAA. Chitosan was treated at the stationary phase which leads to the highest accumulation of hydrolysable tannin compared to the untreated control. Hydrolysable tannin was observed and compared using HPLC at the Rt of 4.91 in both chitosan treated and untreated cells. This is the first ever report where use of chitosan has been done to enhance the production of the hydrolysable tannin in P. debilis using cell suspension culture technique.

Ex situ conservation of Phyllanthus fraternus Webster and evaluation of genetic fidelity in regenerates using DNA-based molecular marker.

Germplasm storage of Phyllanthus fraternus by using synseed technology has been optimized. Synseeds were prepared from nodal segments taken from in vitro-grown plantlets. An encapsulation matrix of 3 % sodium alginate and 100 mM calcium chloride with polymerization duration up to 15 min was found most suitable for synseed formation. Maximum plantlet conversion (92.5 ± 2.5 %) was obtained on a growth regulator-free ½-strength solid Murashige and Skoog (MS) medium. Multiple shoot proliferation was optimum on a ½ MS medium containing 0.5 mg/l 6-benzylaminopurine (BAP). Shoots were subjected to rooting on MS media containing 1 mg/l α-naphthaleneacetic acid (NAA) and acclimatized successfully. Encapsulated nodal segments can be stored for up to 90 days with a survival frequency of 47.33 %. The clonal fidelity of synseed-derived plantlets was also assessed and compared with that of the mother plant using rapid amplified polymorphic DNA and inter-simple sequence repeat analysis. No changes in molecular profiles were observed among the synseed-derived plantlets and mother plant, which confirms the genetic stability of regenerates. This synseed production protocol could be useful for in vitro multiplication, short-term storage, and exchange of germplasm of this important antiviral and hepatoprotective plant.

High frequency shoots regeneration for mass multiplication of Phyllanthus fraternus Webster--an important antiviral and hepatoprotective plant.

An efficient, rapid, and highly reproducible regeneration protocol was successfully developed for Phyllanthus fraternus from the field-derived mature nodal segments. The explants induced multiple shoots on cytokinin containing medium. The highest frequency (99 %) and maximum number of shoots (19.75) were induced on Murashige and Skoog's (MS) medium supplemented with 2.22 µM 6-benzylaminopurine after 3-4 weeks of culture initiation. The elongated shoots were rooted on MS medium supplemented with indol-3-butyric acid (IBA) or α-naphthalene acetic acid. Pulse treatment of microshoots promoted significant increase in the percentage of rooting and number of root regeneration per shoot. The highest rooting (100 %) and maximum number of roots (8.75) per shoot was obtained when shoots were dipped in IBA solution (0.98 mM) for 5 min and further subcultured on MS basal medium. Plantlets were successfully acclimatized and established in soil. Regenerated plants were grown normally in the field without showing any morphological variations. This cost-effective protocol will help the mass multiplication of P. fraternus for commercial propagation and high biomass production of this valuable medicinal plant.

Chromium-induced changes in ultramorphology and secondary metabolites of Phyllanthus amarus Schum & Thonn. - an hepatoprotective plant.

Chromium (Cr) concentration in the environment is increasing day by day due to its excessive use in leather processing, refractory steel, and chemical manufacturing industries. Excess Cr in the environment causes hazardous effects on all living beings including plants. In this context attempts have been made to see the effect of Cr on a very important medicinal plant Phyllanthus amarus Schum and Thonn. It has focused as a hepatoprotective plant curing hepatitis 'B' in modern research. The P. amarus seedlings of approximately same height and weight were grown in similar conditions in experimental garden. After 30 days plants were harvested and chlorophyll, protein, sugar, starch and secondary metabolites concentration were estimated in these plants as per standard methods. Besides ultramorphological variations in leaves and Cr accumulation in roots and aerial parts were also checked with the help of scanning electron microscope and atomic absorption spectrophotometer respectively. The study revealed that Cr causes significant decrease in fresh and dry weight, length of root and shoot, protein, sugar, chlorophyll and carotenoids in P. amarus. On the other hand it is interesting to note that the concentration of therapeutically active secondary metabolites - phyllanthin and hypophyllanthin increased up to certain level of Cr. It may be due to stress caused by the accumulation of Cr. Besides, ultramorphological changes viz. wide stomatal opening and less wax deposition on both the surfaces of leaves were also observed.

NaCl as a physiological modulator of proline metabolism and antioxidant potential in Phyllanthus amarus.

Some medicinal plants need to be cultivated commercially in order to meet the ever-increasing demand for medicinal plants for the indigenous systems of medicine as well as for the pharmaceutical industry; in this regard, it seems significant to test the important medicinal plants for their salt-tolerance capacity, with a view to exploiting the saline lands for medicinal plant cultivation. Phyllanthus amarus plants were grown in the presence of NaCl in order to study the effect of NaCl (80 mM NaCl) in the induction of oxidative stress in terms of lipid peroxidation (TBARS content), H2O2 content, osmolyte concentration, proline(PRO)-metabolizing enzymes, and antioxidant enzyme activities. Groundwater was used for irrigation of control plants. Plants were uprooted randomly on 90 days after sowing (DAS). NaCl-stressed plants showed increased TBARS, H2O2, glycine betaine (GB), and PRO contents, whereas NaCl uptake decreased proline oxidase (PROX) activity and increased gamma-glutamyl kinase (gamma-GK) activity when compared to control. The antioxidant enzymes superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) were increased under salinity.

Chemical basis of plant leaf movement.

Nyctinastic plants open and close leaves with a circadian rhythm. Here we discuss chemical aspects of the mechanism of nyctinastic leaf movement. Nyctinastic plants from five different genera are known to contain species-specific leaf-opening and leaf-closing factors. The relative concentrations of leaf-closing and leaf-opening factors of the nyctinastic plant Phyllanthus urinaria change circadianly, suggesting that nyctinastic movement is regulated by two classes of circadianly regulated factors with opposing functions. A closing and an opening factor of Albizzia, when linked to a fluorescent dye, both specifically labeled motor cells of pluvini. A membrane fraction of pluvini contains proteins of 210 and 180 kDa that bind to a leaf-opening factor of Cassia mimosoides. The molecular identification of these proteins is underway.

Lignans from cell suspension cultures of Phyllanthus niruri, an Indonesian medicinal plant.

Cell suspension cultures of Phyllanthus niruri were used to study the lignan profiles and biosynthesis. Suspension cultures yielded two lignans: the new cubebin dimethyl ether (1) and urinatetralin (2), a new lignan from P. niruri, but reported earlier from P. urinaria. This is the first report of cell suspension cultures of P. niruri that successfully produce lignans. Feeding 0.5 mM ferulic acid or 0.5 mM caffeic acid, being early precursors of lignan biosynthesis, resulted in an increase up to 0.7 mg g(-1) DW of 1 (control value 0.1 mg g(-1) DW) and up to 0.3 mg g(-1) DW of 2 (control value 0.2 mg g(-1) DW). Comparison of the lignan profiles of cell suspensions, callus cultures, aerial plant parts, roots, and seeds showed significant differences.

Effect of cadmium on growth, ultramorphology of leaf and secondary metabolites of Phyllanthus amarus Schum. and Thonn.

The pollution is increasing in the environment by different kinds of human activities, which results in the accumulation of heavy metals including cadmium in the soil and water and it causes different types of problems to living beings. As the plants are utilized by human being as food and medicine, therefore, it is mandatory to see the effect of metals on plants. In this context, efforts have been made to observe the effect of different concentration of Cadmium (Cd) on Phyllanthus amarus Schum. and Thonn., because Cd is the widespread metal and the plants response to low and high level of exposure is a complex phenomenon. P. amarus is mostly grown as weed in agricultural and waste lands. It is a reputed plant used in Indian indigenous systems of medicine with hepatoprotective, diuretic, stomachic properties and is recently being used for the treatment of hepatitis B. The study revealed that Cd causes significant decrease in fresh and dry weight, length of root and shoot, protein, chlorophyll, carotenoids and sugar and increase in starch content. It is interesting to note that the therapeutically active compounds-phyllanthin and hypophyllanthin, enhanced at certain levels of Cd due to abiotic stress. Besides, the ultramorpholical changes were also observed in stomatal opening and wax deposition on both the surfaces of leaves.

Antioxidative and cardioprotective effects of Phyllanthus urinaria L. on doxorubicin-induced cardiotoxicity.

Cardiac toxicity is a major adverse effect caused by doxorubicin (DOX) therapy. Many recent studies have shown that DOX toxicity involves generation of reactive oxygen species (ROS). Although protection or alleviation of DOX toxicity can be achieved by administration of antioxidant vitamins such as ascorbic acid and vitamin E, their cardioprotective effect remains controversial. Thus alternative naturally occurring antioxidants may potentially be candidates for antioxidant therapy. In this study, we investigated the antioxidative and cytoprotective effects of Phyllanthus urinaria (PU) against DOX toxicity using H9c2 cardiac myoblasts. The total antioxidant capacity of PU (1 mg/ml) was 5306.75+/-461.62 FRAP value (microM). DOX IC50 values were used to evaluate the cytoprotective effects of PU ethanolic extract (1 or 10 microg/ml) in comparison with those of ascorbic acid (VIT C, 100 microM) and N-acetylcysteine (NAC, 100 microM). PU treatments (1 or 10 microg/ml) dose dependently caused rightward DOX IC50 shifts of 2.8- and 8.5-fold, respectively while treatments with VIT C and NAC increased DOX IC50 by 3.3- and 4.2-fold, respectively. Additionally, lipid peroxidation and caspase-3 activity were parameters used to evaluate cytoprotective effect. All antioxidants completely inhibited cellular lipid peroxidation and caspase-3 activation induced by DOX (1 microM). Endogenous antioxidant defense such as total glutathione (tGSH), catalase and superoxide dismutase (SOD) activity was also modulated by the antioxidants. PU treatment alone dose dependently increased tGSH, and this effect was retained in the presence of DOX. Similar effect was observed in the assessment of catalase and SOD enzyme activity. The nuclear factor kappaB (NFkappaB) transcription factor assay demonstrated that all antioxidants significantly inhibited DOX-induced NFkappaB activation. Our results suggest that PU protection against DOX cardiotoxicity was mediated through multiple pathways and this plant may serve as an alternative source of antioxidants for prevention of DOX cardiotoxicity.