Identification of species-diagnostic inter simple sequence repeat markers for ten Phyllanthus species

Phyllanthus has been widely used in traditional medicine as an antipyretic, a diuretic, and to treat liver diseases and viral infections. Correct genotype identification of medicinal plant material remains important for the botanical drug industry. Limitations of chemical and morphological approaches for authentication have generated the need for newer methods in quality control of botanicals. In the present study, attempts were made to identify species-diagnostic markers for ten Phyllanthus species using the inter simple sequence repeat-polymerase chain reaction (ISSR-PCR) fingerprinting method. PCR amplification using seven ISSR primers resulted in significant polymorphism among the populations from different species. P. angustifolius and P. urinaria showed monomorphic frequency of maximum (63.88%) and minimum (20.64%), respectively. Seventeen species-diagnostic markers were identified for seven species (P. acidus, P. emblica, P. fraternus, P. urinaria, P. rotundifolius, P. amarus, and P. angustifolius) while no marker was detected for P. reticulatus, P. nivosus, and P. virgulatus. A maximum of six species-diagnostic markers were identified for P. acidus and a minimum of only one of 755 bp was available for P. amarus. Among the seventeen markers, nine were present in all individuals of particular species. The species-specific differences in fragment numbers and sizes could be used as diagnostic markers to distinguish the Phyllanthus species quickly.     

Chemoprotective activity of an extract of Phyllanthus amarus against cyclophosphamide induced toxicity in mice.

The effect of 75% methanolic extract of the plant Phyllanthus amarus (P. amarus) was studied against cyclophosphamide (CTX) induced toxicity in mice. Administration of CTX (25 mg/kg b.wt, i.p.) for 14 days produced significant myelosuppression as seen from the decreased WBC count and bone marrow cellularity. Administration of P. amarus extract at doses 250 and 750 mg/kg b.wt significantly reduced the myelosuppression and improved the WBC count, bone marrow cellularity as well as the number of maturing monocytes. CTX treatment also reduced the activity of glutathione system and increased the activity of phase I enzyme that metabolize CTX to its toxic side products. P. amarus administration was found to decrease the activity of phase I enzyme. Administration of P. amarus also increased the cellular glutathione (GSH) and glutathione-S-transferase (GST), thereby decreasing the effect of toxic metabolites of CTX on the cells. Administration of P. amarus did not reduce the tumor reducing activity of CTX. In fact, there was a synergistic action of CTX and P. amarus in reducing the solid tumors in mice. Results indicated that administration of P. amarus can significantly reduce the toxic side effects of CTX and is not interfering with the antitumor efficiency of CTX.     

Development of species-specific SCAR markers for identification of three medicinal species of Phyllanthus

Phyllanthus amarus Schum. & Thonn. has been widely used in traditional medicine in Thailand as an antipyretic, a diuretic, to treat liver diseases and viral infections. Two closely related species, P. debilis L. and P. urinaria Klein ex Willd., with different and less effective medicinal properties, are less commonly used. These three species are similar in morphology and often occur in overlapping populations in nature. The latter two species can easily be mistaken for P. amarus and collected for medicinal uses, which can lead to undesirable results. DNA fingerprints of these species were obtained using RAPD-PCR techniques. RAPD markers specific for each species were identified. Primers for highly specific sequence-characterized-amplified-regions (SCAR) were then designed from nucleotide sequences of specific RAPD markers. These primers efficiently amplified SCAR markers of 408, 501 and 319 bp unique to P. amarus, P. debilis and P. urinaria respectively. This method of plant identification was rapid and highly specific when tested against DNA of several closely related species and was able to amplify specific markers from mixed DNA samples.     

South–East Asian Phyllantheae II. Some Phyllanthus of subsect. Swartziani

In South–East Asia, Phyllanthus sect. Phyllanthus subsect. Swartziani Webster, presently includes P. amarus Schum. & Thonn., P. debilis Klein ex Willd. and P. airy–sha–wii sp. nov. The last species is found in North Thailand and has until now been confused with P. debilis. The Asian distribution of P. debilis is discussed.     

华南植物的增补

报道华南地区3种植物：山柚子科的茎花山袖（Champereiamanillanavar．longistamined）是新组合的变种,其花序和嫩叶可供蔬食;大朝科的美洲珠子草（Phyllanthusamarus）是具有药用价值的归化种;台湾核果木（Drypetesformosana）生长于珠江口东侧的海岛,为广东新纪录种。     

Cultivation ofPhyllanthus amarus and evaluation of variables potentially affecting and the inhibition of viral DNA polymerase

Phyllanthus amarus (Euphorbiaceae) possesses activity against hepatitis B virus and related hepadnaviruses. One such activity, the inhibition of endogenous hepadnavirus DNA polymerase, differed little between cultivatedP. amarus and plants collected from the wild. Inhibitory activity equivalent to that in wild plants was obtained from both shoots and roots sown at different times of the year in a subtropical region (Dade County, Florida, U.S.A.). Plant size and two levels of fertilization did not significantly affect activity. Plots were planted using a cellulose gel to evenly disperse the small seeds. Gel amendments used at sowing had no significant effect on the antiviral activity of harvested plants. Drip irrigation permitted successful cultivation during the dry season. Plastic mulch was used to control weeds.Phyllanthus amarus grows slowly, and reaches a maximum size and vigor at about 5–7 months after sowing. Under south Florida conditions, the greatest biomass ofP. amarus was produced when seeds were sown in mid-winter for a summer harvest. With weed control by the mulch, water and fertilization via the drip irrigation, six and a half month old plants (from sowing) reached an average dry weight of approximately 40 glplant when harvested in July or August.     

Development of species-specific SCAR markers for identification of three medicinal species of Phyllanthus

Phyllanthus amarus Schum.& Thonn.has been widely used in traditional medicine in Thailand as an antipyretic.a diuretic.to treat liver diseases and viml infections.Two closely related species,P. debills L.and P.urinaria KIein ex Willd.,with different and less effective medicinal properties,are less commonly used.These three species are similar in morphology and often Occur in overlapping populations in nature.The latter two species can easily be mistaken for P.amarus and collected for medicinal uses, which can lead to undesirable results.DNA fingerprints of these species were obtained using RAPD-PCR techniques.RAPD markers specific for each species were identified.Primers for highly specific sequence-characterized-amplified-regions (SCAR) were then designed from nucleotide sequences of specific RAPD markers.These primers efficiently amplified SCAR markers of 408,501 and 319 bp unique to P.amarus,P.debilis and P.urinaria respectively.This method of plant identification Was rapid and highly specific when tested against DNA of several closely related species and was able to amplify specific markers from mixed DNA samples.     

e antigen in acute hepatitis B.

To examine the association between e antigen and hepatitis-B surface antigen (HBs Ag) we studied 90 inpatients with acute viral hepatitis type B. e Antigen was present in 24 of the patients; these patients had detectable levels of HBs Ag for significantly longer than the 66 with no e antigen in their serum. The HBs Ag subtypes D (adw) and Y (ayw) were similarly distributed among patients with e antigen and among those without, and no differences in the results of biochemical liver function tests were observed between the two groups during the acute phase of illness. Three of the five patients who developed clinical and histological signs of chronic liver disease were positive for e antigen, a finding which supports the hypothesis that e antigen has a prognostic value in hepatitis B.     

Subtypes of Hepatitis B Antigen in Blood Donors and Post-transfusion Hepatitis: Clinical and Epidemiological Aspects

Subtyping of hepatitis B antigen (HBA) in blood donors revealed subtype ad in 56% while patients with icteric post-transfusion hepatitis from the same centre showed subtype ay in the majority of the cases (75%). Donors with subtype ad in serum were mostly asymptomatic long-term carriers of the antigen with normal liver function (83%), while 70% of donors with subtype ay in serum had signs of acute or chronic liver disease. Healthy long-term carriers of HBA seem to present little risk of transmitting hepatitis irrespective of subtype. It is, however, possible that these differences in blood donors with subtype ad and patients with post-transfusion hepatitis with subtype ay might reflect epidemiological circumstances rather than biological differences in the two viral strains.     

Phyllanthus urinaria extract attenuates acetaminophen induced hepatotoxicity: Involvement of cytochrome P450 CYP2E1

Acetaminophen is a commonly used drug for the treatment of patients with common cold and influenza. However, an overdose of acetaminophen may be fatal. In this study we investigated whether mice, administered intraperitoneally with a lethal dose of acetaminophen, when followed by oral administration of Phyllanthus urinaria extract, may be prevented from death. Histopathological analysis of mouse liver sections showed that Phyllanthus urinaria extract may protect the hepatocytes from acetaminophen-induced necrosis. Therapeutic dose of Phyllanthus urinaria extract did not show any toxicological phenomenon on mice. Immunohistochemical staining with the cytochrome P450 CYP2E1 antibody revealed that Phyllanthus urinaria extract reduced the cytochrome P450 CYP2E1 protein level in mice pre-treated with a lethal dose of acetaminophen. Phyllanthus urinaria extract also inhibited the cytochrome P450 CYP2E1 enzymatic activity in vitro. Heavy metals, including arsenic, cadmium, mercury and lead, as well as herbicide residues were not found above their detection limits. High performance liquid chromatography identified corilagin and gallic acid as the major components of the Phyllanthus urinaria extract. We conclude that Phyllanthus urinaria extract is effective in attenuating the acetaminophen induced hepatotoxicity, and inhibition of cytochrome P450 CYP2E1 enzyme may be an important factor for its therapeutic mechanism.     

Amplification and excision of integrated polyoma DNA sequences require a functional origin of replication

Cells transformed by Polyoma virus (Py) can undergo a high rate of excision or amplification of integrated viral DNA sequences, and these phenomena require the presence of homology (i.e., repeats) within the viral insertion as well as a functional viral large T antigen (T-Ag). To determine whether the main role of large T-Ag in excision and amplification was replicative or recombination-promoting, we studied transformed rat cell lines containing tandem insertions of a ts-a Py molecule (encoding a thermolabile large T-Ag) with a deletion of the origin of viral DNA replication. Culturing of these cells at the temperature permissive for large T-Ag function did not result in any detectable excision or amplification of integrated Py sequences. We then introduced into origin-defective lines a recombinant plasmid containing the viral origin of replication and the gene coding for resistance to the antibiotic G418. All G418-resistant clones analyzed readily amplified the integrated plasmid molecules when grown under conditions permissive for large T-Ag function, showing that these cells produced viral large T-Ag capable of promoting amplification in trans of DNA sequences containing the Py origin. These observations strongly suggest that Polyoma large T antigen promotes excision or amplification of viral DNA by initiating replication at the integrated origin, providing a favorable substrate for subsequent recombination.     

Interaction and replication activation of genotype I and II hepatitis delta antigens

The nucleotide sequences of hepatitis D viruses (HDV) vary 5 to 14% among isolates of the same genotype and 23 to 34% among different genotypes. The only viral-genome-encoded antigen, hepatitis delta antigen (HDAg), has two forms that differ in size. The small HDAg (HDAg-S) trans-activates viral replication, while the large form (HDAg-L) is essential for viral assembly. Previously, it has been shown that the packaging efficiency of HDAg-L is higher for genotype I than for genotype II. In this study, the question of whether other functional properties of the HDAgs are affected by genotype differences is addressed. By coexpression of the two antigens in HuH-7 cells followed by specific antibody precipitation, it was found that HDAgs of different origins interacted without genotypic discrimination. Moreover, in the presence of hepatitis B virus surface antigen, HDAg-S was incorporated into virion-like particles through interaction with HDAg-L without genotype restriction. As to the differences in replication activation of genotype I HDV RNA, all HDAg-S clones tested had some trans-activation activity, and this activity varied greatly among isolates. As to the support of HDV genotype II replication, only clones of HDAg-S from genotype II showed trans-activation activity, and this activity also varied among isolates. In conclusion, genotype has no effect on HDAg interaction and genotype per se only partly predicts how much the HDAg-S of an HDV isolate affects the replication of a second HDV isolate.     

Performance of Tetranychus urticae and Neoseiulus californicus on strawberry cultivars and assessment of the effect of glandular trichomes

The performance of Tetranychus urticae and its predator Neoseiulus californicus on ten strawberry cultivars was determined in the laboratory. Development time and survival of T. urticae from egg to adult were recorded on Albión, Aromas, Camarosa, Diamante, Festival, Kp, Sabrosa, Selva, Sweet Charlie, and Whitney. Fecundity of newly molted and mated females was recorded during the first 10 days of oviposition. Predation rate and fecundity of N. californicus were tested on Albión, Aromas, Festival, Kp, Sabrosa, and Whitney. Predator females reared on each cultivar were placed individually in experimental units, and the number of eggs per day was counted during 3 days. Cultivars with high hairiness (Albión, Aromas, and Festival) and cultivars with low hairiness (Sabrosa, Whitney and Kp) were identified, to assess the effect on the performance of both species. Development time, survival from egg to adult, and fecundity of T. urticae differed among cultivars. Festival was classified as moderately resistant, Aromas and Kp were moderately susceptible, and the others were intermediate. The number of prey consumed per day per female of N. californicus differed between cultivars and time. Fecundity of N. californicus did not differ among cultivars; however, it did over time. The development time and fecundity of T. urticae did not differ among high and low hairiness cultivars. The glandular hairiness affected neither consumption nor fecundity of N. californicus. According to detrimental and propitious effect on T. urticae and N. californicus performance, respectively, we concluded that Festival and Albión could be used along with this predator in T. urticae management programs.     

Thidiazuron-induced shoot organogenesis and production of hepatoprotective lignan phyllanthin and hypophyllanthin in <Emphasis Type="Italic">Phyllanthus amarus</Emphasis>

Callus cultures from nodal and leaf explants of Phyllanthus amarus were established on Murashige and Skoog (MS) medium with various combinations of auxins and cytokinins. The leaf-derived callus induced on 2.26 μM 2,4-dichlorophenoxyacetic acid (2, 4-D) + 2.32 μM Kinetin (Kin) upon transfer to medium containing thidiazuron (TDZ) exhibited higher shoot regeneration (32.4 ± 1.3 shoots per culture). Four-week-old shoots rooted readily on 1.5 μM Indol acetic acid (IAA)-containing medium and were successfully acclimatized with 98% survival. The lignans, Phyllanthin (PH) and Hypohyllanthin (HPH), of leaf extracts from naturally grown plants were identified by using TLC, HPLC and H1-NMR. The PH and HPH production in the regenerated shoots was compared to their production in callus cultures, plants under field conditions and in naturally grown plants. The regenerated shoots on MS + 2.27 μM TDZ produced about two times higher PH and HPH than the leaves of naturally grown plant. The present study provides a useful system for further studies on in vitro morphogenesis, elicitor-assisted production of PH and HPH and A. rhizogenes-mediated genetic transformation in Phyllanthus amarus.     

Quantitative determination of phyllanthin and hypophyllanthin in Phyllanthus species by high-performance thin layer chromatography

A simple, precise and rapid high-performance thin-layer chromatographic method has been developed for the estimation of phyllanthin (1) and hypophyllanthin (2), the important lignans of Phyllanthus species, especially Phyllanthus amarus. Separation of 1 and 2 was carried out on silica gel 60 F254 layers eluted with hexane:acetone:ethyl acetate (74:12:8), and the analytes were visualised through colour development with vanillin in concentrated sulphuric acid and ethanol. Scanning and quantification of spots was performed at 580 nm. Recoveries of 1 and 2 were 98.7 and 97.3%, respectively. The method was validated and the peak purities and limits of detection and quantification were determined.     

Isolation and characterization of temperature-sensitive mutants of measles virus.

Nine temperature-sensitive (ts) mutants of nonattenuated Edmonston strain measles virus were isolated from wild-type virus which was grown in the presence of 5-fluorouracil. Adsorption, temperature shift, and complementation experiments indicated that all these mutants were restricted at an intracellular stage of infection. However, all the mutants were more rapidly inactivated at 41 C than was wild-type virus, suggesting that the ts product of each mutant either influences or is a structural component of the virus. Three complementation groups were found to be represented among the mutants. Group A contained one mutant and it did not induce synthesis of detectable amounts of viral antigen at the nonpermissive temperature (39 C). Group B consisted of six mutants which did not induce viral antigen synthesis at 39 C and one mutant which did. Group C was represented by one mutant and it induced viral antigen synthesis at 39 C. The two mutants which induced sythesis of viral antigen also induced synthesis of relatively small amounts of virus-specific RNA at 39 C. These mutants, while producing cytoplasmic and nuclear accumulations of viral antigen at 39 C, were restricted in production of syncytia and hemadsorption. All the mutants were less neurovirulent than wild-type virus, as indicated by their inability to produce acute disease in newborn hamsters.     

Ornithine carbamoyltransferase genes and phaseolotoxin immunity in Pseudomonas syringae pv. phaseolicola

Two different DNA fragments encoding ornithine carbamoyltransferase (OCTase) were cloned from Pseudomonas syringae pv. phaseolicola NPS3121. These fragments did not cross-hybridize and encoded OCTases which differed with respect to their sensitivity to purified phaseolotoxin, an OCTase inhibitor produced by this phytopathogenic bacterium. Recombinant plasmids carrying these DNA fragments complemented OCTase-deficient strains of Escherichia coli and Pseudomonas aeruginosa. Extracts of the complemented E. coli strain contained OCTase enzyme activities with similar degrees of sensitivity to purified phaseolotoxin as extracts of P.s.phaseolicola grown at either 20 or 30°C. The OCTase activity detectable in extracts of P.s.phaseolicola grown at 20°C is insensitive to phaseolotoxin while that detectable in extracts of cells grown at 30°C is sensitive to the toxin. E.coli HB101 harboring recombinant plasmids carrying the gene(s) encoding the phaseolotoxin-insensitive enzyme activity exhibited resistance to purified phaseolotoxin. The results of Tn5 mutagenesis and Southern blotting and the pattern of complementation of OCTase-deficient and Tox^- mutant strains suggest that the gene(s) encoding the phaseolotoxin-insensitive OCTase is part of a gene cluster involved in phaseolotoxin production.     

Development of a potent <Emphasis Type="Italic">in vitro</Emphasis> source of <Emphasis Type="Italic">Phylanthus amarus</Emphasis> roots with pronounced activity against surface antigen of the hepatitis B virus

Summary In vitro culture of hairy roots of Phyllanthus amarus induced by Agrobacterium rhizogenes was established. Their growth and ability for in vitro inactivation of hepatitis B virus surface antigen was studied and compared with adventitious roots grown in vitro. The selected hairy root clone HR-1 was capable of growing at a very fast rate, and an approximately 900-fold increase in weight of root biomass was achieved after 4 wk of culture in hormone-free quarter-strength liquid Murashige and Skoog medium with continuous agitation. Non-transformed roots cultured in the presence of 1.0 mg l⁻¹ (5.71 μM) indole-3-acetic acid increased by 330-fold. The immuno-inactive property of roots was maximal in the crude extract. The hairy roots were shown to possess 85% inhibition (in contrast to 15% in the control) in binding of hepatitis B surface antigen (HBsAg) to its antibody (anti-HBs) after 24 h of incubation with HbsAg-positive sera in vitro at 37°C. Out of three fractions selected on the basis of molecular weight components of the extract, the Fraction III containing comparatively lower molecular weight substances (≤3500) yielded the highest activity. The extract from non-transformed roots was found to possess similar efficiency (87% inhibition). The levels of activity in both types of in vitro-raised roots were higher than those of naturally occurring roots and leafy shoots. The ability of P. amarus hairy root cultures to yield high biomass with the anti-viral property at high levels may provide an alternative source of raw material for more detailed study in the field of pharmaceutical research.