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Protection of the hemopoietic system by Podophyllum hexandrum against gamma radiation-induced damage.

A semi-purified extract of low-altitude Podophyllum hexandrum (REC-2001) containing a relatively low content of podophyllotoxin (3.25 %) exhibited potent antioxidant ability in lipid media (at 1000 microg/mLagainst 0.25 kGy) and significant (p < 0.05) hydroxyl ion scavenging potential (78.83 % at 500 microg/mL). In vitro investigations revealed the ability of REC-2001 to significantly (p < 0.05) reduce radiation-induced hemolysis (2 microg/mL; 46.184 %) and nitric oxide scavenging levels (IC (50): 792 +/- 1.25 microg/mL). Protection of the hemopoietic system of Strain 'A' mice administered 20 mg/kg BW REC-2001 30 min prior to lethal irradiation (10 Gy) was recorded and was mediated by free radical scavenging and lowering of lipid oxidation. Further studies investigating the effects of REC-2001 on stem cell modulation are warranted.

Animals↗

Enhanced podophyllotoxin production from Agrobacterium rhizogenes transformed cultures of Podophyllum hexandrum.

Podophyllotoxin, a potent chemotherapeutic agent is obtained from Podophyllum hexandrum Royle. Embryos of P. hexandrum were transformed using different strains of Agrobacterium rhizogenes viz. A4, 15834, K599. Transformed nature of the calli was ascertained and the cultures were further maintained as individual clones. HPLG analysis of transformed cultures depicted a three-fold increase in podophyllotoxin content in comparison to controls.

Cell Line, Transformed↗

Cytotoxicity of in vitro produced podophyllotoxin from Podophyllum hexandrum on human cancer cell line.

Podophyllotoxin was produced by cell culture of Podophyllum hexandrum under in vitro culture conditions. A maximum of 4.26 mg/L of podophyllotoxin was produced when P. hexandrum was cultivated in 3 L stirred tank bioreactor. The compound extracted from the cell culture was applied to the human breast cancer cell line (MCF-7) and 1 nM podophyllotoxin was able to inhibit the growth of the cancer cells by 50%. The most profound inhibitory effect of podophyllotoxin was observed when it was applied in the beginning of cell growth.

Breast Neoplasms↗

Determination of the aryltetralin lignan content of podophyllum resins and roots/rhizomes.

A simple, quantitatively reproducible method for determining the aryltetralin lignan content of the resins and roots/rhizomes of Podophyllum hexandrum and P. peltatum is described. The method confirms that P. hexandrum resins and roots/rhizomes contain approximately four times the quantity of lignans as do those of P. peltatum and also that there is a significant variation in the lignan content of P. hexandrum resins.

Chromatography, Thin Layer↗

Enhancement of radiation-induced apoptosis by Podophyllum hexandrum.

The aqueous extract of Podophyllum hexandrum (RP-1), which has been recently reported to manifest radioprotective and anti-tumour properties, has been investigated for its mode of action. RP-1, under in-vitro conditions dose-dependently chelated metal ions, inhibited radiation or metal ion-induced hydroxyl radicals and lipid peroxidation and scavenged superoxide anions. Intraperitoneal administration of RP-1 to mice pre-irradiation (10 Gy) induced more DNA fragmentation and lipid peroxidation in thymocytes maximally at 4 and 8 h, respectively, in comparison with RP-1 treatment or irradiation. Flow-cytometric quantification of sub-diploid peak, oligonucleosomal cleavage assay (ladder) and depletion of total thiols also corroborated the ability of RP-1 to enhance radiation-induced apoptosis. RP-1 in presence of 100 microM CuSO(4) induced strand breaks in plasmid DNA and addition of metal chelators (EDTA and deferoxamine) inhibited the strand scission. Treatment with a major constituent of RP-1, podophyllin, did not cause strand breaks, but isolated constituents of RP-1, quercetin or podophyllotoxin, induced strand breaks. Depending on its concentration in the milieu, RP-1 acted as a pro- or antioxidant modifying the radiation-induced apoptosis and therefore could be exploited for cancer management.

Animals↗

Optimization of culture parameters for production of podophyllotoxin in suspension culture of Podophyllum hexandrum.

The root explants of the germinated seedlings of Podophyllum hexandrum were grown in MS medium supplemented with indole acetic acid (IAA) (2 mg/L) and activated charcoal (0.5%), and healthy callus culture was obtained after incubation for 3 wk at 20 degrees C. The cultivation of plant cells in shake flask was associated with problems such as clumping of cells and browning of media, which were solved by the addition of pectinase and polyvinylpyrrolidone. The effect of major media components and carbon source was studied on the growth and podophyllotoxin production in suspension culture. It was found that glucose was a better carbon source than sucrose and that NH4+:NO3- ratio (total nitrogen concentration of 60 mM) and PO4(3-) did not have much effect on the growth and product formation. The relative effect of culture parameters (inoculum level, pH, IAA, glucose, NH4+:NO3- ratio, and PO4(3-)) on the overall growth and product response of the plant cell suspension culture was further investigated by Plackett-Burman design. This indicated that inoculum level, glucose, IAA, and pH had significant effects on growth and production of podophyllotoxin. To identify the exact optimum concentrations of these parameters on culture growth and podophyllotoxin production, central composite design experiments were formulated. The overall response equations with respect to growth and podophyllotoxin production as a function of these culture parameters were developed and used to determine the optimum concentrations of these parameters, which were pH 6.0, 1.25 mg/L of IAA, 72 g/L of glucose, and inoculum level of 8 g/L.

Biotechnology↗

3-O-beta-D-Galactopyranoside of quercetin as an active principle from high altitude Podophyllum hexandrum and evaluation of its radioprotective properties.

The aqueous-ethanolic extract (AEE) of high altitude Podophyllum hexandrum has earlier been reported to render a radioprotective effect against lethal gamma radiation in in vitro model. AEE has also been reported to possess metal chelating and DNA protecting properties. The present study was undertaken to isolate and characterize the bioactive principle present in AEE and investigate its role in radiation protection. A novel molecule was found to be present in AEE and was assigned as 3-O-beta-D-galactoside of quercetin by acid hydrolysis, LC-MS, LC-APCI-MS/MS and 13C NMR spectra. Various biological activities were investigated at in vitro level. The antioxidant potential of AEE in lipid and aqueous phase was determined against numerous stresses. AEE was found to be significantly (p < 0.05) protective, i.e., against Fe2+ and Cu2+-induced linoleic acid degradation, respectively. Radiation-induced lipid oxidation studies revealed that AEE maximally works at a [lignan]/0.25 kGy ratio 400 (ratio of concentration of AEE divided by the radiation dose, i.e., 0.25 kGy) and no drug-induced lipid oxidation at all concentrations tested was found. In a time-dependent study, total antioxidant activity was maximally exhibited at 1 mg/ml. The site-specific and non-site-specific deoxyribose degradation assay exhibited a dose-dependant hydroxyl scavenging potential of AEE (0.05-500 microg/ml). The anti-lipid peroxidation ability of AEE against radiation (0.25 kGy)-induced lipid peroxidation was higher in case of neural tissue homogenate as compared to kidney homogenate [activity ratio: 0.039 (brain) < 0.24 (kidney)]. The protein protection study using bovine serum albumin was also done for two time intervals (2 h and 4 h) and significant (p < 0.05) protection was observed at 500 microg/ml (> 97%). This study implies that 3-O-beta-D-galactoside present in AEE renders radioprotection by protecting lipids, proteins in renal and neural model system against supra-lethal (0.25 kGy) gamma radiation.

Altitude↗

Anti-inflammatory effects of Podophyllum hexandrum (RP-1) against lipopolysaccharides induced inflammation in mice.

PURPOSE: Down-regulation of lipopolysaccharide (LPS) induced hyper-inflammatory response by non-toxic pharmacological agents acquires paramount importance for countering bacterial sepsis. Anti-inflammatory potential of aqueous extract of Podophyllum hexandrum, a plant well documented in Ayurvedic literature for various therapeutic purposes, was investigated. METHODS: In vivo studies were performed on Balb/c mice pre-treated with supra-lethal dose of LPS endotoxin (E.coli 055:B5) with or without treatment with P. hexandrum extract (RP-1). Mouse peritoneal macrophage cultures were used to understand ex vivo effects of RP-1 on LPS generated nitric oxide (NO), secretion of IFN-gamma, IL-6 and TNF-alpha. Griess assay and sandwich ELISA method were used to quantify inducible NO and cytokines respectively. RESULTS: Minimal dose of LPS that rendered 100% mortality to mice was found to be 450 microg/kg b.w. Administration of RP-1 (200 mg/kg b.w., i.p.) one hour before lethal LPS treatment (0.5 mg/kg b.w.) rendered maximum (78%) survival. Ex vivo study revealed that RP-1 (50 microg/ml) treatment to peritoneal macrophages inhibited LPS (5 microg/ml) induced nitrite generation to 37%, IFN-gamma secretion to 5%, IL-6 secretion to 50% and TNF-alpha secretion to 50 % of LPS treated control values. CONCLUSION: This study has demonstrated anti-inflammatory potential of aqueous extract of P. hexandrum.

Animals↗

[Study on extraction methods of activity components from Podophyllum emodi wall].

Supercritical fluid Extraction (SFE) with subsequent HPLC analysis was utilized to extract activity components such as podophyllotoxin, deoxypodophyllotoxin, kaempferol and quercetin from the roots and stems of Podophyllum emodi Wall. The extraction yields obtained by SFE were compared to those obtained by organic solvent extraction and were found to be comparable or lower. In addition, we used the orthogonal experiment which conducts with 3 factors and 4 levers in order to find the best dynamic extraction conditions.

Carbon Dioxide↗

AFLP analysis of nephthytis (Syngonium podophyllum Schott) selected from somaclonal variants.

This study analyzed genetic differences of 19 cultivars selected from somaclonal variants of Syngonium podophyllum Schott along with their parents as well as seven additional Syngonium species and six other aroids using amplified fragment length polymorphism (AFLP) markers generated by 12 primer sets. Among the 19 somaclonal cultivars, 'Pink Allusion' was selected from 'White Butterfly'. Tissue culture of 'Pink Allusion' through organogenesis resulted in the development of 13 additional cultivars. Self-pollination of 'Pink Allusion' obtained a cultivar, 'Regina Red Allusion', and tissue culture propagation of 'Regina Red Allusion' led to the release of five other cultivars. The 12 primer sets generated a total of 1,583 scorable fragments from all accessions, of which 1,284 were polymorphic (81.9%). The percentages of polymorphic fragments within 'White Butterfly' and 'Regina Red Allusion' groups, however, were only 1.2% and 0.4%, respectively. Jaccard's similarity coefficients among somaclonal cultivars derived from 'White Butterfly' and 'Regina Red Allusion', on average, were 0.98 and 0.99, respectively. Seven out of the 15 cultivars from the 'White Butterfly' group and three out of six from the 'Regina Red Allusion' group were clearly distinguished by AFLP analysis as unique fragments were associated with respective cultivars. The unsuccessful attempt to distinguish the remaining eight cultivars from the 'White Butterfly' group and three from the 'Regina Red Allusion' group was not attributed to experimental errors or the number of primer sets used; rather it is hypothesized to be caused by DNA methylation and/or some rare mutations. This study also calls for increased genetic diversity of cultivated Syngonium as they are largely derived from somaclonal variants.

Araceae↗

Separation of diastereoisomers of podophyllum lignans by micellar electrokinetic chromatography.

Seven pairs of diastereoisomers of podophyllum lignans at 2-position, including two pairs of spin-labeled compounds, were separated by micellar electrokinetic chromatography with 5 mM sodium tetraborate-20 mM NaH2PO4-120 mM sodium dodecylsulfate-30% (v/v) 2-propanol (pH 6.5-7) within 35 min. The migration behaviors of the compounds with different types and concentrations of organic modifiers were studied. The method can be used to identify the purification of the lignans, monitor the 2-H configuration transformation, and determine the 2-H configuration of the spin-labeled derivatives.

Chromatography, Micellar Electrokinetic Capillary↗

High yield of podophyllotoxin from leaves of Podophyllum peltatum by in situ conversion of podophyllotoxin 4- O-beta-D-glucopyranoside.

Rehydration of powdered tissues of Podophyllum peltatum L. prior to extraction with an organic solvent allows endogenous beta-glucosidases to hydrolyze lignan 4-O-beta-D-glucosides in situ and increase the yield of podophyllotoxin. Aqueous extraction of rhizomes and leaves of P. peltatum yielded 4- to 10-fold greater quantities of podophyllotoxin than the traditional ethanolic extraction. Most significantly, leaves were shown to contain over 52 mg of podophyllotoxin per g of dry weight (5.2%), exceeding levels previously reported from any source. These results point to the use of leaves harvested from cultivated P. peltatum as an attractive alternative to the destructive collection of natural populations.

Glucosides↗

Guinea pig maximization test: Podophyllum lignans.

The guinea pig maximization test was used to study the sensitizing potential of Podophyllum lignans; 0.5% of each drug in propylene glycol was used for induction and 0.01%, 0.05%, 0.1% and 0.5% preparations for topical challenge. The 0.1% and 0.5% preparations caused non-allergic toxic reactions associated with the cutaneous destructive influence of these drugs; allergic responses did not occur.

Animals↗

Intralesional interferon alfa-2b treatment of condylomata acuminata previously resistant to podophyllum resin application.

The safety and efficacy of intralesionally administered interferon alfa-2b were assessed in 11 patients suffering from condylomata acuminata for four to 10 months for whom application of podophyllum resin had proved unsuccessful. Three warts from each patient were injected with 10(6) IU interferon alfa-2b three times a week for three weeks. Treatment was followed by a 13 week observation period. Interferon alfa-2b treatment resulted in a highly significant (p less than 0.0001) reduction in the mean size of the treated warts, which decreased from an initial size of 29 mm2 to 2-3 mm2 by week 16. In six out of the 10 patients completing the trial, both the test condylomata and adjacent control warts cleared completely; a recurrence was observed in one of these six patients. Influenza like symptoms (headache and myalgia) were the most common side effects reported, though they were mild in nature and not disabling. These results corroborate those of previous trials with interferon preparations and indicate that its antiviral activity makes it a possible treatment for this sexually transmitted disorder.

Adult↗

Production of podophyllotoxin by plant cell cultures of Podophyllum hexandrum in bioreactor.

Submerged cultivation of Podophyllum hexandrum for the production of podophyllotoxin was carried out in a 3l stirred tank bioreactor fitted with a low-shear Setric impeller. The specific requirements of the medium, such as carbon source (sugar) and light, were established for the growth of and podophyllotoxin production by P. hexandrum in suspension cultures. Substitution of sucrose by glucose resulted in higher growth and podophyllotoxin production. The biosynthesis of podophyllotoxin was favored when plant cells were cultivated in the dark. An agitation speed of 100 rpm was sufficient to mix the culture broth in the bioreactor without causing any significant cell damage. Biomass and podophyllotoxin accumulation in 3 l bioreactor under batch growth conditions were 6.5 g/l and 4.26 mg/l, respectively, in 22 d. This resulted in an overall podophyllotoxin productivity of 0.19 mg/(l.d), which represented an increase of 27% in comparison to its productivity in a shake flask. Podophyllotoxin production was found to be a combined growth-associated and non-growth associated process.

Journal Article↗

[Bacterial leaf blight affecting Syngonium podophyllum in Argentina].

Bacterial leaf blight of Syngonium podophyllum caused by Xanthomonas campestris pv. syngonii is recorded for the first time in Argentina. The first symptom of the disease was an interveinal watersoaking of leaves, the tissues became chlorotic and finally necrotic over areas of about 4 cm. The identification of the causal microorganism was based on disease symptoms, morphological, physiological and biochemical characteristics and pathogenicity test.

Argentina↗