Podophyllotoxin Content Research Articles

Accumulation of podophyllotoxin in the root culture of Hyptis suaveolens (L.) Poit: A potential natural lignan for clinically useful anticancer drugs

Podophyllotoxin (PTOX) is a natural antiviral, antirheumatic and anticancer molecule, but it is expensive to chemically synthesize. The present study aimed to evaluate growth and PTOX accumulation in root cultures of Hyptis suaveolens treated with different concentrations of auxins, vitamins and myo-inositol. Root cultures grown in liquid medium supplemented with different concentrations of indole-3-butyric acid (IBA), 1-naphthaleneacetic acid (NAA), vitamins and myo-inositol were used to measure biomass production and PTOX content. PTOX quantitation was performed via high-performance liquid chromatography with diode-array detection (HPLC-DAD) using a newly developed analytical method after successful validation. Root culture in MS medium supplemented with 1 mg L−1 IBA +0.5 mg L−1 NAA resulted in the highest root dry weight (248.76 mg) and the highest PTOX concentration in the roots (179.97 μg g−1 root). The roots cultured in liquid MS medium supplemented with 1 mg L−1 IBA +0.5 mg L−1 NAA presented the greatest increase in root biomass and PTOX content. This adequate balance of vitamin and myo-inositol supplementation in liquid MS culture medium increased the root dry weight and PTOX content.

Integrated transcriptome and metabolome analysis reveals the podophyllotoxins accumulation and formation mechanisms in Juniperus sabina L. leaves

AbstractPodophyllotoxin is an important secondary metabolite because of its remarkable anticancer and anti‐Condyloma acuminatum activities. Juniperus sabina L. (J. sabina) has been proven as a new plant source of podophyllotoxins. However, the mechanism of podophyllotoxins accumulation and synthesis in J. sabina is unclear, which greatly limits their applications. Based on this, transcriptomic and metabolomic analyses were performed on different developmental stages of J. sabina leaves to determine the molecular mechanism of the biosynthesis of podophyllotoxins in this study. Firstly, 8 podophyllotoxins and 54 differentially accumulated compounds were identified from metabolomic analysis of J. sabina leaves at different developmental stages. The total lignans and podophyllotoxin contents were determined by HPLC, clarifying that May to September was the critical period for the accumulation of podophyllotoxins in J. sabina leaves. Transcriptome and metabolic analysis showed that 47 DEGs encoded 16 key enzymes involved in podophyllotoxin biosynthesis, among which 8 key enzyme genes (PAL, HCT, CCR, C4H, DIR, PLR, OMT and CYP82) were identified. Furthermore, JsPLR was proven to be a key rate‐limiting enzyme cytoplasmic‐localized. JsPLR overexpression significantly increased matairesinol and podophyllotoxin content.. The findings of qRT‐PCR study indicated that the differential expression of JsPLR gene might be a key factor leading to the differential accumulation of podophyllotoxin. Overall, the study brings new insight into the accumulation and formation mechanisms of podophyllotoxins and the comprehensive development and utilization of J. sabina resources.

Green Ultrasound-Assisted Extraction of Podophyllotoxin from Juniperus scopulorum Needles

Background—Podophyllotoxin, a polyphenolic compound with major applications in cancer treatment, is in short supply, as its source is now an endangered plant species. It is therefore essential to find another available plant that produces high levels of podophyllotoxin. Some Juniperus species are known to contain podophyllotoxin, more exactly within their needles, but are still unused in this regard. Objective—The aim is to develop an efficient green ultrasound-assisted extraction protocol for podophyllotoxin from commercially available Juniperus scopulorum varieties. Methodology—To highlight optimal extraction conditions, a single-factor experiment was performed to identify the parameters (extraction duration, frequency, temperature extraction, preliminary grinding, and plant material concentration) influencing extraction. A multifactorial approach using the Box–Behnken design was then applied to determine the exact optimal extraction conditions. Results—The conditions for extracting the maximum amount of podophyllotoxin consist of 51.5 min of extraction time, in ethanol at 69.3%, and at 58.8 °C. This is the highest podophyllotoxin extraction yield ever obtained from Juniperus scopulorum needles. Compared with the reference protocol for the extracting of podophyllotoxin from Juniperus, ultrasonic extraction is an eco-friendly protocol, safe for humans, and the podophyllotoxin extracted by this method has much greater stability, allowing its purification. The analyses also indicate a variation in the podophyllotoxin content of Juniperus scopulorum needles, (from 7.02 to 10.34 mg/g DW) depending on the variety and year of purchase. Conclusion—Extraction in ethanol at 69.3% and at 58.8 °C for 51.5 min allows the extraction of up to 10.34 mg podophyllotoxin/g from freeze-dried Juniperus scopulorum needles.

Enhanced podophyllotoxin production of endophyte Fusarium proliferatum TQN5T by host extract and phenylalanine.

Fermentation technology using endophytes is considered a potential alternative approach for producing pharmaceutical compounds like podophyllotoxin (PTOX). In this study, fungus TQN5T (VCCM 44284) was selected from endophytic fungi isolated from Dysosma versipellis in Vietnam for PTOX production through TLC. The presence of PTOX in TQN5T was further confirmed by HPLC. Molecular identification indicated TQN5T as Fusarium proliferatum with 99.43% identity. This result was asserted by morphological characteristics such as white cottony, filamentous colony, layer and branched mycelium, and clear hyphae septa. Cytotoxic assay indicated both biomass extract and culture filtrate of TQN5T presented strong cytotoxicity on LU-1 and HepG2 with IC50 of 0.11, 0.20, 0.041, and 0071, respectively, implying anti-cancer compounds were accumulated in the mycelium and secreted into the medium. Further, the production of PTOX in TQN5T was investigated in the fermentation condition supplemented with 10µg/ml of host plant extract or phenylalanine as elicitors. The results revealed a significantly higher amount of PTOX in the PDB + PE and PDB + PA at all studied time points in comparison with PDB (control). Especially, after 168h of culture, PTOX content in the PDB with plant extract reached the peak with 314µg/g DW which is 10% higher than the best yield of PTOX in previous studies, denoting F. proliferatum TQN5T as a promising PTOX producer. This is the first study on enhancing the PTOX production in endophytic fungi by supplementing phenylalanine-a precursor for PTOX biosynthesis in plants into fermented media, suggesting a common PTOX biosynthetic pathway between host plant and endophytes. KEY POINTS: • Fusarium proliferatum TQN5T was proven for PTOX production. • Both mycelia extract and spent broth extract of Fusarium proliferatum TQN5T presented strong cytotoxicity on cancer cell lines LU-1 and HepG2. • The supplementation of 10µg/ml host plant extract and phenylalanine into fermentation media of F. proliferatum TQN5T improved the yield of PTOX.

Variation of Podophyllotoxin with the age in different plant parts of Podophyllum hexandrum

In the present study, variation in podophyllotoxin content was studied with growth and development in different parts of Podophyllum hexandrum, i.e., leaf lamina, petiole and rootstock using High Performance Liquid Chromatography (HPLC). It was found that podophyllotoxin content in rootstock and leaf lamina increased with increase in age of the plant while it was recorded vice-versa in petiole of the plant. The rootstock was found to contain the highest content of podophyllotoxin in comparison to other parts.

Effect of ploidy level on podophyllotoxin content and expression of genes related to its biosynthesis in callus cultures of Linum album

Effect of ploidy level on podophyllotoxin content and expression of genes related to its biosynthesis in callus cultures of Linum album

Essential Oil Composition and Bioactivity of Two Juniper Species from Bulgaria and Slovakia.

Juniperus excelsa M. Bieb and J. sabina L. contain essential oil (EO), while J. sabina also contains podophyllotoxin, which is used as a precursor for anti-cancer drugs. Two studies were conducted. The first assessed the variability in the EO profile and podophyllotoxin concentration of the two junipers, depending on the location and tree gender. The main EO constituents of J. excelsa were α-cedrol, α-limonene and α-pinene, while the constituents in J. sabina were sabinene, terpinen-4-ol, myrtenyl acetate and α-cadinol. The podophyllotoxin yield of 18 J. sabina accessions was 0.07–0.32% (w/w), but this was not found in any of the J. excelsa accessions. The second study assessed the effect of hydrodistillation (Clevenger apparatus) and steam distillation (in a semi-commercial apparatus) on the EO profile and bioactivity. The extraction type did not significantly alter the EO composition. The EO profiles of the two junipers and their accessions were different and may be of interest to the industry utilizing juniper leaf EO. Breeding and selection programs could be developed with the two junipers (protected species) in order to identify chemotypes with (1) a high EO content and desirable composition, and (2) a high concentration of podophyllotoxin in J. sabina. Such chemotypes could be established as agricultural crops for the commercial production of podophyllotoxin and EO.

ROS-Activated homodimeric podophyllotoxin nanomedicine with self-accelerating drug release for efficient cancer eradication

Although podophyllotoxin (POD) demonstrates high efficiency to inhibit various cancers, its clinic application is limited to poor bioavailability. Nanoparticles derived from homodimeric prodrugs with high drug loading potential are emerging as promising nanomedicines. However, complete intracellular drug release remains a major hindrance to the use of homodimeric prodrugs-based nanomedicine. We sought to develop a reactive oxygen species (ROS) responsive POD dimeric prodrug by incorporating vitamin K3 (VK3) and Pluronic F127 to synthesize a spheroid nanoparticle (PTV-NPs). PTV-NPs with high POD content could release drugs under the ROS enrichment microenvironment in cancer cells. The released VK3 could produce abundant ROS selectively in tumor cells catalyzed by the overexpressed NAD(P)H: quinone oxidoreductase-1 (NQO1) enzyme. In turn, the resultant high ROS concentration promoted the conversion of POD dimeric prodrug to POD monomer, thereby achieving the selective killing of cancer cells with weak system toxicity. In vitro and in vivo studies consistently confirmed that PTV-NPs exhibit high drug loading potential and upstanding bioavailability. They are also effectively internalized by tumor cells, induce abundant intracellular ROS generation, and have high tumor-specific cytotoxicity. This ROS-responsive dimeric prodrug nanoplatform characterized by selective self-amplification drug release may hold promise in the field of antitumor drug delivery.

Effects of Arbuscular Mycorrhizal Fungi Glomus mosseae on the Growth and Medicinal Components of Dysosma versipellis Under Copper Stress.

In order to investigate the effects of arbuscular mycorrhizal fungi (AMF) (Glomus mosseae) inoculations and additions of copper (Cu) ion at gradual concentrations (0, 200, and 400mgkg-1) on different parameters of Dysosma versipellis such as growth, lipid peroxidation (MDA and MRP), antioxidation enzymatic (SOD, POD and CAT) activities, and active medicinal components. Pot experiments have been conducted. The results showed that additions of Cu could inhibit growth and the activity of antioxidant enzymes, increase the degree of membrane lipid peroxidation, and decrease the podophyllotoxin content of D. versipellis compared with the control. Meanwhile, inoculations with AMF enhanced its antioxidant capacity and reduced the degree of membrane lipid peroxidation in leaves of D. versipellis under Cu stress. Besides, AMF inoculations significantly increased the biomass and content of podophyllotoxin in roots of D. versipellis, while it decreased Cu absorption content in roots. Thus, inoculations with AMF could effectively alleviate the Cu stress and improve the active components content of D. versipellis, which might be important for Cu stress adaptation and the improved productivity and quality of D. versipellis.

Effects of exogenous IBA and fungal elicitor on growth of in vitro roots culture of Dysosma versipellis and production of podophyllotoxin

Using the White as basic medium, the effects of the exogenous IBA and endophytic fungal elicitor on the growth of in vitro roots cultures of Dysosma versipellis and production of podophyllotoxin were investigated in this study. The results showed that the IBA and the endophytic fungus Zasmidium syzygii elicitor could increase the content of podophyllotoxin of in vitro roots of D. versipellis after 3 weeks. The White medium added with 3 mg·L~(-1) IBA induced the highest increase of podophyllotoxin(1 830.86 μg·g~(-1)), which was 2.07 folds greater than the control, and followed by 1.5 mg·L~(-1) IBA, fungal elicitor, 1 mg·L~(-1) IBA, 0.5 mg·L~(-1) IBA and 4.5 mg·L~(-1) IBA, which was 1.82, 1.71, 1.63, 1.43 and 1.1 folds greater than the control, respectively. The results also showed that the growth of roots was certain positively correlated with the change of IBA concentration. Therefore, 3 mg·L~(-1) IBA was the most suitable for the production of podophyllotoxin in the in vitro roots of D. versipellis, and the stimulating effect of Z. syzygii fungal elicitor was between 1.5 mg·L~(-1) and 1 mg·L~(-1) IBA, which was a potential natural elicitor to induce the accumulation of podophyllotoxin in future production.

Effect of Precursor and Phytohormones on Podophyllotoxin Production in Juniperus virginiana Suspension Cultures

Our results showed that cinnamic acid can increase podophyllotoxin production in Juniperus virginiana suspension cultures. The best effect was manifested after a 24-hours application of a 10 mmol/L concentration. The highest podophyllotoxin content was determined at 1.47 mg/g DW and the production was statistically significantly stimulated by about 444% in comparison with the control. Comparison of podophyllotoxin production in the cinnamic acid- and salicylic acid-elicited J. virginiana suspension cultures confirms that the maximum increase in both cases was induced by the 24-hours application of the 10 mmol/L concentration. In contrast, the best effect of jasmonic acid was manifested after the longest 168-hours application of a 5 mmol/L concentration.

The effect of colored plastic films on the photosynthetic characteristics and content of active ingredients of Dysosma versipellis

Light intensity and quality affect photosynthesis, plant morphology, and the synthesis of primary and secondary metabolites. Dysosma versipellis (Hance) M. Cheng is an endangered species endemic to China, and a highly valued medicinal and ornamental plant. In this study, we discuss the effects of different light spectrums conferred by colored plastic films on photosynthesis and the contents of active ingredients of D. versipellis. D. versipellis plants were cultured for 90 days under white, red, yellow, or blue film. The blue film treatment generally increased the chlorophyll content, photosynthetic rate (Pn), maximal photochemical efficiency of PSII (Fv/Fm), actual photochemical efficiency of PSII (ФPSII), photochemical quenching (qP), and the podophyllotoxin content of the rhizomes. The blue film treatment also decreased the maximum net photosynthetic rate (Amax), apparent photosynthetic quantum efficiency (AQY), and podophyllotoxin contents of the stems. The yellow film treatment resulted in a decline of the Amax, AQY, Fv/Fm, ФPSII, qP, chlorophyll contents, and podophyllotoxin contents of the leaves and rhizomes; however, the light compensation point (LCP), light saturation point, and minimum fluorescence (Fo) were increased. There were no significant differences in chlorophyll content, Amax, LCP, AQY, Fv/Fm, ФPSII, or qP between the white and red film treatments. These results suggest that in D. versipellis, blue film treatments promote photosynthesis and the accumulation of podophyllotoxin, while yellow film treatments inhibit photosynthesis and the accumulation of podophyllotoxin.

Comparing aryltetralin lignan accumulation patterns in four biotechnological systems of Linum album

Linum album is a herbaceous plant with medical interest due to its content of podophyllotoxin (PTOX), an aryltetralin lignan with cytotoxic activity. Previous studies in our laboratory showed that cell suspension cultures of L. album produced more PTOX than methoxypodophyllotoxin (6-MPTOX), both lignans being formed from the same precursor after divergence close to the end of the biosynthetic pathway. In contrast, the hairy roots produced more 6-MPTOX than PTOX.Taking into account this variability, we were interested to know if the lignan profile of an in vitro PTOX-producing L. album plant changes according to the biotechnological system employed and, if so, if this is due to cell dedifferentiation and/or transformation events. With this aim, we established four biotechnological systems: (1) Wild type cell suspensions, (2) transformed cell suspensions, (3) adventitious roots and (4) hairy roots.We determined the production of four aryltetralin lignans: PTOX, 6-MPTOX, deoxypodophyllotoxin (dPTOX) and β-peltatin.The results show that in vitro plantlets, WT cells and transformed cells predominantly produced PTOX, production being 11-fold higher in the plantlets. Otherwise, the adventitious and hairy roots predominantly produced 6-MPTOX, the adventitious roots being the most productive, with MPTOX levels 1.58-fold higher than in transformed roots.We can infer from these results that in the studied plants, cell differentiation promoted the formation of 6-MPTOX over PTOX, while transformation did not influence the lignan pattern.

Abiotic factors influencing podophyllotoxin and yatein overproduction in Leptohyptis macrostachys cultivated in vitro

This work describes an in vitro propagation protocol for the large-scale cultivation of Leptohyptis macrostachys (Benth.) Harley & JFB Pastore and the influence of abiotic factors on podophyllotoxin and yatein production. The plant was established from seeds collected in Chapada Diamantina, BA, Brazil and submitted to different growth mediums and physical conditions. The podophyllotoxin and yatein contents were quantified by HPLC/DAD and with pure standards and these two lignans were present in all experiments. The lignan quantities were evaluated using the Sisvar Program, compared by Tukey’s test and hierarchical cluster analysis and principal component analysis. In all experiments, podophyllotoxin and yatein were detected at different concentrations. The best protocol cultivar of L. macrostachys was established from seeds in MS ½ medium supplemented with 1.5% sucrose and 11.55μM of gibberellic acid A3 (GA3) at 30°C, which yielded the highest concentration of podophyllotoxin (5.831mgg−1). These results are important findings for the production of podophyllotoxin from the tissue culture.

Transcriptomes of Podophyllum hexandrum unravel candidate miRNAs and their association with the biosynthesis of secondary metabolites

Podophyllotoxin is a high value metabolite having anticancerous activity and procured from a medicinal plant Podophyllum hexandrum. The escalating demand for podophyllotoxin necessitates development of alternate production platforms. Cell cultures have been tried in the past, however, podophyllotoxin yields were very low (0.3%). Multiple genes of biosynthetic pathway have been correlated with podophyllotoxin content. Understanding regulation of podophyllotoxin is, therefore, essential to design a suitable genetic intervention strategy. Six potential miRNAs were identified in NGS transcriptomes of P. hexandrum for which eight potential mRNA targets were discerned. The identified miRNAs and their mRNA targets (UDP glycosyltransferase, flavonol synthase, glyceraldehyde 3-phosphate dehydrogenase, peroxidase, malate dehydrogenase, phosphoenolpyruvate carboxylase, WRKY 37 and MYBF1 transcription factors) were further tested for any correlation through qRT-PCR. miR396b, miR2673a, miR828b and miR2910 established negative correlation with their mRNA targets through downregulation via complementary miRNA:mRNA base pairing. This is the first report on identification, characterisation, validation and expression analysis of miRNAs and their targets in P. hexandrum.

Production of Podophyllotoxin by Plant Tissue Cultures of Juniperus virginiana

Plant tissue cultures are a potential source of secondary metabolites. However, their production, when compared with intact plants, is usually lower. Phenylalanine, a biogenetic precursor of podophyllotoxin, was used to stimulate podophyllotoxin production in callus and suspension cultures of Juniperus virginiana L. The best phenylalanine effect on podophyllotoxin production was manifested in three-years-old callus cultures after a 21-days application of a 10 mmol/L concentration. A podophyllotoxin content of 0.15 mg/g DW was determined, which was about 400% higher in comparison with the control. The maximum content (0.48 mg/g DW) in newly derived suspension cultures (the 4' passage) was induced by 14-days application of a I mmol/L concentration; this was about 243% higher than the control. In one-year-old suspension cultures the highest podophyllotoxin content (0.56 mg/g DW) was recorded also after 14-days application of a I mmol/L concentration; this was about 211% higher than in the control cultures.

Identification, validation, and expression of ABC transporters in Podophyllum hexandrum and their role in podophyllotoxin biosynthesis

Podophyllum hexandrum Royle is an important medicinal herb of North-Western Himalayas, and podophyllotoxin, being its major metabolite, has been used extensively in the preparation of several anticancer drugs. Podophyllotoxin accumulates in rhizomes; however, no information exists on the role of ATP-binding cassette (ABC) transporters vis-a-vis podophyllotoxin content. The present study reports identification, validation, and expression analysis of ABC transporter genes from P. hexandrum. Total 252 ABC transporter genes were identified as unigenes out of which 22 were further validated using real time qPCR in different tissues of varying podophyllotoxin content. Differential expression analysis and Pearson’s correlation coefficient revealed two candidate genes PhABC6 and PhABCIII having a positive correlation with the podophyllotoxin content. PhABCIV showed the highest expression in rhizomes (20.53-folds compared to shoots) suggesting its possible role in transport and accumulation of podophyllotoxin.

Methyl Jasmonate Induces Enhanced Podophyllotoxin Production in Cell Cultures of Thracian Flax (Linum thracicum ssp. thracicum)

The Linum thracicum ssp. thracicum cell lines developed in this study are a feasible source for the sustainable production of podophyllotoxin, a lignan with an aryltetralin skeleton that is used for the manufacture of the chemotherapeutic drugs etopophos and teniposide. We used mass spectrometry to confirm the presence of the aryltetralin lignan in the thracian flax cell cultures. Next, we explored how changes in the culture medium influenced the podophyllotoxin content. Out of six developed cell lines, four were selected for further experiments and challenged with elicitors. The selected cell lines clustered into two groups: developed in full strength medium (Li) vs developed in half strength medium (HS). While podophyllotoxin production in the Li cell lines was boosted by 80% upon administration of the elicitor methyl jasmonate, the HS lines produced high amounts of the target metabolite triggered by reduced concentration of nutrients and were only slightly influenced by the elicitor.

Effect of topographical factors on podophyllotoxin content in Sinopodophyllum hexandrum and study on ecological suitability

In order to find the optimal topographical factor for regionslization, the content of cimetidine in 116 Sinopodophyllum hexandrum sample collected from Sichuan, Qinghai, Gansu, Tibet, Yunnan and Shaanxi provinces, was determined. Using mathematical statistics and geographical spatial analysis of GIS analysis, the relationship between content of podophyllotoxin and influencing factors including altitude gradient and gradient position was analyzed. It is found that the optimal altitude was 2 800 m to 3 600 m, the aspect of slope north or northeast and northwest and the slope 12 degrees to 65 degrees with a high suitability degree. Considering the artificial planting, the suitable planting area for S. hexandrum is comfirmed. The topographical factor is important for S. hexandrum regionalization, but has hardly effect on podophyllotoxin content. The results of the study provide an important scientific basis for S. hexandrum production development. But there are many factors which affect suitability index and podophyllotoxin content of S. hexandrum, it is necessary to consider other factors like climate and soil while exploitation and protection of S. hexandrum.

Correlation research on contents of podophyllotoxin and total lignans in Sinopodophyllum hexandrum and ecological factors

To explore the correlation between the ecological factors and the contents of podophyllotoxin and total lignans in root and rhizome of Sinopodophyllum hexandrum, podophyllotoxin in 87 samples (from 5 provinces) was determined by HPLC and total lignans by UV. A correlation and regression analysis was made by software SPSS 16.0 in combination with ecological factors (terrain, soil and climate). The content determination results showed a great difference between podophyllotoxin and total lignans, attaining 1.001%-6.230% and 5.350%-16.34%, respective. The correlation and regression analysis by SPSS showed a positive linear correlation between their contents, strong positive correlation between their contents, latitude and annual average rainfall within the sampling area, weak negative correlation with pH value and organic material in soil, weaker and stronger positive correlations with soil potassium, weak negative correlation with slope and annual average temperature and weaker positive correlation between the podophyllotoxin content and soil potassium.