Podophyllum Species Research Articles

Assessment of genetic diversity and interspecific relationships among three species of Podophyllum using AFLP markers and podophyllotoxin content

The genus Podophyllum (common name: May Apple) has high medicinal value due to the presence of anticancer molecule, podophyllotoxin. A total of 35 individuals belonging to three species of Podophyllum viz. P. hexandrum Royle, P. sikkimensis R. Chatterjee and Mukherjee both Indian species, and their American counterpart, P. peltatum L. have been investigated with a view to ascertain variation in their (1) podophyllotoxin content, and (2) molecular profiles generated through AFLP markers. The active principle content varied within the representative individuals of different populations of a species and between species; the species-wise podophyllotoxin content (% of dry wt) ranged as follows: P. hexandrum-Munsyari populations: 0.39–1.20 %, P. hexandrum-Kullu populations: 0.58–1.50 % (highest), P. peltatum: 0.50–1.30 %, and P. sikkimensis: 0.06–0.73 % (lowest). Detection of podophyllotoxin in P. sikkimensis, although at low levels, would appear to be the first report of its occurrence in this species. The genetic diversity and relationship amongst 35 sampled individuals of three species have been analyzed using 20 AFLP markers, which resulted into 1,358 loci of which 595 were polymorphic revealing 44 % polymorphism. High level of genetic diversity was observed (percent of polymorphic bands, PPB = 88.01 %; PIC = 0.813) among the species, while it was low within the individual species (PIC = 0.57 %; Marker index, MI = 4.77). Genetic similarity among the species (calculated with Euclidean coefficient) showed two major clusters. Cluster one contained all the individuals of P. peltatum (American May Apple) whereas cluster two grouped together individuals representing various populations, belonging to both the species of Indian May Apple (P. hexandrum, and P. sikkimensis). The observed paired relationship (45–50 % similarity; calculated from AFLP data) of intercontinental species in the Podophyllum group (P. hexandrum, and P. sikkimensis vs. P. peltatum) would appear to be paraphyletic. The AFLP data of the analyzed representatives have been used to examine the sister relationships among these species, and would be beneficial to find ways to strengthen the gene flow among populations to maintain the natural genetic variation within the populations of Podophyllum species.

Next Generation Sequencing in Predicting Gene Function in Podophyllotoxin Biosynthesis

Podophyllum species are sources of (-)-podophyllotoxin, an aryltetralin lignan used for semi-synthesis of various powerful and extensively employed cancer-treating drugs. Its biosynthetic pathway, however, remains largely unknown, with the last unequivocally demonstrated intermediate being (-)-matairesinol. Herein, massively parallel sequencing of Podophyllum hexandrum and Podophyllum peltatum transcriptomes and subsequent bioinformatics analyses of the corresponding assemblies were carried out. Validation of the assembly process was first achieved through confirmation of assembled sequences with those of various genes previously established as involved in podophyllotoxin biosynthesis as well as other candidate biosynthetic pathway genes. This contribution describes characterization of two of the latter, namely the cytochrome P450s, CYP719A23 from P. hexandrum and CYP719A24 from P. peltatum. Both enzymes were capable of converting (-)-matairesinol into (-)-pluviatolide by catalyzing methylenedioxy bridge formation and did not act on other possible substrates tested. Interestingly, the enzymes described herein were highly similar to methylenedioxy bridge-forming enzymes from alkaloid biosynthesis, whereas candidates more similar to lignan biosynthetic enzymes were catalytically inactive with the substrates employed. This overall strategy has thus enabled facile further identification of enzymes putatively involved in (-)-podophyllotoxin biosynthesis and underscores the deductive power of next generation sequencing and bioinformatics to probe and deduce medicinal plant biosynthetic pathways.

Podophyllotoxin, a medicinal agent of plant origin: past, present and future

ABSTRACT The aryltetralin-type lignan podophyllotoxin, the main agent present in the resin extracted from the rhizomes of different podophyllum species, possesses important antineoplastic and antiviral properties and has been widely used in diverse cultures since ancient times for medicinal purposes as a cathartic and antihelminthic agent. Today, podophyllotoxin serves as the starting material for the preparation of the well-known cytostatic agents: etoposide and teniposide which are used in combination therapies with other drugs for the treatment of a variety of malignancies. The present review focuses on the lead compound, that is, podophyllotoxin and summarizes its structural characteristics, natural sources, biological activities, novel derivatives, medicinal applications and future perspectives.

Mayapple: A review of the literature from a horticultural perspective

Species of the Podophyllum genus contain important anti-cancer compounds: podophyllotoxin, �peltatin, and �-peltatin. A Podophyllum species found in India, Podophyllum emodi Wall. (Syn. P. hexandrum Royale, Berberidaceae), has been over-harvested to meet pharmaceutical industry demand. The Indian species P. emodi Wall, is considered an endangered species because the compound is found in the roots and whole plant was harvested. On the other hand, American Mayapple is a rhizomatous herbaceous perennial found in the wild throughout North America from Quebec and Minnesota to Florida and Texas and source of compound is leaves. Commercial plantings of mayapple have the potential to reduce harvest pressure on this species. In a review of the scientific literature, few references about commercial production or field establishment were found. To begin to understand mayapple as a horticultural crop, this review presents a summary of the plant’s botany, climate and soils, growth and development, sexual and asexual propagation, pests and diseases, and medicinal uses. Sixty references are listed.

Chemometric Evaluation of the Anti‐cancer Pro‐drug Podophyllotoxin and Potential Therapeutic Analogues in Juniperus and Podophyllum Species

Podophyllotoxin, deoxypodophyllotoxin, demethylpodophyllotoxin and podophyllotoxone are four therapeutically potent secondary metabolites. There is a dearth of information on the holistic analysis of their distribution pattern in both phylogenetic and ecological contexts. To analyse the continuum of the above metabolites in Juniperus and Podophyllum species collected from natural populations in Himalayan environments and the botanical gardens of Rombergpark and Haltern (Germany) using multi-component LC-ESI-MS/MS, coupled with statistically relevant chemometric assessment. We evaluated the individual and holistic metabolite profiles and chemometrically correlated the phytochemical loads between various species (infraspecific), organic and aqueous extracts, and populations of the same species from different locations, different species from same location, different species from different locations and infrageneric populations from same and different locations. Multivariate analysis revealed Juniperus x-media Pfitzeriana as a suitable alternative to Podophyllum hexandrum for commercial exploitation. A significant positive correlation of podophyllotoxone with both podophyllotoxin and demethylpodophyllotoxin, and a negative correlation of podophyllotoxin with both deoxypodophyllotoxin and demethylpodophyllotoxin (infraspecific among Podophyllum), were observed by Kruskal's multidimensional scaling and corroborated by principal component analysis, indicating probable similarity and/or difference between the biosynthetic pathways, and synergistic and/or antagonistic principles, respectively. Finally, linear discriminant analysis and hierarchical agglomerative cluster analysis revealed considerable infrageneric and infraspecific variability in secondary compound spectra and load of the different populations under study. Such holistic studies of plants and their therapeutic metabolites ought to assist in selecting plants, geographical areas and environmental conditions for bioprospecting and global-scale phytochemical and phylogenetic diversity studies in the future.

Podophyllotoxin: Current approaches to its biotechnological production and future challenges

AbstractMany plant‐derived agents are being used to treat cancer, including taxol, vinblastine, vincristine, or camptothecin and podophyllotoxin derivatives, among others. Plant biotechnology can provide a new tool for the production of anticancer agents but in spite of considerable efforts to produce vinblastine and vincristine in cell cultures and knowledge of the biosynthetic pathway of Catharanthus roseus alkaloids, the biotechnological production of taxol has only been achieved at an industrial level by companies such as Phyton Biotech and Cytoclonal Pharmaceutics. Podophyllotoxin was isolated as the active antitumor agent from the roots of Podophyllum species and more recently from the genus Linum and others. Etoposide, teniposide, and etophos are semi‐synthetic derivatives of podophyllotoxin and are used in the treatment of cancer. Biotechnological approaches, including the use of cell cultures, biotransformation, or metabolic engineering techniques to manipulate the biosynthetic pathway, represent an alternative for the production of podophyllotoxin and are discussed in this review.

Updated biotechnological approaches developed for 2,7′-cyclolignan production

2,7'-Cyclolignans constitute a group of compounds regularly employed in clinical contexts due to their antiviral and anticancer properties as expressed in a number of already available designed drugs. Possibilities for other therapeutic developments are indicated. All commercial preparations actually originate from a single compound, podophyllotoxin, currently extracted from a few limited-in-abundance species of Podophyllum. This supply problem has stimulated considerable interest in the development of alternative strategies offering greater sustainable availability of the compound at affordable costs. Approaches such as plant breeding and micropropagation of the high-producing Podophyllum species have been explored with the aim of establishing production of these compounds. Since 20 years of exploration of the total chemical synthesis of podophyllotoxin has not proved economically viable, extensive research has now been undertaken to find methods for stimulating the accumulation of 2,7'-cyclolignans using tissue cultures. Both undifferentiated and differentiated cell cultures, mainly from Linum, Podophyllum, Juniperus, Callitris, Anthriscus and Forsynthia genera, have been reported. Although considerable progress has been made concerning the concentration and productivity of certain strains of Linum album, no economically viable production system has been established. Exploration and development of biosynthetical pathways leading to 2,7'-cyclolignans may open paths for future metabolic engineering, such as the bioconversion of deoxypodophyllotoxin to epipodophyllotoxin employing a human hepatic enzyme heterologously expressed in Escherichia coli.

Germplasm conservation and quantitative estimation of podophyllotoxin and related glycosides of Podophyllum hexandrum

We have done comparative morphological and chemotaxonomic studies to investigate the phylogenetic relationship of different accessions within the Podophyllum species. HPLC profiles revealed that all Podophyllum hexandrum accessions collected from different geographical locations are chemically highly diverse. Also, we have observed that chemotaxonomic studies clearly demonstrated that chemical characters of the Podophyllum hexandrum are suitable to generate essentially the same relationship as revealed by RAPD analysis.

In vitro embryogenesis and marker guided analysis of Podophyllum hexandrum: an endangered medicinal plant.

For in vitro embryogenesis, excised embryos germinate within a week of inoculation on Murashige and Skoog basal medium supplemented with different concentrations of Plant growth regulators, BA (1.0 - 4.0 mg/l) and IAA (0.5 - 2.0 mg/l). Basal medium with BA concentration (0.5 mg/l) and IAA (1.0 mg/l) showed better results than other combinations and was therefore adopted for further studies. The combination of MS + BA (0.5 mg/l + IAA 1.0 mg/l) supplemented with activated charcoal (0.5 - 1.0%) result in optimum growth ofPodophyllum hexandrum plantlets. Comparative chemotaxonomic studies were done to investigate the phylogenetic relationship of different accessions within the Podophyllum species. Chemical profiles demonstrated that all Podophyllum hexandrum accessions collected from different geographical regions are chemically diverse. Chemotaxonomic data showed that chemical characters of the investigated species were able to generate essentially the same relationship as revealed by RAPD analysis. The study has revealed that maximum amount of the podophyllotoxin (5.97%) and podophyllotoxin b-D glycoside (5.72%) was present in the Podophyllum population collected from Keller (Shopian) and Khilanmarg (Gulmarg) area of Jammu and Kashmir, respectively. Key words: Podophyllum, podophyllotoxin, quantitative, metabolite, chemotaxonomic.

Secoisolariciresinol dehydrogenase: mode of catalysis and stereospecificity of hydride transfer in Podophyllum peltatum

Secoisolariciresinol dehydrogenase (SDH) catalyzes the NAD+ dependent enantiospecific conversion of secoisolariciresinol into matairesinol. In Podophyllum species, (-)-matairesinol is metabolized into the antiviral compound, podophyllotoxin, which can be semi-synthetically converted into the anticancer agents, etoposide, teniposide and Etopophos. Matairesinol is also a precursor of the cancer-preventative "mammalian" lignan, enterolactone, formed in the gut following ingestion of, for example, various high fiber dietary foods, as well as being an intermediate to numerous defense compounds in vascular plants. This study investigated the mode of enantiospecific Podophyllum SDH catalysis, the order of binding, and the stereospecificity of hydride abstraction/transfer from secoisolariciresinol to NAD+. SDH contains a highly conserved catalytic triad (Ser153, Tyr167 and Lys171), whose activity was abolished with site-directed mutagenesis of Tyr167Ala and Lys171Ala, whereas mutagenesis of Ser153Ala only resulted in a much reduced catalytic activity. Isothermal titration calorimetry measurements indicated that NAD+ binds first followed by the substrate, (-)-secoisolariciresinol. Additionally, for hydride transfer, the incoming hydride abstracted from the substrate takes up the pro-S position in the NADH formed. Taken together, a catalytic mechanism for the overall enantiospecific conversion of (-)-secoisolariciresinol into (-)-matairesinol is proposed.

Lignans in plant cell and organ cultures: An overview

Lignans are found in a wide variety of plant species. The lignan podophyllotoxin is of special interest, since its derivatives like e.g. etopophos® are used in anticancer therapy. As chemical synthesis of podophyllotoxin is not yet economic, it still has to be isolated from wild growing Podophyllum species, some of which are considered to be endangered species. Therefore plant in vitro cultures may serve as alternative sources for podophyllotoxin and for other types of lignans as well. This review describes the establishment of plant cell and tissue cultures for lignan production and the experiments to improve product yields by changing the cultivation parameters, addition of elicitors and feeding of precursors. It also summarizes the use of plant cell and organ cultures to study the biosynthesis of lignans on enzymological level. Abbreviations: DOP – deoxypodophyllotoxin; LARI – lariciresinol; MATAI – matairesinol; 6MPTOX – 6-methoxypodophyllotoxin; PINO – pinoresinol; PTOX – podophyllotoxin; SECO – secoisolariciresinol

Synthesis of Stereospecifically Deuterated Matairesinol, Podorhizol, Epipodorhizol, and Yatein

(2,5,6,2',5',6'- 2 H 6 )-Matairesinol [6], (7'α- 2 H 1 )-yatein [13], (7- 2 H 1 )epipodorhzol [14], and (7- 2 H 1 )-podorhizol [17] have been prepared as a preliminary to conducting incorporation studies in plants of the Podophyllum species in order to further understand the biosynthesis of podophyllotoxin

The separation of acyl protection groups in alkali-sensitive glucosides. Synthesis of podophyllotoxin-beta-D-glucoside

AbstractThe synthesis of podophyllotoxin‐β‐D‐glucoside (III), an antimitotic lignan compound isolated from Podophyllum species, is reported. Reaction of podophyllotoxin (I) with tetra‐O‐acetyl‐α‐D‐glucopyranosyl bromide in acetonitrile in the presence of Hg(CN)2 yields tetra‐O‐acetyl‐podophyllotoxin‐α‐D‐glucoside (II), which is converted into podophyllotoxin‐β‐D‐glucoside (III) by ZnCl2‐catalysed methanolysis. This transesterification is an advantageous method for the preparation of glycosides, sensitive to base and acid, from their corresponding acetyl derivatives. Scope and conditions of the reaction are discussed.

Podophyllum‐ Lignane: Struktur und Absolutkonfiguration von Podorhizol‐β‐D‐glucosid ( = Lignan F). 19. Mitt. über mitosehemmende Naturstoffe [1

AbstractPodorhizol‐β‐D‐glucoside (1) ( = lignan F), C28H34O13, a new lignan from the roots and rhizomes of Podophyllum species, represents one of the rare glucosides which are cleaved by acids as well as by bases. Acid hydrolysis furnished the intact aglycone podorhizol (3) and in addition two cyclisation products characterized as desoxy‐podophyllotoxin (10) and (−)‐isodesoxypodophyllotoxin (11), respectively. Base catalysed fission of the glucoside 1 yielded D‐glucose and anhydropodorhizol (9). Podorhizol was smoothly oxidized to the corresponding ketone podorhizone (5), which upon treatment with NaOCH3 underwent cleavage into methyl 3,4,5‐trimethoxy‐benzoate (8) and the benzylbutyrolactone derivative 7. These findings combined with spectroscopical evidence revealed the structure of podorhizol as a 2,3‐dibenzylbutyrolactone derivative.

The chemistry of Podophyllum.

Podophyllum is the dried roots and rhizomes of species of Podophyllum (Fam. Berberidaceae). It is derived commercially from two species of plants, namely, Podophyllum peltatum L., the American species, and P. emodi Wall. (P. hexandrum Royle), the Indian. Both are herbaceous perennials. The species do not have an ancient history compared with that of many other medicinal plants. P. peltatum is indigenous to the United States and Canada and grows commonly in eastern North America from Quebec through Florida and westward to Minnesota and Texas. It is usually called in the United States “May apple” or “American mandrake”; older names are “Indian apple”, “wild lemon”, “duck’s foot”, “vegetable calomel”, etc.

Podophyllum pleianthum HANCE. A Morphological Study, with Supplementary Notes on Allied Plants

1. A morphological study of Podophyllum pleianthum HANCE was made, particularily in respects to the leaf arrangement, the branching system of the inflorescence and the behaviour of the medullary bundles.2. The foliar cauline leaves are not arranged in the 1/2 divergence in the strict sense, if observed in the early developmental stage of the stem.3. The inflorescence which is apparently a pleiochasium or umbel, belongs to a modified type of the cincinnus or scorpioid cyme.4. The medullary bundles are similar to those of P. peltatum in their course and origin, but the medullary cauline bundles are more complicated in behaviour.5. Some remarks were given regarding the opinion of WOODSON that P. pleianthum belongs to the monotypic genus Dysosma separated from other species of Podophyllum.6. The cauline leaves of Podophyllum, Diphylleia, Glaucidium and Hydrastis are arranged as slight modifications of the typical 1/2 divergence in the early stage of their development. The scale leaves of the winter bud are also arranged in two slightly-wounded rows and do not show the exact 1/2 divergence in Glaucidium and Hydrastis. Therefore, the apparent 1/2 divergence of the cauline leaves, observed in these four genera, may be derived from the spirally-wounded or nearly 2/5 divergence accompanied by the decrease in the number of leaves.7. Some supplementary notes and corrections were given to the previous paper of the writer regarding the morphology of these genera.