Podophyllotoxin Content Research Articles

Variation in podophyllotoxin concentration in leaves and rhizomes of American mayapple ( Podophyllum peltatum L.)

Podophyllotoxin is a precursor for compounds used in the synthesis of commercially available anticancer drugs and their precursors etoposide, teniposide, and etopophos. Podophyllotoxin is currently obtained from the underground plant parts (roots and rhizomes) of Himalayan mayapple ( Podophyllum emodii Wall.) but is also found in several other species, including American mayapple. There is no information in the literature on podophyllotoxin concentration in the roots or rhizomes of the American mayapple, and it was not clear if podophyllotoxin concentration in underground plant parts is correlated with the concentration in aboveground plant parts. The objective of this study was to estimate podophyllotoxin concentration in the leaves and rhizomes of 28 accessions of American mayapple under natural conditions (wild) and compare this to podophyllotoxin concentrations of the same accessions in the leaves when cultivated. Podophyllotoxin concentration in the rhizomes was positively correlated to soil organic matter content and to the concentrations of soil-available P and Na. Podophyllotoxin in the leaves was negatively correlated to soil-available Mg concentrations. The concentration of podophyllotoxin in the leaves varied from undetectable amounts to 2.52%, whereas podophyllotoxin in rhizomes varied from 0.14% to 0.42%. Most of the accessions had greater than 0.45% podophyllotoxin concentration in the leaves under both wild and cultivated conditions. There was no significant correlation between the podophyllotoxin concentrations in rhizomes and leaves; nine accessions had the highest podophyllotoxin concentration in leaves under natural environment. Another nine accessions had the highest podophyllotoxin in the leaves under cultivated conditions, and an additional eight accessions had greater podophyllotoxin concentrations in roots than in leaves irrespective of the environment. The results from this study may contribute toward developing American mayapple into a new cash crop for U.S. farmers.

Molecular cloning and characterization of the secoisolariciresinol dehydrogenase gene involved in podophyllotoxin biosynthetic pathway from Tibet Dysosma.

Secoisolariciresinol dehydrogenase catalyzes the conversion of secoisolariciresinol into matairesinol that is a central precursor of antiviral and anti-tumor podophyllotoxin. The full - length cDNA encoding secoisolariciresinol dehydrogenase (designated as DtSD) was cloned and characterized from Tibet Dysosma, Dysosma tsayuensis Ying. The full-length DtSD cDNA was 994 bps containing an 837-bp open reading frame encoding a 278-amino-acid polypeptide with a calculated molecular mass of 29.2 kDa and an isoelectric point of 6.32. Comparative analysis indicated that DtSD was similar with other plant SDs at the level of sequence with the highly conserved catalytic motif, Ser-X13-Tyr-X2-Lys. The homology - based structural modeling showed that DtSD was similar to the SD enzyme from Podophyllum peltatum. Tissue expression pattern analysis indicated that DtSD expressed in root, rhizome, petiole and fruit but at different levels. The highest expression level was found in petiole, and the lowest in fruit. The expression of DtSD could not be detected in leaf and flower. Finally, the podophyllotoxin were detected in all the six tested tissues including root, rhizome, leaf, petiole, flower and fruit at different levels. The highest content of podophyllotoxin (180.5±2.74 μg/g) was found in rhizome, and then followed by root, flower, leaf and petiole. The lowest content of podophyllotoxin (6.03±0.12 μg/g) was found in fruit. The expression levels of DtSD were not consistent with the content of podophyllotoxin in the six tested tissues. So, this might mean that the biosynthetic tissues of podophyllotoxin precursors were not the storage tissues. Key words: Dysosma tsayuensis Ying, secoisolariciresinol dehydrogenase, cloning, expression profile, podophyllotoxin, content.

Enhanced production of podophyllotoxins by co-culture of transformed Linum album cells with plant growth-promoting fungi

To investigate the plant growth-promoting effect and stimulation of lignan biosynthesis, the effect of culture filtrates/live co-culture of two arbuscular mycorrhizae-like fungi, Piriformospora indica and Sebacina vermifera, on growth of Linum album cells and on production of podophyllotoxin (PT) and 6-methoxypodophyllotoxin (6-MPT) was studied. For elicitation studies, different volumes of culture filtrates (autoclaved and membrane-filtered) of P. indica/S. vermifera were added to suspension cultures of L. album. The culture filtrates of both the fungi exhibited a positive effect on product formation. For co-culture experiments, both fungi were individually co-cultivated at different concentrations with L. album in suspension cultures for different time periods. This resulted in significant enhancement of PT and 6-MPT content in the plant cells. The activity of phenylalanine ammonia lyase (PAL) was observed to be related to the lignan accumulation, indicating its role as the key enzyme of the phenylpropanoid pathway. The study resulted in total lignan (PT and 6-MPT) production of 745.6 mg/l with a very high PT productivity of 52.4 mg/(l.d).

Impact of Soil Nutrients and Environmental Factors on Podophyllotoxin Content among 28 Podophyllum Hexandrum Populations of Northwestern Himalayan Region Using Linear and Nonlinear Approaches

Podophyllotoxin is the active ingredient in the rhizome of an endangered Indian medicinal herb, Podophyllum hexandrum. Podophyllotoxin content in the P. hexandrum differs greatly in different natural habitats. The podophyllotoxin content reached more than 6.62% when soil pH value was about 4.82, soil organic carbon (C) was more than 3.23%, and nitrogen (N) content was more than 2.7% of soil dry weight. Available phosphorous (P) content of more than 0.419% and potassium (K) content of more than 1.56% resulted in low podophyllotoxin content. The linear relationship detected between podophyllotoxin and soil nutrients, environmental factors, and altitude suggested that further optimization of these factors are important in the conservation and exploitation of P. hexandrum in the northwestern Himalayan region, Himachal Pradesh, India. In this regard, like artificial neural network (ANN) and multiple linear regression (MLR), the prediction model used in this study to map the effect of these factors on podophyllotoxin yield will be helpful.

Lignan and Nutrient Concentrations in American Mayapple (Podophyllum peltatum L.) in the Eastern United States

Podophyllotoxin is an anticancer compound found in Indian mayapple (Podophyllum emodii Wall.), American mayapple (Podophyllum peltatum L.), and other species. Podophyllotoxin and its derivatives are used in several commercially available pharmaceutical products such as the anticancer drugs etoposide, teniposide, and etopophos. Currently, the commercial production of podophyllotoxin is based on Indian mayapple. The objective of this study was to estimate podophyllotoxin concentration in American mayapple across its natural habitats in the eastern United States and to identify high podophyllotoxin types that could be used for further selection and cultivar development. Analyses of American mayapple leaves collected from 37 mayapple colonies across 18 states indicated a significant variation in podophyllotoxin, α-peltatin, and β-peltatin content and the presence of chemotypes. Overall, the concentrations of podophyllotoxin, α-peltatin, and β-peltatin in the collected accessions ranged from below detectable levels to 45.1, 47.3, and 7.0 mg·g−1 dry weight, respectively. We classified American mayapple accessions into seven groups: 1) with very high concentration of podophyllotoxin (greater than 20 mg·g−1) and no α- or β-peltatin; 2) high podophyllotoxin (greater than 10 mg·g−1) and no α-peltatin but trace amounts of β−peltatin; 3) medium podophyllotoxin (1 to 10 mg·g−1) and no α- or β-peltatin; 4) low podophyllotoxin (0.05 to 1 mg·g−1) and high α-peltatin; 5) trace amounts of podophyllotoxin and high concentration of α-peltatin and α-peltatin; 6) high α-peltatin and trace amounts of podophyllotoxin or β−peltatin; and 7) high α−peltatin and no podophyllotoxin or β-peltatin. American mayapple was found to grow on various soil types with a range of pH (4.6 to 7.6) and dissimilar concentrations of phytoavailable soil nutrients. Tissue zinc concentration was positively correlated to podophyllotoxin, whereas soil and tissue phosphorus was positively correlated to the concentration of α-peltatin. The results from this study may contribute toward the development of high podophyllotoxin-containing varieties of American mayapple and the development of a new cash crop for American farmers.

Bioprospection of Eastern red cedar from nine physiographic regions in Mississippi

Juniperus virginiana L. (Family Cupressaceae), commonly called Eastern red cedar, is a widely distributed species in the United States and parts of Canada. It produces two important chemical products, the anticancer compound podophyllotoxin and essential oil. The objective of this study was to evaluate variations in podophyllotoxin concentration in Eastern red cedar, across nine physiographic regions in Mississippi: Tishomingo Hills, Bluff Region, Blackland Prairie, Jackson Prairie, Delta, Pontotoc Ridge, Flatwoods, North Central Plateau, and Piney Woods. The concentration of podophyllotoxin in red cedar needles across different physiographic regions varied from 0.11% to 0.36%. This variation was not statistically significant in 2007 nor in 2008, suggesting that soil nutrient concentrations and topography within Mississippi may not affect the synthesis and the accumulation of podophyllotoxin in Eastern red cedar. Podophyllotoxin accumulation seems to be a stable trait. The results demonstrated that J. viriginiana could be used as a consistent source of podophyllotoxin and that the collection of plant material for commercial extraction of podophyllotoxin can be done independent of the site of collection within the state.

PH‐sensitive Podophyllotoxin carrier for cancer cells specific delivery

AbstractA pH‐sensitive drug targeting system for solid tumors was established based on N‐isopropylacrylamide (NIPAAm) and chitosan conjugates. The mass ratio of NIPAAm and chitosan was adjusted to obtain super pH‐sensitive characteristic and the structure was studied by using Fourier transform infrared spectroscope to confirm the successful synthesis of the nanoparticles. The pH‐sensitive and drug release characteristics in vitro were studied as well. Human lung cancer cells A‐549 and human fibroblast were used to test the biocompatibility of blank and Podophyllotoxin (POD) loaded nanoparticles further to certificate the reliability of targeting acidic tumor extracellular pH. Results revealed that when charge ratio between NIPAAm and CS achieve 4:1(w/w), the drug‐loaded nanoparticles, which diameters ranged from 50 to 150 nm, exhibited super pH‐sensitive responses to tumor pH. Encapsulation and loading efficiencies were 63.7% and 2.4%, respectively. The cumulative release rate of POD, which significantly enhanced at pH 6.8 while decreased rapidly either below pH 6.5 or above pH 6.9 at 37°C. At pH 6.8, POD‐loaded nanoparticles showed cytotoxicity in MTT test and fluorescence microscopic study, comparable to that of free POD at the same POD concentrations, whereas at pH 7.4 there was little cytotoxicity at the tested concentration range. Thereby, the atoxic PNIPAAm‐g‐chitosan nanoparticle has the potentiality as a novel anticancer drugs carrier. POLYM. COMPOS., 2010. © 2009 Society of Plastics Engineers

Characterization of Podophyllotoxin Yielding Cell Lines ofPodophyllum hexandrum

Abstract Callus cultures of Podophyllum hexandrum were initiated and established from juvenile explants isolated from axenic seedlings and mature explants isolated from 1 year old plants on B5 basal medium (at half strength) and modified MS medium supplemented with growth regulators. For initiating cell lines, cell suspension cultures were established with selected callus lines in B5 basal medium supplemented with growth regulators and cell lines were established by cell plating technique. Eight cell lines (RC1, RC2, RC3, RC4, RC5, RC6, RC7 and ZE) maintained as callus stocks in the laboratory have been characterized and compared on the basis of growth rate, morphology, podophyllotoxin content, ploidy, karyotype analysis and RAPD analysis. Morphologically the cell lines were of six different types like globular and dark brown, friable with rough surface and light brown, compact and yellow, compact and creamish, compact and dark brown in colour and creamish and highly friable in nature. The cell lines had ...

Improved podophyllotoxin production by transformed cultures of Linum album

Various cell and hairy root cultures of L. album were developed and analyzed for podophyllotoxin content. Transformed callus and hairy root cultures developed from infection of stem portions of in vitro-germinated L. album plant with Agrobacterium rhizogenes NCIM 5140 strain were selected on the basis of high podophyllotoxin content and growth. Based on the integration of Ri T(L)-DNA and T(R)-DNA, integration of only the ags and not the rol gene in transformed cell culture indicated fragmented integration pattern. The effect of different cultivation media and carbon source on growth and podophyllotoxin production were studied in shake-flask suspension cultures. Detailed batch growth and production kinetics with sugar consumption profile were also established. Maximum volumetric productivity of 4.40 and 2.75 mg/L per day was obtained in cell suspension and hairy root cultures, respectively.

Characterization of genetic structure of Podophyllum hexandrum populations, an endangered medicinal herb of Northwestern Himalaya, using ISSR-PCR markers and its relatedness with podophyllotoxin content

To obtain accurate estimates of genetic structure for purpose of conservation planning for wild Indian May apple (Podophyllum hexandrum) in the Northwestern region of Himalayas, Himachal Pradesh, genetic diversity among and within 28 populations were analyzed. Eleven microsatellite DNA markers were isolated and used to quantify genetic structure. Out of 68 ISSR loci tested, 88.23% were polymorphic. The genetic diversity was high (percentage of polymorphic bands, PBB = 83.82%; Shannon’s information index, I = 0.4413) at the population level, but low within individual study populations (PBB = 34.22%; Shannon’s information index I = 0.1879). The mean coefficient of gene differentiation (Gst) was 0.7484, indicating that 29.44% of the genetic diversity resided within the population. Analysis of molecular variance (AMOVA) indicated that 48% of the genetic diversity among the study populations was attributed to geographical location while 29% was attributed to differences in their habitats. An overall value of mean estimated number of gene flow (Nm = 0.1618) indicated that there was limited gene flow among the sampled populations. We found a clear tendency for higher Gst values and podophyllotoxin levels between the populations with increasing geographical altitude. However, the existing variation in podophyllotoxin content among the populations was proved to be coupled with geographical altitude (r = 0.922) but not with genetic variations (r = - 0.273). Hence we recommend that any further cultivation of this species requires optimization of environmental factors in order to increase the rate of production of podophyllotoxin from any collected population.

Assessment of diversity in Podophyllum hexandrum by genetic and phytochemical markers

For successful conservation and domestication of a species, evaluation of its genetic diversity by different markers is important. Morphological characteristics, phytochemical variation and random amplified polymorphic DNA (RAPD) profiles were generated in different accessions of Podophyllum hexandrum in order to determine the genetic diversity. Random amplified polymorphic DNA (RAPD) analysis revealed a high degree of genetic diversity among the accessions used in the study. There was also high diversity in the concentration of marker compounds in the collected samples as revealed by HPLC analysis. It is shown that the approaches used in the work successfully discriminate between the accessions of this species and thus they constitute interesting tools to analyze molecular, biochemical and phenotypic diversity within this species. Similarity measurement using UPGMA followed by cluster analysis resulted in formation of many groups based on geographical distribution that generally reflected expected trends between the genotypes. There were also some important exceptions like PW-S, an accession from Wastoorwan, Khrew showing close resemblance to PG-S and PG-B collected from Gulmarg but grown at two different gene banks at Srinagar and Bonera. Further an accession PSH-B from Keller was significantly diverse from the rest of the native genotypes phytochemically, morphologically and at molecular level. RAPD data analysis was found to be significant predictor of phytochemical markers in cultivated P. hexandrum germplasm. Twelve accessions grown in gene bank repository were subjected to RAPD analysis and were assessed for content of podophyllotoxin and podophyllotoxin β- d-glycoside by HPLC. Individual regressions of podophyllotoxin and podophyllotoxin β- d-glycoside by RAPD analysis against HPLC has been found to determine linear values. Strong correlation and a strong association of values of the phytochemical variables and the DNA polymorphism data has been recorded.

Production of lignans in callus culture of <i>Podophyllum hexandrum</i>

Purpose: Podophyllum hexandrum Royle, a source of highly valued podophyllotoxin has been subjected to heavy collection from the wild. The ever-increasing demand of podophyllum is mainly due to two semi synthetic derivatives of podophyllotoxin that is etoposide and teniposide, which are used in the treatment of various types of cancer. The anti cancer lignan derivative podophyllotoxin in Podophyllum hexandrum is biosynthesized at very low quantities in intact plant, so the biotechnological production of podophyllotoxin has been considered essential. Method: The aseptically germinated embryos of Podophyllum hexandrum were developed on solid nutrient agar slab. For the growth of callus culture, Murashigae and Skoog media (MS media)) with various concentrations of BAP, NAA and GA3 adjusted to pH 5.8 was used. Podophyllotoxin content in the alcoholic extract of calli and plant root was analysed by HPLC and HPTLC and was also compared with cultivated Podophllum hexandrum root extracts. Result: A fully defined MS medium supplemented with Naphthalene acetic acid and 6benzylaminopurine (BAP) were effective for both initiation and sustained growth of callus tissue. The relative proportion of callus was markedly influenced by presence of plant growth regulators. The amount of Podophyllotoxin obtained from callus was 0.78 and 0.79 percent as characterized by HPLC and HPTLC respectively. Conclusion: The study revealed that callus culture may be a fruitful tool for the production of Podophyllotoxin resin, an anticancer entity.

Podophyllotoxin Content, Above- and Belowground Biomass in Relation to Altitude in Podophyllum hexandrum Populations from Kumaun Region of the Indian Central Himalaya

The morphological features of Podophyllum hexandrum Royle, a 'critically endangered' medicinal herb and a source of podophyllotoxin, were studied in populations growing in different parts of the Kumaun region of the Indian Central Himalaya. Plant growth performance in terms of biomass accumulation and podopyllotoxin levels in the rhizomes collected from eleven natural populations (P1 to P11, altitude ranging from 2740 to 3350 m) were analyzed. Morphological features, e. g., plant height, stem diameter and leaf area were, in general, negatively correlated with an increase in the altitude. Maximum aboveground (8.46 g/individual) and belowground (48.18 g/individual) biomass values were recorded from a population (P9) at the lowest altitude (2740 m) and, in general, the species was found to perform better at the lower altitudes. The podophyllotoxin content of rhizomes ranged between 0.36-1.08% (on dry wt. basis) in different populations, and a positive correlation was observed between podophyllotoxin content and an increase in the altitude.

Podophyllotoxin content in Podophyllum hexandrum Royle plants of known age of seed origin and grown at a lower altitude

Podophyllum hexandrum Royle, an important alpine herb, and a source of the highly valued aryltetralin-type lignan, podophyllotoxin, has been subjected to heavy collection from the wild due to ever increasing demand. The present study deals with an attempt to bring this plant under cultivation at a relatively lower altitude and to evaluate (1) various growth parameters including above and below ground biomass accumulation, net assimilation rate and relative growth rate, etc., and (2) podophyllotoxin content from the resulting above and below ground biomass in seed raised plants of a known age series of 1–5 years. The podophyllotoxin content was estimated on the basis of HPLC analyses. The levels were found to increase with the plant age and the maximum amount was found in 5-year old plants. This study demonstrates that (1) seeds can be conveniently used for raising healthy propagules in easily approachable locations at a relatively lower altitude, and that the plants can be maintained in such sites over long periods, and (2) this approach of “conservation through cultivation” can be suggested as an effective tool for the management of this “critically endangered status” species.

Protection of the Hemopoietic System byPodophyllum hexandrumagainst 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.

Frequency and Timing of Leaf Removal Affect Growth and Podophyllotoxin Content ofPodophyllum peltatumin Full Sun

Podophyllotoxin is a pharmaceutical compound found in leaves and rhizomes of American mayapple (P. peltatum L.), a species being investigated as an alternative to that of the Indian mayapple (P. emodi). Leaves alone can serve as a renewable source of podophyllotoxin (and other lignans) leaving rhizomes undisturbed to produce leaf biomass in subsequent years. It is not known, however, how frequently or severely plants can be defoliated without adversely affecting future plant growth, lignan content, or podophyllotoxin yield (g.m(-2)). This study compared harvest strategies that were mild to severe in frequency and timing of leaf removal. A wild population in full sun was subjected to leaf removal treatments of varying frequency (every year, every 2nd or 3rd year) and timing (early or late). Control plots not previously harvested were included every year. Plots were 1.0 m2 and established during spring of 2001. Duration of the study was four years. P. peltatum plants did not tolerate the most severe harvest treatment: annual harvest frequency in combination with early harvest time. Early annual harvests reduced total leaf dry mass and total leaf area in a consistent and linear manner. In contrast, plants tolerated annual harvests when conducted late in the growing season and tolerated early harvests when conducted every 2nd or 3rd year. The number of sexual shoots was reduced to zero by early annual harvests. Podophyllotoxin content was 2.7 to 6.5 times greater in leaves harvested early compared to those harvested late, though content was significantly greater in only two out of four years. In conclusion, we can recommend leaf removal every year from well-established P. peltatum populations grown in full sun if harvests are conducted late in the growing season. This harvest strategy ensures maximum podophyllotoxin yield without jeopardizing future leaf biomass yield. Leaves harvested early appear to have greater podophyllotoxin content, but we discourage early harvest every year. Instead, our results indicate that leaves can be harvested early every other year without reducing long-term performance of P. peltatum populations.

Frequency and Timing of Leaf Removal Affect Growth and Podophyllotoxin Content of American Mayapple in Shade

Leaves of American mayapple (Podophyllum peltatum) are being investigated as an alternative and renewable source of podophyllotoxin, a pharmaceutical compound used in the manufacture of several drugs. This study examined long-term performance of mayapple populations subjected to different harvest strategies. A naturally occurring population in shade was subjected to leaf removal treatments of frequency (every year, every 2nd or 3rd year) or timing (early or late season). Plots were 1.0 m2, established during Spring 2001, and treatments were applied from 2001 to 2004. Control plots not previously harvested were also included each year. Plants did not tolerate the severest of leaf removal treatments: early harvest time in combination with annual harvest frequency. Early annual harvests reduced total leaf dry mass and total leaf area in a quadratic manner. Late harvest conducted annually, and early harvest conducted every other year, also reduced leaf dry mass and area but not as much as early annual harvest. Plants harvested every year, early, or early every year produced fewer sexual shoots than other treatment combinations. Contents of α-peltatin, β-peltatin, and total lignans were higher for leaves harvested early than those harvested late during each year of the study, demonstrating that lignan contents were affected by leaf age and not treatment. In conclusion, our results for plants grown in shade show that leaves can be removed late in the growing season every 2nd or 3rd year or early every 3rd year without reducing long-term performance of the population. This is more restrictive than that reported for populations in full sun where plants tolerated late harvests every year.

Molecular Identification ofSinopodophyllum hexandrumandDysosmaSpecies using cpDNA Sequences and PCR-RFLP Markers

Rhizomes of Sinopodophyllum hexandrum and Dysosma species, which have long been used in the traditional Chinese herbal medicine, have similar morphology and chemical composition. However, the podophyllotoxin content is higher in the rhizomes of S. hexandrum than in those of Dysosma species. The PCR-amplified fragments of trnT -trnL showed length variation between S. hexandrum and Dysosma species, and sequence comparison indicated that the length variation resulted from differential indels. There were species-specific PCR-RFLP markers of the chloroplast trnD -trnT region. Our results suggest that both chloroplast intergenic regions can be used for the identification of S. hexandrum and Dysosma Rhizoma medicines on the market.

Post-harvest and scale-up extraction of American mayapple leaves for podophyllotoxin production

Leaves of American mayapple ( Podophyllum peltatum L. ) are of interest to the pharmaceutical industry as an alternative source of podophyllotoxin, an aryltetralin lignan that is the precursor of widely used anticancer drugs etoposide, teniposide, and etopophos. In this study, the effects of post-harvest handling were evaluated by inflicting physical damage to leaves of mayapple to simulate rough handling. The effects of storage conditions before and after being dried were also evaluated. In addition, techniques for conducting large-scale extractions of podophyllotoxin from bulk samples were investigated. Crushing injury, damaging the leaves in more than 70% of its area, has improved podophyllotoxin content of leaves when dried at 40 °C within 24 h of harvest. In contrast, podophyllotoxin content was greatly reduced when the leaves were dried at room temperature at 15% relative humidity and 24 °C. Podophyllotoxin was stable with no significant changes over time when the leaves were dried, ground, and stored under different conditions for up to 60 days. Based on these findings, mayapple leaves do not require careful handling at harvest. In fact, leaves can be handled in a manner consistent with mechanical injury as long as leaves are dried at 40 °C within 24 h. Leaves can then be stored for up to 60 days, and probably much longer, when dry. If leaves cannot be dried in a timely manner, they can be stored at 4 °C for up to 4 weeks without significant loss of podophyllotoxin.

Development of an enzyme-linked immunosorbent assay for podophyllotoxin

A competitive indirect enzyme-linked immunosorbent assay (ciELISA) for podophyllotoxin was developed by using polyclonal antibody, and its suitability for the determination of this analyte in spiked water samples was studied. To avoid antibody production to the linker, the succinoyl-podophyllotoxin (hapten) mimicking the analyte was synthesized and conjugated with the carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) by mixed anhydride reaction (MAR) and active ester method (AEM). Polyclonal antibodies raised against Hapten–BSA 1 synthesized by MAR and Hapten–BSA 2 by AEM were screened and selected for the ciELISA. One set of antibodies from the rabbit M4440 immunized with Hapten–BSA 1 showed an I 50 value of 2.21 μg/mL with a detection limit of 0.12 μg/mL, and the other set from the rabbit M4469 immunized with Hapten–BSA 2 had an I 50 value of 0.7897 μg/mL with a detection limit of 0.0056 μg/mL. This assay showed the cross-reactivities with the structurally closely related compounds. Recoveries from the podophyllotoxin-fortified tap water in the assay were in the range of 72–115%. A good correlation between podophyllotoxin concentration measured by the ELISA and HPLC ( R 2 = 0.9924, Y = 1.195X − 0.257) was obtained from linear regression analysis. These results indicate that the ELISA could be a convenient and supplemental analytical tool for monitoring podophyllotoxin and its analogues in waters without previous extraction or cleanup.