Source: MISSISSIPPI STATE UNIV submitted to NRP
AMERICAN MAYAPPLE AND EASTERN RED CEDAR AS POTENTIAL DOMESTIC SOURCES OF THE ANTI-CANCER COMPOUND PODOPHYLLOTOXIN
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0193218
Grant No.
2003-35504-12852
Cumulative Award Amt.
$220,000.00
Proposal No.
2002-01525
Multistate No.
(N/A)
Project Start Date
Nov 1, 2002
Project End Date
Oct 30, 2007
Grant Year
2003
Program Code
[71.2]- (N/A)
Recipient Organization
MISSISSIPPI STATE UNIV
(N/A)
MISSISSIPPI STATE,MS 39762
Performing Department
NORTH MISSISSIPPI RESEARCH & EXTENSION CENTER
Non Technical Summary
Leaves of the American mayapple contain podophyllotoxin, a pharmaceutical compound currently being used, or tested in clinical trials, for treatment of cancer, arthritis, malaria, psoriasis, and genital warts. Since leaves are a renewable resource, American mayapple appears to be a candidate for commercial exploitation by growers of specialty crops in North America. Leaves of another plant, Eastern red cedar, also contain podophyllotoxin but at substantially lower concentrations. Eastern red cedar is more available than American mayapple because it readily volunteers in open, nonagricultural areas. There is a need to determine which of these species has the greatest potential as a domestic source of podophyllotoxin. Currently, podophyllotoxin is available from roots and rhizomes of the Indian mayapple, an endangered species of the Himalayas. Mississippi State University will grow American mayapple under field and greenhouse conditions to determine the effects of fertilization, soil pH, time of planting, propagation techniques, shade, pest control, and harvest strategy on biomass yield and podophyllotoxin content. Studies will also determine biomass yield and podophyllotoxin content of wild populations of Eastern red cedar. The University of Mississippi will quantify podophyllotoxin from these studies and develop extraction procedures for bulk, commercial-scale quantities. With these data, production budgets for American mayapple and Eastern red cedar will be estimated, providing economic comparisons with the current source of podophyllotoxin, the Indian mayapple.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20522201060100%
Knowledge Area
205 - Plant Management Systems;

Subject Of Investigation
2220 - Medicinal crops, non-narcotic;

Field Of Science
1060 - Biology (whole systems);
Goals / Objectives
Develop cultural practices to establish American mayapple, Podophyllum peltatum, in field plantings. Estimate biomass and podophyllotoxin yield of leaves of American mayapple grown as a cultivated crop. Determine the extent to which cultural practices influence biomass and podophyllotoxin content. Estimate biomass and podophyllotoxin yield of leaves of Eastern red cedar, Juniperus virginiana, harvested from the wild. Determine the extent to which plant maturity, genotype, and location influence podophyllotoxin content. Compare costs associated with each potential source of podophyllotoxin, (a) resin from the current source of podophyllotoxin: rhizomes of the Indian mayapple, P. emodi, (b) leaves of American mayapple, and (c) leaves of Eastern red cedar.
Project Methods
Due to extreme variability of podophyllotoxin content of wild populations, accessions of American mayapple of known podophyllotoxin content will be established in field and greenhouse plantings. A wide range of propagation and cultivation techniques will be examined to determine their effects on survival, growth, drug content, and biomass yield. Podophyllotoxin content of leaves of Eastern red cedar will be determined from micro- and bulk samples of plants from wild populations. The extent of natural variability of podophyllotoxin content in Eastern red cedar will be determined from plants of different maturities and sexes and from samples taken at different times and locations. Costs associated with production (or collection), handling, extraction, and purity of product will be assessed for American mayapple and Eastern red cedar and compared to that of Indian mayapple.

Progress 11/01/02 to 10/30/07

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objectives were met.

Publications


    Progress 01/01/06 to 12/31/06

    Outputs
    In 2006, 37 mayapple colonies were sampled from 18 states using GPS. The states that were sampled include: Mississippi, Alabama, Arkansas, Georgia, Kentucky, Louisiana, Maryland, Massachusetts, New York, New Hampshire, North Carolina, Ohio, Pennsylvania, South Carolina, Tennessee, Texas, Virginia, and West Virginia.The samples were analyzed by HPLC (Canal et al., 2001). Analyses of mayapple leaves collected from 18 states indicated a significant variation in podophyllotoxin, a-peltatin and b-peltatin content. Overall, the concentrations of podophyllotoxin, alpha-peltatin and beta-peltatin in the collected accessions ranged from below detectable levels to 45.04, 47.30, and 7.01 mg/g dry weight, respectively. These results demonstrated the presence of chemotypes and confirm earlier studies by Moraes et al. (2002) that there are significant resources of American mayapple in various states. The mayapple accessions can be used for the development of high-podophyllotoxin cultivar of mayapple. In 2007, we will continue with bioprospecting of mayapple in other locations. In addition, the high-podophyllotoxin accessions identified in the 2006 bioprospecting will be sampled again, and will be established for further study on productivity and chemical profiling at the North Mississippi Research and Extension Center in Verona, MS. We also sampled eastern red cedar (Juniperus virginiana) from 12 sites. The samples are currently being analyzed for podophyllotoxin content.

    Impacts
    Two major outcomes are expected: (1) A database and a GIS map of available germplasm of American mayapple (P. peltatum) and Eastern red cedar (J. virginiana) in the US will be generated, that can be used for recollection by plant breeders and herbarium curators; (2) High-podophyllotoxin accessions of these plants will be identified, and the mayapple accessions will be further tested for variety development.

    Publications

    • Zheljazkov, V.D., B. Avula, V.M. Maddox, M.P.A. Jones, R. Moraes, and I. Khan. 2007. Bioprospecting of American mayapple. SAAS and Annual Meeting Amer. Soc. Agron. Southern Branch. Feb 3-7, Mobile, AL.
    • Zheljazkov, V.D., B. Avula, V.M. Maddox, M.P.A. Jones, R. Moraes, and I. Khan. 2007. Bioprospecting of American mayapple. under preparation for Planta Medica.


    Progress 01/01/04 to 12/30/04

    Outputs
    Podophyllum species are the major source of arytetralin lignans and among those podophyllotoxin is the compound of commercial interest since it is the starting material for the semi-synthesis of the anti-cancer pharmaceuticals etoposide, teniposide and etopophos. These chemotherapeutic drugs have been used for the treatment of lung and testicular cancers as well as certain leukemias. In addition to the anti-cancer drugs, podophyllotoxin is also the precursor of new derivatives in clinical trials for the treatment of rheumatoid arthritis in Europe. Podophyllotoxin preparations are also sold in drugstores for dermatological use to treat genital warts. The commercial source of podophyllotoxin, Podophyllum emodi Wall (syn. P. hexandrum Royle) a native species of the alpine Himalayas in India that has been declared endangered. Previous search for an alternative source of podophyllotoxin that the content in leaves of Podophyllum peltatum L. are equal to that of rhizomes of P. emodi and this content is a stable genetic trait and has not changed drastically altered over time. The proposed research has focused on cultivation of mayapple in the fields. In fact, our field results have confirmed that cultivation of P. peltatum is possible, easy. Herein are some of our conclusions: 1-Type of the planting and propagule type have influenced field plantings; growth and performance. 2- Low temperature exposure as treatment of to break dormancy was effective, and shoots can be induced to grow at any time of the year. 3- Growers of maypple can establish and cultivate American mayapple under full sun, thus providing leaf biomass with high rpodophyllotoxin content, while avoiding the cost of expensive shade structure. We have concluded after a two-year of field trial that mayapple is a shade tolerant plant. Currently we are analyzing data to evaluate the effects early vs. late harvest in the season; yearly harvest vs. after two consecutive growing season on yield of podophyllotoxin and also on the sustainability of the crop. The results will be submitted to publication in a near future.

    Impacts
    American Mayapple has proven to be surprisingly easy to establish and cultivate in field plantings and it appears to have a high potential to be domesticated. Shade studies showed mayapple should be preferable cultivated under full sun. Leaf removal late in the growing season will maintain plant vigor and allow the underground rhizomes to produce harvestable leaf material every year.

    Publications

    • Maqbool, M., K.E. Cushman, R.M. Moraes and P.D. Gerard. 2004. Overcoming dormancy of mayapple rhizome segments with low temperature exposure. HortScience. 39(2):307-311.
    • Cushman,K., M. Maqbool, I. Khan, H. Lata, E. Bedir, and R.M. Moraes. 2004. Shade affects growth and podophylotoxin content of mayapple Second year results. Annual Report 2003 of the North Mississippi Research & Extension Center, Mississippi Agricultural & Forestry Experiment Station Information Bulletin 405:317-322.
    • Maqbool, M., K. Cushman, and R. Moraes. 2004. Mulch type, mulch depth, and planting depth for field grown mayapple Second year results. Annual Report 2003 of the North Mississippi Research & Extension Center, Mississippi Agricultural & Forestry Experiment Station Information Bulletin 405:323-331.
    • Maqbool, M., K.E. Cushman, H. Lata, and R.M. Moraes. 2004. Variation of lignan content in American mayapple leaves by time of harvest and shoot type: A preliminary report. Annual Report 2003 of the North Mississippi Research & Extension Center, Mississippi Agricultural & Forestry Experiment Station Information Bulletin 405:332-334.
    • Maqbool, M., K. Cushman, I. Khan, and R.M. Moraes. 2004. Lignan content and leaf biomass of mayapple under different growing conditions. Annual Report 2003 of the North Mississippi Research & Extension Center, Mississippi Agricultural & Forestry Experiment Station Information Bulletin 405:335-341.
    • Maqbool, M., K. Cushman, H. Lata, and R.M. Moraes. 2004. Propagule type and planting time affect mayapple growth Third year results. Annual Report 2003 of the North Mississippi Research & Extension Center, Mississippi Agricultural & Forestry Experiment Station Information Bulletin 405:342-347.


    Progress 01/01/03 to 12/31/03

    Outputs
    Several two-year projects were brought to completion in 2003. A two-year study about the effects of increasing levels of shade on growth and podophyllotoxin content of American mayapple was completed. A manuscript describing the research was prepared and accepted for publication in HortScience. A two-year study of the effects of mulch type, mulch depth, and transplant depth of mayapple rhizome segments was also completed in 2003. A manuscript describing the research was prepared and accepted for publication in HortScience. Several four-year field studies involving mayapple will be completed during the spring of 2004.

    Impacts
    As individual research studies are completed, the ease to which American mayapple can be domesticated is becoming apparent. It is a rather slow-growing plant, but it establishes easily in field plantings when using rhizome segments as propagules. It has few pests and grows well in full sun. Eastern red cedar was found to be a stable, yet low-yielding, source of podophyllotoxin.

    Publications

    • Cushman, K.E., Maqbool, M. and Moraes, R.M. 2003. Mulch type, mulch depth, and planting depth for field-grown mayapple: First-year results. Annual Report 2002 of the North Mississippi Research & Extension Center. Mississippi Agricultural & Forestry Experiment Station Information Bulletin 398:351-357.
    • Maqbool, M., Cushman, K.E., Bedir, E., Khan, I.A. and Moraes, R.M. 2003. The effect of shade on growth and podophyllotoxin content of mayapple. Annual Report 2002 of the North Mississippi Research & Extension Center. Mississippi Agricultural & Forestry Experiment Station Information Bulletin 398:358-362.
    • Cushman, K.E., Maqbool, M., Bedir, E., Khan, I.A. and Moraes, R.M. 2003. Growth and lignan content of field-established mayapple. Annual Report 2002 of the North Mississippi Research & Extension Center. Mississippi Agricultural & Forestry Experiment Station Information Bulletin 398:363-366.
    • Maqbool, M., Cushman, K.E. and Moraes, R.M. 2003. Growth of transplanted mayapple: Effect of propagule type and planting time. Annual Report 2002 of the North Mississippi Research & Extension Center. Mississippi Agricultural & Forestry Experiment Station Information Bulletin 398:367-371.
    • Cushman, K.E., Maqbool, M., Gerard, P.D., Bedir, E., Lata, H. and Moraes, R.M. 2003. Variation of podophyllotoxin in leaves of Eastern red cedar (Juniperus virginiana). Planta Medica 69:477-478.


    Progress 01/01/02 to 12/31/02

    Outputs
    Rhizome segments of the American mayapple were successfully harvested from the wild, transplanted to greenhouse and field plantings, and grown under cultivated conditions using common horticultural practices. The effect of these practices on biomass production and podophyllotoxin content were quantified. In addition, foliage of immature and mature plants of Eastern red cedar was sampled, dried, and then extracted for podophyllotoxin content, once a month over a sampling period of one year.

    Impacts
    Results of our studies to date demonstrate that domestication of the American mayapple is clearly possible. Preliminary results have shown, however, that mayapple is a slow-growing plant that may not withstand the stress of repeated harvests of leaf biomass. These are important issues of horticultural relevance to growers of specialty crops. Though lower in podophyllotoxin content, foliage of Eastern red cedar may be a more practical and abundant source than mayapple. It is yet to be determined which plant, mayapple or Eastern red cedar, might produce the highest and most easily obtainable yields of podophyllotoxin when handled in bulk.

    Publications

    • Maqbool, M., Cushman, K.E., and Moraes, R.M. 2002. Propagule type and planting time for field-established mayapple. p. 533-536. In J. Janick and A. Whipkey (eds.). Trends in New Crops and New Uses. Proceedings of the Fifth National Symposium, New Crops and New Uses: Strength in Diversity. ASHS Press, Alexandria, Va.
    • Moraes, R.M., Lata, H., Bedir, E., Maqbool, M. and Cushman, K. 2002. The American mayapple and its potential for podophyllotoxin production. p. 527-532. In J. Janick and A. Whipkey (eds.). Trends in New Crops and New Uses. Proceedings of the Fifth National Symposium, New Crops and New Uses: Strength in Diversity. ASHS Press, Alexandria, Va.
    • Cushman, K.E., Maqbool, M. and Moraes, R.M. 2002. First-year growth of transplanted mayapple (Podophyllum peltatum) rhizomes: Influence of propagule type and planting time. Annual Report 2001 of the North Mississippi Research & Extension Center. Mississippi Agricultural & Forestry Experiment Station Information Bulletin 386:303-305.
    • Cushman, K.E., Maqbool, M. and Moraes, R.M. 2002. Harvest time and shoot type affect leaf biomass and lignan content of mayapple (Podophyllum peltatum) in sun or shade. Annual Report 2001 of the North Mississippi Research & Extension Center. Mississippi Agricultural & Forestry Experiment Station Information Bulletin 386:306-307.
    • Maqbool, M., Cushman, K.E., Moraes, R.M. and Gerard, P.D. 2002. Overcoming dormancy of mayapple (Podophyllum peltatum) rhizome propagules with low temperature exposure. Annual Report 2001 of the North Mississippi Research & Extension Center. Mississippi Agricultural & Forestry Experiment Station Information Bulletin 386:300-301.
    • Maqbool, M., Cushman, K.E., Moraes, R.M., Bedir, E. and Gerard, P.D. 2002. Podophyllotoxin content of leaves of Eastern red cedar (Juniperus virginiana). Annual Report 2001 of the North Mississippi Research & Extension Center. Mississippi Agricultural & Forestry Experiment Station Information Bulletin 386:302.