Lignans, as natural products, are distributed widely in the plant kingdom. More than 200 compounds in this general class have been identified. Lignans have aroused considerable interest because some of them display antitumor activities. This is particularly true of the podophyllotoxin group of lignans, which are constituents of the medical resin extracted from Podophyllum species. Podophyllotoxins are a particularly instructive class of natural products for consideration in the design and synthesis of potential anticancer agents based upon natural product prototypes.
The medical use of Podophyllum species dates back over 1000 years. At that time the roots of wild chervil were used in a salve for treating cancer in England. About 400-600 years ago, the natives of the Himalayas and the American Indians independently discovered that the aqueous extracts of the roots (podophyllin) from Podophyllum species was a canthartic and poison. After the American Indians introduced the use of podophyllin to the American colonists, it became such a popular drug that it was included in the US Pharmacopoeia in 1820 as a canthartic and cholagog and remained until 1942, when it was removed because of its severe toxicity.
However, Kaplan (1942) revived interest in podophyllin when he cured the venereal wart (condyloma acuminatum) with topical application of podophyllin oil. This led to studies of the action of podophyllin on tumor tissue and to intensive chemical examinations of the constituents of podophyllin. King and Sullivan demonstrated that podophyllin produced this therapeutic effect by acting as a mitotic poison, and Hartwell and Shear first reported that podophyllin inhibited the growth of experimental cancer cells in animals.
At the same time, the chemical structure of podophyllotoxin was established by Hartwell and Schrecker. In the 1960’s, Stahelin and his coworkers at Sandoz tested many semi-synthetic podophyllotoxin derivatives as anticancer agents. They found two derivatives, VP-16-213 and VM-26, which had potential anticancer activity with minimal toxicity. VP-16-213 (Etoposide) has passed governmental approval for use in lung cancer and testicular cancer in several European countries and approved by the FDA in the USA in 1983 and in Japan in 1986.
Podophyllum species are the perennial plants in the family Berberidaceae, and they have the chromosome number 2n = 12. One of the most common species is Podophyllum peltatum, (May apple), which grows in gregarious groups in the oak-hickory forests all over the eastern United States and southern Canada. It has whitish nodding flowers with parts in whorls of three between palmately dissected peltate leaves. Plants flower from March to May. Meijer surveyed the distribution of P. peltatum in eastern North America and identified the optimum conditions for its cultivation on a commercial scale.
A second species, P. hexandrum (P. emodi), distributed from the Himalayas to southwest China (India, Bhutan, Pakistan, China, and Formosa), has properties and uses similar to the May apple. It has white or pale red rotate flowers with palmately parted peltate leaves. The flowering time is May and the ripe fruits are red, ellipsoidal, and edible.
A third species, Podophyllum pleianthum (Dysosma pleianthd), grown in the southern part of China and Formosa, has two palmately lobate peltate leaves and has five to eight dark red nodding flowers at the forked point of the petioles.
Furthermore, Dipphylleia sinensis (China) and D. cymosa (Japan) and Podophyllum japonica (Ranzania japonica, Japan) are known to contain some podophyllotoxins.
In addition to the Podophyllum species, Polygala polygama (), Juniperus virginiana (), Linum album (), and Linum flavum () contain some podophyllotoxins.
Extraction and Structure of Podophyllotoxins
Analysis of nonglucosidic lignans present in an alcoholic extract of the dried root/rhizome of Podophyllum species showed that the range of lignans observed in P. peltatum, P. pleianthum, and P. hexandrum was the same except that no peltatins were detected in P. pleianthum ().
Medicinal Components and Demand on the World Market
Etoposide, VP-16-213, 4′-demethyl epipodophyllotoxin ethylidene β-D-glucose, a semi-synthesitic derivative of podophyllotoxin, has been shown to be an effective anti tumor agent in the treatment of lung cancer, a variety of leukemias, and other solid tumors. Etoposide is administered by injection or orally as a capsule. Etoposide is used mainly in the U.S.A. and Europe by the trade name of Vepeside.
The estimated sold units (1 unit = 100 mg) in the U.S.A. and Europe were more than 830,000 units in 1986 and the gross sale of Vepeside in the U.S.A. and Lastet (trade name in Japan) were estimated to be 100 million $ and 3.8 billion yen in 1989, respectively.
Podophyllum: Conclusions and Prospects
Callus induction, suspension culture, plant regeneration from callus and protoplast culture of three Podophyllum species (P. pleianthum, P. peltatum, and P. hexandrum) were examined under various culture conditions. 2,4-D or NAA in the presence of BA promoted callus induction and callus growth. The best medium for callus culture was B5 medium supplemented with 0.2 ppm BA and 2 ppm 2,4-D or NAA. Suspension culture was established from both P. pleianthum and P. peltatum callus in MS liquid medium supplemented with 0.2 ppm kinetin and 1 ppm 2,4-D. Plant regeneration from callus tissue of P. pleianthum was accomplished by culturing the callus on a cytokinin-rich medium. The best medium for plant regeneration was B5 medium containing 2 ppm BA and 0.2 ppm NAA. Podophyllotoxin contents of the regenerated plant leaves was three times higher than that of original plant leaves, but the regenerated plant roots contained 1 /4 podophyllotoxin compared to that in original plant roots.
Etoposide is one of the most successful medicines derived from plant material. A major direction for future studies of podophyllotoxins should focus on the mechanism of action and on the development of new podophyllotoxin analogs which not only have different pharmacological potencies but also possess unique biological properties. There are several studies demonstrating that Etoposide acts in acute nonlymphoblastic leukemia and lymphoma, and furthermore Etoposide has therapeutic value in the treatment of AIDS-associated Kaposis sarcoma.
It is estimated that the world market of chemicals produced by plants is over 30 billion $, and about 20 of these chemicals will be produced by tissue culture or by genetically improved plants, by 1995. For industrial production, highly valuable chemicals (> 1000 $/kg) such as antitumor substances must be chosen, largely due to the low and/or unstable productivity of many undifferentiated cultures. To some degree, this deficiency can be overcome by screening for cell clones derived from individual high-producing cells, as with the production of ajmalicine by Catharanthus roseus cultures and shikonin by cultures of Lithospermum erythrorhizon ().
A development that may revolutionize the role that in vitro culture plays in fine chemical synthesis is the discovery of rapidly growing, productive, and stable “hairy root” cultures obtained by the genetic transformation of plant tissue by the pathogenic soil bacterium, Agrobacterium rhizogenes (). The induction of hairy root and production of secondary metabolites were established in the formation of pigments and alkaloids such as shikonin, betacyanin, atropine, scopolamine, and hyoscyamine. These hairy roots produced as many alkaloids as normal roots and the alkaloid pattern was similar to that of the original plants. Thus, hairy root cultures may be a useful system for large-scale production of secondary metabolites, yet they are amenable to the techniques of selection and genetic manipulation that have been developed with dispersed cell systems. The infection of Agrobacterium rhizogenes to Podophyllum species was tried in our laboratory, but no hairy roots have been formed yet.
The large-scale production of podophyllotoxins by tissue culture has not succeeded at present; however, plant regeneration from callus, suspension culture, and protoplast culture were partially successful.
Regarding the protection of natural resources, large-scale propagation of plants using a technique of plant regeneration which can produce multiple clonal plants in vitro is strongly desired.
Selections from the book: “Medicinal and Aromatic Plants III”, 1991.