Trevor Smart and Brad Geary, Plant and Wildlife Sciences
Recent research has been published determining the presence of anticancerous withanolides in the plant Datura wrightii. These withanolides have been found to be antiproliferative towards cancers such as breast cancer, gliobastoma, and head and neck cell carcinoma [1,2]. There also have even been some studies showing withanolide potential for neuritic and synaptic regeneration. These withanolides, which are oxygenated C-28 steroid hydrocarbons, are undergoing further research from Datura wrightii, but the associated endosymbionts of Datura wrightii are yet to be the subject of current studies. Endosymbionts, being fungi living within the plant, have commonly been found to produce either the same or very similar chemicals as are isolated from plant materials [3]. Often, when chemicals are found to be similar this is because of metabolic transformation, often termed biotransformation, which acts to alter the structure and function of the chemical, either organic or protein based.
The first step in understanding the relationship between endosymbiont and D. wrightii produced withanolides is to identify the origin of the compound. The origin of the compound will help us understand this relationship, specifically for metabolic transformational effects, because endosymbionts and plants both produce exoenzymes capable of degrading potentially harmful compounds. It is these exoenzymes that are responsible for the degradation and change in chemical structure commonly seen in many plant-endosymbiont relationships [4]. Therefore, it is entirely possible that the withanolide products isolated from D. wrightii have already undergone metabolic transformation and differ from their original chemistry which may cause radical structural and functional changes.
Comparing plant and endosymbiont derived withanolides involved the collection of plant samples from a site near Ivins, UT. Roots, rhizome, and plant stems were surface sterilized in order to eliminate all non-endosymbiont derived fungi and bacteria. Each endosymbiont isolated was cultured separately onto both water agar and 10% potato dextrose agar. Once grown adequately, the endosymbionts were transferred into bottles of 50% potato dextrose broth and subsequently screened using mass spectrometry to determine if withanolides are synthesized by the endosymbionts.
All mixtures suspected to contain withanolides underwent an extraction series method as outlined by the most recently published procedure of withanolide extraction [5]. Each endosymbiont suspected of producing withanolides was characterized morphologically. Mass spectrometry and thin layer chromatography were used at each step in the extraction series to confirm or disconfirm the presence of withanolides.
The results of this project were inconclusive in determining the presence of antiproliferative withanolides in native Datura wrightii. Several mass spectra seemed to show promise in indicating withanolide presence however they were either not present or lost in later dilutions. Because no withanolides were determined to be in D. wrightii samples from a site near Ivins, UT a second plant collection was taken from a site near Primm, NV in further search for withanolide presence. No withanolides were located from the Primm, NV site either.
Nonetheless, successes can be drawn from this experiment in a number of areas especially in refining protocol which will serve as the basis for future experimentation. Already, several experiments in our research laboratory have stemmed off of this project and future successes in these projects will be directly linked our experience from this project.
The results of this project were, to say the least, disappointing. When analyzing our outcomes there were several aspects of this project which could explain our results. The most likely scenario is that endosymbionts of D. wrightii do not produce withanolides. Another is that over time, as the endosymbionts are recultured on the agar plates, is that there was an attenuation, or loss of production, of withanolide biosynthesis. Furthermore, withanolides could have been lost in our separation techniques however I believe this to be the least likely circumstance. Whatever the case, our screening of endosymbionts of D. wrightii did not indicate the presence of withanolides.
By our results, we conclude that it is unlikely that antiproliferative withanolides are synthesized by Datura wrightii. It is most likely that withanolides are solely produced by the plant Datura wrightii. This seems to suggest that there is not a symbiotic relationship in which endosymbionts of D. wrightii produce withanolides however there could still be a relationship involving metabolic transformation.
1) Zhang H, Bazzill J, Gallagher R, et al. Antiproliferative Withanolides from Datura wrightii. Journal of Natural Products. 2013. 76 (3) 445–449.
2) Machin R, Veleiro A, Nicotra V, et al. Antiproliferative Activity of Withanolides against Human Breast Cancer Cell Lines. Journal of Natural Products. 2010. 73(5) 966-968.
3) Borges K, Borges W, Pupo M, Bonato P. Endophytic Fungi as Models for the Stereoselective Biotransformation of Thioridazine. Applied Microbiology Biotechnology. 2007. 77(3) 669-674.
4) Wang Y, Dai C. Endophytes: a Potential Resource for Biosynthesis, Biotransformation, and Biodegradation. Annals of Microbiology. 2011. 62(3) 207-215.
5) Kaufmann B, Christen P, Veuthey J. Parameters Affecting Microwave-assisted Extraction of Withanolides. Phytochemical Analysis. 2001. 12(5) 327-331.