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1 This Journal of Environmental Horticulture article is reproduced with the consent of the Horticultural Research Institute (HRI which was established in 1962 as the research and development affiliate of the American Nursery & Landscape Association (ANLA HRI s Mission: To direct, fund, promote and communicate horticultural research, which increases the quality and value of ornamental plants, improves the productivity and profitability of the nursery and landscape industry, and protects and enhances the environment. The use of any trade name in this article does not imply an endorsement of the equipment, product or process named, nor any criticism of any similar products that are not mentioned. Copyright, All Rights Reserved

2 Research Reports: Flavonoid Shifts During Developmental Stages of Spathiphyllum Inflorescences 1 Roseann s. White 2 University of Central Florida Department of Biological Sciences Orlando, FL r Abstract " Flavonoids of spadix and spathe samples representing 6 different stages of maturation were extracted in methanol and analyzed by paper chromatography. Although the flavonoid composition appeared similar for the different developmental spadix stages, flavonoid diversity increased during maturation. Similar results were obtained from spathe samples. The flavonoid chromatographic profile obtained with leaf blades also differed markedly from that of either the spathe or spadix. These preliminary chromatographic studies demonstrate phytochemical diversity not only between different plant organs but within the same organ at different developmental stages. Index words: Araceae, flavonoids, Spatiliphyllum, inflorescence development, phytochemistry, plant fingerprinting " Introduction Sizable research funds are presently being expended in the private development of new plant varieties. Stable and definitive protection for developers of these varieties and the parts thereof becomes important both for the developer who must recover costs and the allied landscape and nursery industries which seek new and better stock (7). Classical methods of description of morphological and horticultural attributes have been used to characterize new cultivars to lrec~ived for publication February 6, 1985; in revised form April 2, The research reported here was supported in part by a grant from the Horticultural Research Institute and a University of Central Florida Graduate Studies and Research Summer Faculty Development Award. The au~hor acknowledges with gratitude the assistance giv.en by H.A. Miller and D.H. Vickers. 2Associate Professor of Biological Sciences. be protected by piant patent law (3). Patent disclosures so prepared are often so incomplete that extensive litigation has been the only recourse. In addition to the traditional methods of description incorporating colors, numbers, shapes, etc., scientists have recently used phytochemical methods to aid with identification. Most useful in this regard are the natural chemical products called flavonoids which are part of the phenolic group. Flavonoids constitute one of the largest and most diverse groups of naturally occurring phenolics (6). It has been estimated that about 2% of all carbon photosynthesized by plants is converted into flavonoids or closely related compounds (6). To characterize individual flavonoids the methanolicextract from dried plant parts is subjected to chromatography. The method may use paper, column, and thin layer chromatographic methods as well as high performance liquid Copyright 1985 Horticultural Research Institute 1250 I Street, N.W., Suite 500 Washington, D.C Reprints and quotations of portions of this publication are permitted on condition that full credit be given to both the HRI Journal and the author(s), and that the date of publication be stated. The Horticultural Research Institute is not responsible for statements and opinions printed in the 'Journal of Environmental Horticulture; they represent the views of the authors or persons to whom they are credited and are not binding on the Institute as a whole. Where trade names, proprietary products, or specific equipment is mentioned, no discrimination is intended, nor is any endorsement, guarantee or w'arranty implied by the researcher(s) or their respective employer or the Horticultural Research Institute. The Journal ofenvironmental Horticulture (USPS Publication No ) is published quarterly in March, June, September, and December by the Horticultural Research Institute. Subscription rate is $25.00 per year in USA; $40.00 per year for others.second-class postage paid at Washington, D.C. and at additional mailing office. Send address changes to HRI, 1250 I Street, N.W., Suite SOO, Washington, D.C. 2000S. J. Environ. Hort. 3(3): September

3 chromatographic (HPLC) systems. Chromatography produces a chemical "fingerprint" which is useful for protection of patented plant varieties. Recently, Asen (2) has extended the use of chemical markers for chemotaxonomy from the species level to the cultivar level by separating indistinguishable white flowers of New Guinea Impatiens sister seedlings on the basis of flavonoid profiles obtained by liquid chromatographic methods. However, for longlived inflorescences in foliage plants, flavonoid variation in response to developmental stage has not been well documented. Our studies on Spathiphyllum and other foliage plants were initiated in response to requests from several central Florida foliage growers that we develop chemical characterizations for their patented horticultural varieties. Spathiphyllum is one of the more primitive Araceae and has not been previously studied phytochemically. Extraction, purification and structural identification of all classes of flavonoids in Spathiphyllum would serve to elucidate the systematic relationships within the family, but our purpose was to develop a standardized approach to chemical comparison of diverse clones. Standardization is especially important for, as Asen (2) demonstrated, differences between cultivars are often quantitative as well as qualitative. To be confident that the chemical description could be met repeatedly and be of value to patent protection, we tested inflorescences in several developmental stages which can be recognized with considerable precision. Materials and Methods Preparation ofplant Extracts. Samples of Spathiphyllum 'Mauna Loa' vegetative leaves and inflorescences at progressive developmental stages were harvested simultaneously from a large sheltered, monoclonal planting on the University of Central Florida campus in Orlando. Six stages were defined by spathe color matched to a Munsell Color Cascade and by morphological condition. Spathe, spadix and peduncle were separated for each inflorescence before drying at 45 C (110 F). Dried structures from each stage were then extracted in absolute methanol. Additional extractions were made into an increasingly polar solvent series. Each extract was spotted on Whatman 3MM (46 x 57 cm) paper for descending two dimensional chromatography using methods described previously by Mabry et a1. (5). Samples were chromatographed in the first dimension for approximately 22 hr using 3: 1: 1 (by Vol) t-butanol:acetic acid:water. After air drying the chromatograms were rotated and refolded for descending chromatography in the second dimension using 15:85 (by Vol) acetic acid:water. Developed chromatograms were examined initially in a darkroom under long wavelength ultraviolet (UV) light with and without ammonia fumes. Treatment of dried chromatograms with ammonia vapor heightened the sensitivity of detection and produced color changes of structural significance. Methanolic aluminum chloride spray increased intensity of visualization of some compounds and reacted predictably to flavonoids that have a free 5-hydroxy group. All visualized spots were circled in pencil and color annotated for comparison of stages and consideration of chemical nature. Results and Discussion Preliminary examination of chromatograms from each structure in each of the six stages (Table 1) for the presence of flavonoids revealed spots visible. under long wave UV 94 Table 1. Descriptive developmental stages of Spathiphyllum defined by morphological condition and color characteristics. Stage Number *Color of Spathe Description of Morphology White with slight tendency to 22-1 Spathe enrolled prior to OPening to 22-4 Recently OPened spathe; Mostly two stamen stage to to 24-2 (center) Pollinated stage Spathe and ovaries becoming pigmented to 22-7 Spathe greening and all anthers are dehised to Spathe greening and spadix mostly pigmented to Spathe nearly green saturated as well as spadix. *Colors described according to Munsell Color Cascade System. and UV-ammonia fumes. The methods employed assure that phenolics and flavonoids account for the great majority of such ultraviolet visible spots. Some differences were found among spadix developmental stages 1-6 (Fig. 1). A composite diagram of the UV/ UV ammonia visualized components for spadix stages 1-6 showed those components whose presence varied in different stages (Fig. 3). Comparison of flavonoid profiles for all six spadix stages showed that a fluorescent blue violet component present in stages 2-6 was absent in spadix stage 1, the immature or newly emerging spadix. A yellow (UV) component absent in stages 1-3 was present as a trace in stage 4 and in high concentration in stages 5 and 6. A component not visible with UV was visualized with UV/ ammonia fumes and found as a trace in stage 4 and in higher concentration in stages 5 and 6. In contrast, a dark UV region which appeared yellow with ammonia fumes was present in spadix stages 1-3 but showed only a trace in stage 4 and was absent in chromatograms of stages 5 and 6. Probable flavonoid types for those whose presence varied with developmental stage were determined from their color characteristics, Rf-values, and reagent responses (Table 2). Although the total flavonoid composition was largely similar in various spadix stages, component A appeared as a possible isofl~vone mono- or di~lycoside. This component is lacking in anthesis and increases in concentration after total dehiscence of anthers. Compound A could also be cinnamic acid or a cinnamic acid ester. With the exception of the dark component, B, present in stages 1-3, flavonoid diversity increases with developmental maturation as evidenced by the presence of components C and D appearing in stages 4, 5, and 6. Chromatograms of spathe stages 1-6 demonstrated that flavonoid diversity in the spathe also increases after anthesis (Table 3) with at least 3 (possibly 5) compounds found after total dehiscence of anthers. Comparison of chromatographic spot appearance of stages 1, 2 with stage 3, stage 4, and stages 5, 6 revealed that the total number of presumed flavodoids in these profiles increases with increasing developmental maturity (Fig. 2). A dark UV component, E, was slow moving in both dimensions and first appeared as a trace in stage 3 becoming increasingly concentrated i~ stages J. Environ. Hort. 3(3): September 1985

4 (~~~ ~ Y{NH 3 Y(NH 3 ) ~~ ~,-. ~D~:=.:...:::...J, ' EtfNH ) \.:.~~~. Fig. 1. Chromatographic profiles of spadix developmental stages 1, 3, 6. Upper-Stage 1; Middle-Stage 3; Lower-Stage 6. Color code for figures I, 2, 3: D (Dark to Dark Violet), V (Violet), B (Blue), Br (Brown), G (Green), Y (YeUow), R (Red), P (Pink), W (White), L (Light), f (Fluorescent), vis (visible), NB) (ammonia fume color), and._.- not visible with UV alone or present in very low concentration. Fig. 2. Chromatographic profiles of spathe developmental stages 1, 3, 6. Upper-Stage 1; Middle-Stage 3; Lower-Stage 6. See Fig. 1 for explanation of symbols. J. Environ. Hort. 3(3): September

5 4,5 and 6. Another dark UV component, F, turned yellow/ red-brown with ammonia fumes first appeared as a trace in 4 and in higher concentrations in 5 and 6. Blue-violet (UV) component G first appeared in stage 4 and in increasing concentration in stages 5 and 6. In stages 5 and 6 two new UV visualized components appeared-violet component H (yellow with ammonia fumes) and overlapping yellow-green component I. The flavonoid chromatographic profile obtained with leaf blades differed markedly from that of either the spathe or spadix. Both a violet component and a pink component present in the chromatograms of leaf blades were absent in both spathe and spadix (Fig. 3). A number of components designated with dashed lines in the profile of blades (Fig. 3) were absent in chromatograms of blades but were found in one or more stages of spadix and/or spathe. These preliminary chromatographic studies demonstrate phytochemical diversity not only between different plant organs but within the same organ at different developmental stages. Consideration must be given to the developmental stage sampled in phytochemical characterization of any plant species or cultivar. The absence of flavonoid components on our chromatograms may have been due to their complete absence from the plant sample or to marked differences in concentration. If the differences observed in our chromatograms were due to quantitative rather than qualitative differences in flavonoid composition, the quantitative differences in concentrations were very large because all samples were analyzed from very high to low concentrations. Differences in flavonoid content observed with developmental stage may be due to different aglycones attached to the same or different sugars or to similar aglycones with variations in the nature of sugars attached. The actual purification and identification of the flavonoids of Spathiphyllum detected with paper chromatography in this study will require the more refined methods of HPLC (1, 3, 4), gas chromatography, UV/visible spectrophotometry (5) and nuclear magnetic resonadte (6). However, given such requirements for comparability offsources for the extracts, it is clear from Asen' s work and unpublished on-going research in this laboratory that chromatography for flavonoid "fingerprints" is potentially of great value for recognition of cultivars. Fig ,. ~.~,y.. " l.:omposite chromatographic profiles of spadix and spathe developmental stages 1-6 and blades. Upper-Spadix Composite; Middle-Spathe Composite; Lower-Blades Co~posite. See Fig. 1 for explanation of syrrlbols. Significance to the Nursery Industry The use of chemotaxonomic characterizations as an adjunct to the classical descriptions used for plant cultivars will result in better patent protection of new varieties. Such protection for patented varieties is potentially of economic benefit to the nursery industry by encouraging development and use of high quality, competitively pnced nursery crops. Great strides have been made toward accurate chemical characterization of plant varieties such as the HPLC analyses of different cultivars by Asen (2, 3, 4). Our studies demonstrate the importance of sample selection and control of developmental stage in bringing even greater precision to chemical plant fingerprinting. Those in the horticulture industry can be assured that chemical identification of cultivars is with us today and will become increasingly important for the future of the industry. Literature Cited 1. Anderson, J.M Analysis of plant phenolics by high-performance liquid chromatography. J. Chromatography 259: J. Environ. Hort. 3(3): September 1985

6 2. Asen, S Flavonoid chemical markers as an adjunct for cultivar 5. Mabry, T.J., K.R. Markham, and M.B. Thomas The sysidentification. Hortscience 12: tematic identification of flavonoids. Springer-Verlag, New York, Asen, S Flavonoid chemical markers in Poinsettia bracts. J. Amer. Soc. Hort. Sci. 104: Markham, K.R Techniques of flavonoid identification. Academic Press, New York. 4. Asen, S Identification of flavonoid chemical markers in roses and their high pressure liquid chromatographic resolution and quantitation 7. Williams, S.B Protection of plant varieties and parts as infor cultivar identification. J. Amer. Soc. Hort. Sci. 107: tellectual property. Science 225: Table 2. A summary of the color characteristics, R F values, and possible flavonoid type(s) for components A, B, C, D and the designation of developmental stage(s). Component A UV blue fluorescent/ blue violet Color Characteristics UVammonia R F Values x 100 1st 2nd Possible Flavonoid Type same as UV Isoflavone mono- or diglycoside 2-6 B dark!dark violet yellow Flavonol or flavone glycoside 1-3 C yellow same as UV Flavonols with a free 3-0H and without a free 5-0H Stages 5,6 D not visible blue Isoflavone lacking a low concentration; free 5-0H present in 5, 6 Table 3. A summary of the color characteristics, R F values, and possible flavonoid type(s) for components E, F, G, H, I and the designation of developmental stage(s). Component UV Color Characteristics UVammonia R F Values x 100 1st 2nd Possible F'lavonoid Type E dark yellow H* flavone or flavonol Flavone or flavonol glycoside F dark yellow/brown H* flavone or flavonol Isoflavone 7-0-aminoglycoside G violet/blue same as UV Flavonols lacking free 5-0H but with 3-0H substituted Isoflavones lacking free 5-0H H violet yellow Flavone and flavonol 7-O-glycoside 5,6 yellow/ yellowgreen *AICI 3 -Free 5-0H Flavonoid Yellow UV. yellow/ yellowgreen Flavonols with a free 3-0H and with or without a free 5-0H Stages 3 trace 4,5,6 5,6 4, 5, 6 5,6 J. Environ. Hort. 3(3): September