BIOLOGICALLY ACTIVE NATURAL PRODUCTS: AGROCHEMICALS - CHAPTER 4 ppsx

4 Phytochemical Inhibitors from the Nymphaeceae: Nymphaea odorata and Nuphar lutea Stella D. Elakovich, Stacy Spence, and Jie Yang CONTENTS 4.1 Introduction 4.2 Nuphar lutea 4.3 Nymphaea odorata 4.4 Conclusions Acknowledgments References KEY WORDS: Nymphaeceae, Nymphaea odorata, Nuphar lutea, hydrophytes, allelopathy, growth inhibition, alkaloids, gallotannins. ABSTRACT From a survey for potential allelopathic activity of some 25 herbaceous hydrophytes native to the southeastern U.S., we found two species in the Nymphaeceae to possess the greatest inhibitory activity. Nuphar lutea was the most active species and was some 10 times more active than Nymphaea odorata, the second most active as measured by lettuce seedling and Lemna minor growth assays. Bioassay-directed isolation of plant growth inhibitory constituents from N. odorata led to the isolation of five allelochemicals of moderate activity. These included gallic acid, myricitrin, myricetin, 1,2,3,4,6-pentagalloyl- D-glucose, and 2,3,4,6-tetragalloyl-D-glucose. Examination of alkaloids from N. lutea showed 6,6′-dihydroxythiobinupharidine to be highly inhibitory of lettuce seedling radicle elongation at concentrations greater than 2 ppm. Nupharolutine, a second alkaloid isolated that was of similar structure, was inactive. Literature reports of phytochemicals present in N. odorata and N. lutea, as well as in other members of the Nymphaeceae, are examined. 4.1 Introduction In our work in the area of allelopathy (the influence of one plant on another), we have been working with aquatic and wetland plants native to the southeast U.S. We have examined more than 25 different aqueous extracts from these plants to determine their allelopathic potential. 1-3 The plant species examined are listed in Table 4.1. Species of Eleocharis were included as they often appear in monoculture in their undisturbed natural habitats and © 1999 by CRC Press LLC have been observed to “displace” other hydrophytes. 4 Some species, including Hydrilla ver- ticillata and Myriophyllum aquaticum, were chosen because they are among the most nox- ious of aquatic weeds. Other species were included based on their observed growth patterns and their propensity toward monocultures, and some species were selected based on previous reports of allelopathic activity. Aqueous extracts of each of the plants were sub- jected to lettuce seedling and Lemna minor bioassays. Lettuce seedlings are perhaps the most widely used plant growth inhibition target system. 1,5,6 We pregerminated the seeds, ran each assay at three extract concentrations using 20 seedlings per plate, and ran dupli- cate plates. Radicle lengths were compared to controls. 1 Lemna minor bioassay allows the use of this small floating aquatic plant, with the common name duckweed, as the target species for exploration of the allelopathic nature of aquatic hydrophytes. 7 We maintained axenic L. minor cultures, ran the assay in 24-well sterile tissue culture plates with 1.5 mL of growth medium per well. We then ran each assay at concentrations corresponding to the three concentrations of the lettuce seedling assays, ran six replications per 24-well plate, and ran duplicate plates which agreed to within ± 5%. 1 Control and extract wells were cho- sen such that position was random, and the assay was read when the control wells reached 20 fronds. Advantages of this assay are that it required only 1.5 mL of growth medium to accommodate the entire target L. minor plant, and that aquatic plant targets are more appropriate for evaluation of aquatic plant allelopathy. Results of these initial bioassays showed species in the Nymphaeceae to possess the highest allelopathic potential. World- wide members of the Nymphaeaceae are listed in Table 4.2. Some botanists also include the two genera of Cobombaceae, Brasenia schreberi and Cabomba (about seven species), in the Nymphaeceae. Of the six genera listed in Table 4.2, we examined only Nuphar and Nym- phaea, as these were the only plants readily available to us in the southeastern U.S. The oth- ers are native to Southeast Asia, Australia, or South America. We did, however, also examine Cabomba caroliniana and Brasenia schreberi, two species sometimes included in the Nymphaeceae. Table 4.3 gives lettuce seedling bioassay results of only the most inhibitory TABLE 4.1 Plants Native to the southeastern U.S. Examined for Allelopathic Potential Brasenia schreberi Gmel. Juncus repens Michx. Cabomba caroliniana Gray Limnobium spongia (Bosc.) Steud. Ceratophyllum demersum L. Myriophyllum aquaticum (Vell.) Verdc. Eleocharis acicularis (L.) R. and S. M. spicatum L. E. equisetoides (Ell.) Torr. Najas guadalupensis (Spreng.) Maagus E. flavescens (Poir.) Urban Nuphar lutea (L.) Sibth. and Sm. E. montana (HBK.) R. and S. Nymphaea odorata Ait. E. obtusa (Willd.) Shultes Nymphoides cordata Ell. E. quandrangulata (Michx.) R. and S. Potamogeton foliosus Raf. E. tuberculosa (Michx.) R. and S. Sparganium americanum Nutt. Hydrilla verticillata (L.F.) Royal Vallisneria americana Michx. TABLE 4.2 Worldwide Members of the Genera in the Nymphaeaceae Family Barclaya (two species — tropical Southeast Asia) Euryale ferox (one species — Southeast Asia) Ondinae purpurea (one species — northwestern Australia) Nuphar (7 to 20 species — Northern temperate) Nymphaea (~40 species — cosmopolitan) Victoria (two species — South America) © 1999 by CRC Press LLC extracts tested at 250 ppt. Both N. lutea and N. odorata were divided into above ground and below ground parts before testing. Table 4.4 gives Lemna minor bioassay results of only the most inhibitory extracts tested at 250 ppt. Clearly, Nuphar lutea completely inhibits all growth of both lettuce seedlings and L. minor at this concentration. Brasenia schreberi and Combomba caroliniana, two of the occasional members of the Nymphaeceae, also are among these most inhibitory extracts towards lettuce seedling and Lemna minor as target plants, respectively. Table 4.5 summarizes the results of N. lutea inhibition of lettuce seedlings and L. minor at much lower concentrations than the 250 ppt of Tables 4.3 and 4.4. N. lutea was significantly inhibitory even at 2.5 ppt. It was 10 fold more inhibitory in these two bioassays than the overall next most active plant, Nymphaea odorata. 4.2 Nuphar lutea The genus Nuphar has been examined rather extensively and others have reported anti- fungal activity 8 and the medical utility 9 of Nuphar extracts. The genus is such a rich source of alkaloids that these are known as Nuphar alkaloids. 10 N. lutea produces alkaloids having a regular sesquiterpenic skeleton incorporated into 3-furyl-substituted piperidines or quin- olizidines, as well as C30 alkaloids that contain a sulfur atom along with two 3-furyl sub- stituents. 11-13 As none of these alkaloids had previously been examined for allelopathic TABLE 4.3 Bioassay Results of Inhibition of Lettuce Radicles by Aqueous Extracts of Selected Hydrophytes Nuphar lutea (roots and rhizomes) 100% Nuphar lutea (leaves) 100% Nymphaea odorata (leaves) 95% Juncus repens 86% Vallisneria americana 83% Brasenia schreberi 82% Ceratophyllum demersum 80% Eleocharis acicularis 78% Nymphaea odorata (roots and rhizomes) 78% Note: Tested concentrations were 250 ppt of a filtered solution prepared by extraction of a mass of dried plant material with an equal mass of sterile water. Results are ex- pressed as percent inhibition as compared to the control. TABLE 4.4 Bioassay Results of Inhibition of Lemna minor Growth by Aqueous Extracts of Selected Hydrophytes Nuphar lutea (leaves) 100% Nuphar lutea (roots and rhizomes) 100% Nymphaea odorata (leaves) 98% Myriophyllum aquaticum 83% Nymphaea odorata (roots and rhizomes) 72% Cabomba caroliniana 68% Note: Tested concentrations were 250 ppt of a filtered solution prepared by extraction of a mass of dried plant material with an equal mass of sterile water. Results are ex- pressed as percent inhibition as compared to the control. © 1999 by CRC Press LLC activity, we isolated two of them and examined their allelopathic potential by the lettuce seedling assay. 14 Isolated were 6,6′-dihydroxythiobinupharidine and nupharolutine (Figure 4.1). Of these, nupharolutine was inactive up to 60 ppm, the highest concentration we could test due to solubility constraints of this isolated, water-insoluble compound. The second compound, 6,6′-dihydroxythiobinupharidine, was highly inhibitory of lettuce seedling growth at concentrations greater than 2 ppm as shown in Figure 4.2. It is possible that other of the many Nuphar alkaloids also are plant-growth inhibitory, but they have not yet been isolated and tested. 4.3 Nymphaea odorata Sütfeld and co-workers have recently reported finding resorcinol as one of the exudates of N. lutea seedlings. 15 Although resorcinol-related compounds are widespread products of TABLE 4.5 Inhibition of Lemna minor and Lettuce Seedling Growth by Various Concentrations of Leaf, Root, and Rhizome Extracts of Nuphar lutea Bioassay N. lutea Part Concentration Inhibition (%) (ppt) (control = 0%) Lemna minor leaf 25 66 leaf 12.5 47 leaf 2.5 22 root and rhizome 25 67 root and rhizome 12.5 35 root and rhizome 2.5 22 Lettuce seedling leaf 12.5 100 leaf 2.5 71 root and rhizome 12.5 100 root and rhizome 2.5 69 FIGURE 4.1 The structures of nupharolutine (top) and 6,6′-dihydroxythiobinupharidine (bottom), alkaloids isolated from Nuphar lutea. © 1999 by CRC Press LLC plant metabolism, this is the first report of nonderivatized resorcinol from a higher plant and is the first evidence for exudation of structurally defined phenol from an undisturbed aquatic macrophyte. Activity testing at the concentration resorcinol that is exuded from N. lutea showed the zooplankton Daphnia to be sensitive to resorcinol, but two (of five tested) phytoplankton organisms, Cryptomonas and Rhodomonas, were able to metabolize resorcinol and, in doing so, to increase their starch granules. 15 This suggests that resorcinol acts as an allelochemical in an aquatic medium. Its specificity indicates it may be detrimen- tal to Daphnia but be beneficially used by Cryptomonas and Rhodomonas or other organisms. Three additional species tested were uninfluenced by the levels of resorcinol applied. 15 Sütfeld et al. 15 support the earlier conclusion of Weidenhamer et al. 16 that although a given species may produce and exude only small amounts of allelochemicals, a constant flux of these compounds may produce a significant deleterious effect on target species. 16 Nymphaea odorata has not received the same intensive investigation that Nuphar (with its abundant and important Nuphar alkaloids) has, but some reports exist. The commonly found β-sitosterol and the long chain alcohol 1-hexacosanol have been reported 17 along with reports of the sterols campesterol, stigmasterol, and the triterpenes α- and β-amyrin, lupeol, and taraxasterol. 18 Nymphaea species, other than N. odorata which have been exam- ined, are listed in Table 4.6. Saeed et al. 25 have examined the lipoidal matter of N. hybrida and identified β-amyrin, α-amyrin, campesterol, cholesterol, stigmasterol, and β-sitosterol, as well as the hydrocar- bons squalene and dotriacontane. They also identified a dozen fatty acid methyl esters. The lipoidal material showed antimicrobial activity against some gram-positive bacteria and insecticidal activity against cotton leaf worm. None of the reports of Nymphaea constituents examined the plant growth-inhibiting nature of the identified compounds. We carried out a bioassay-directed examination of the FIGURE 4.2 The results of bioassay of 6,6′-dihydroxythiobinupharidine with lettuce seedlings as the target. Results of replicate runs are shown. Bars with the same letter (within each run) are not significantly different according to the Duncan multiple range test at P < 0.05. The control was different from the inhibitory effect of 6,6′-dihydrox- ythiobinupharidine at >2 ppm. At 0.6 ppm the alkaloid was significantly stimulatory in Run 2. (From Elakovich, S.D. and Yang, J., J. Chem. Ecol., 22:12, 2209-2219, 1996. With permission.) © 1999 by CRC Press LLC plant growth inhibiting (allelopathic) constituents of N. odorata and isolated the compo- nents shown in Table 4.7. 31 Listed are the isolated compounds, their activities in the two bioassays applied, lettuce seedling and Lemna minor, and their millimolar concentration in the assays. Activities are shown for isolated compounds at 100 mg/L or 100 ppm. Struc- tures of these compounds are shown in Figure 4.3. Although the activities are modest, we are not aware of other gallotannins, present in Nymphaea species, reported to possess plant growth inhibitory properties. There are reports of the plant growth inhibitory activity of myricitrin and gallic acid isolated from other sources. Nicollier and Thompson 32 reported myricitrin, a flavonol glycoside, to be the key factor in the multiple bioactivities of Desman- thus illinoensis, a member of the Leguminosae being examined for its allelopathic activity. Myricitrin was allelopathic at less than 10 ppm against tomato root growth and also anti- bacterial against two Bacillus species. Price 33 reported the leaves of Kalanchoe glassfeldiana contained a specific flowering inhibitor shown to be gallic acid. Geraniin, an antimicrobial hydrolyzable tannin, similar in structure to the penta- and tetragalloylglucose compounds we isolated from Nymphaea odorata, was isolated from N. tetragona and found to be active against fish pathogenic bacteria. 34 Tannins have been isolated from Nuphar species, but not from N. lutea. From N. japonicum there have been four dimeric and trimeric hydrolyzable tannins isolated, called nupharins A, B, C, D, E, and F that possess an α-D-glucopyranose core with a hexahydroxydiphenoyl TABLE 4.6 Compounds Reported from Nymphaea Species Other than N. odorata N. alba Linn. Flavonoids including isokaempferide, apigenin, isoquercitrin, hyperoside, quercetin 4′-β-xyloside, 3-methylquercetin 3′-β-xyloside 19 Dienoic, trienoic, and tetraenoic acids 20 α-aminoadipic acid 21 β-sitosterol, gallic acid, 1-hexacosanol 22 Nupharin, nymphaeine, nymphalin 23 Tocopherylesters 24 N. hybrida Lipids 25 Quercetin, myricetin 26 N. mexicana Alkanes of 28 and 29 carbons 27 N. stellata β-sitosterol, coclaurine 28 N. tetragona a Aminopropylhomospermidine 29 N. tuberosa Paine Alkaloids, flavones, triglycerides 30 N. variegatum Engelm. Alkaloids, flavonols, saponins 30 a Spelled Nymphaca tetragona in the original publication. TABLE 4.7 Compounds Identified in Nymphaea odorata by Bioassay-Directed Isolation Compound Concentratio n Percent of Control Lettuce Lemna Gallic acid 0.59 mM 10 50 Myricitrin 0.21 mM Inactive 79 Myricetin 0.31 mM Inactive 60 1,2,3,4,6-PGG 0.11 mM 52 63 2,3,4,6-TGG 0.13 mM 51 52 Note: Bioassay results for lettuce seedling and Lemna minor assays are given. PGG is pentagalloylglu- cose, TGG is tetragalloylglucose. © 1999 by CRC Press LLC group at the 3,6-position. 35-37 These nupharins are similar in structure to the 1,2,3,4,6-penta- galloylglucose we found in Nymphaea odorata. Nishizawa et al. 38 found two gallotannins and two ellagitannins with antibacterial activity in Nuphar variegatum. 4.4 Conclusions This examination of phytochemical inhibitors from the Nymphaeceae leads to the conclu- sions that • Members of the Nymphaeceae appear to possess potent growth-inhibitory com- pounds. • Both Nuphar and Nymphaea possess gallotannins and related compounds of moderate activity. • The Nuphar alkaloid we isolated from N. lutea, 6,6′-dihydroxythiobinupharidine, has by far the greatest plant growth inhibitory properties reported for com- pounds isolated from members of the Nymphaeceae. • Other members of the Nymphaeceae must be examined to determine the extent of family characteristics. • Hydrophytes should be considered as an important source of useful secondary metabolites. ACKNOWLEDGMENTS: The help of Dr. J.W. Wooten is gratefully acknowledged. FIGURE 4.3 Compounds identified from Nymphaea odorata by bioassay-directed isolation. 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Nishizawa, K., Nakata, I., Kishida, A., Ayer, W.A., and Browne, L.M., Phytochemistry, 29, 2491-2494, 1990. © 1999 by CRC Press LLC . gallic acid, myricitrin, myricetin, 1,2,3 ,4, 6-pentagalloyl- D-glucose, and 2,3 ,4, 6-tetragalloyl-D-glucose. Examination of alkaloids from N. lutea showed 6,6′-dihydroxythiobinupharidine to be highly. isoquercitrin, hyperoside, quercetin 4 - -xyloside, 3-methylquercetin 3 - -xyloside 19 Dienoic, trienoic, and tetraenoic acids 20 α-aminoadipic acid 21 β-sitosterol, gallic acid, 1-hexacosanol 22 Nupharin,. Chem., 12, 226, 19 74. 24. Klink, G., Buchs, A., and Gulacar, F.O., Phytochemistry, 36, 81 3-8 14, 19 94. 25. Saeed, A., Omer, E., and Hashem, A., Bull. Fac. Pharm. (Cairo Univ.), 31, 34 7-3 51, 1993, Chem. Abstr.,