BIOLOGICALLY ACTIVE NATURAL PRODUCTS: AGROCHEMICALS - CHAPTER 4 ppsx

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,

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

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.1,5,6We 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.7We 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)

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.

The genus Nuphar has been examined rather extensively and others have reported

anti-fungal activity8 and the medical utility9 of Nuphar extracts The genus is such a rich source

of alkaloids that these are known as Nuphar alkaloids.10N 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.11-13As 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.

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

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

root and rhizome 25 67 root and rhizome 12.5 35 root and rhizome 2.5 22 Lettuce seedling leaf 12.5 100

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.

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.15This 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.15support the earlier conclusion of Weidenhamer et al.16that 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

foundβ-sitosterol and the long chain alcohol 1-hexacosanol have been reported17along with reports of the sterols campesterol, stigmasterol, and the triterpenesα- and β-amyrin, lupeol, and taraxasterol.18Nymphaea 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 identifiedβ-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.)

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 Thompson32 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 Price33 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 tetragonaa 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.

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 PGG is pentagalloylglucose, TGG

is tetragalloylglucose.

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