Pesticide Science Pestic Sci 55 :336–339 (1999)

Molluscicidal and antibacterial compoundsfromPetunia hybrida¹Dieter Mos er, Iris Klaiber, Bernhard Vogler and Wolfgang Kraus *Department of Chemis try , Univers ity of Hohenheim,D-70593 Stuttgart , Germany

Abstract : Three novel and six known petuniasterones were isolated from the aerial parts of Petunia

hybrida (Solanaceae). In addition, chromatography yielded four groups of novel 2,3,4,6-tetra-O-

acylated sucrose ester isomers. The sucrose esters showed molluscicidal and bactericidal activities.

Petuniasterones had diþ erent molluscicidal activities, depending on the side-chains.

1999 Society of Chemical Industry(

Keywords: Petunia hybrida; petuniasterones ; 2,3,4,6-tetra-O-acylated sucrose esters ; molluscicidal activity ;antibacterial activity

1 INTRODUCTION

Schistosomiasis (bilharziasis) is one of the most fre-quent tropical parasitic diseases. Approximately 200million people suþer from this disease, while 600million run the risk of becoming infected. Schisto-somiasis is caused by diþerent species of the trema-tode Schistosoma, which live in the abdominal bloodvessels of infected persons. The schistosomes layeggs which are excreted with faeces or urine andreach water where miracidia larvae hatch out of theeggs and infest freshwater snails. Once in these inter-mediate hosts, the miracidia divide and producethousands of cercaria which are shed into the water.These can then penetrate the skin of humans incontact with the contaminated water and, on reach-ing the blood vessels, develop into adult schis-tosomes.

Although chemotherapy is a valuable method ofcontrolling the disease, the risk of re-infectionremains, and there is still a need for selective andefficient molluscicides to eliminate the snail hostsand interrupt the parasite’s life cycle. In our searchfor naturally occurring pesticides, P hybrida extractshave been found to contain molluscicidal com-pounds. In addition, some of the compoundsexhibited antibacterial properties.

2 MATERIALS AND METHODS

2.1 Analysis

2.1.1 NMR analysisNMR spectra were recorded on a Varian UnityInovaspectrometer (500MHz).

2.1.2 MS analysisMass spectra were recorded on a Finnigan MATTSQ 700 spectrometer.

2.1.3 ChromatographyThe MPLC system consisted of a Bu� chi Chromatog-raphy pump, Waters Diþerential RefractometerR401, Pharmacia Single Path Monitor UV-1(254nm), LKB Radi Rac Fraction Collector, LatekR60 SV injection valve, KPG glass column(400] 45mm) with 300g Merck silica gel 60 (43–60lm), and a glass column (200] 45mm) with 125gMerck RP-18 (25–40lm). The HPLC system con-sisted of a Knauer HPLC PUMP 64, ABI AppliedBiosystems 785A Programmable Absorbance Detec-tor, Philips PM 8222 Dual-Pen Recorder, BischoþHPLC column (250] 4mm) with Merck LiChros-pher 100 RP-18 (5lm) and Rheodyne 7125 injectionvalve with 20ll loop for analytical HPLC; a BischoþHPLC column (250] 20mm) with Merck LiChros-pher 100 RP-18 (5lm), Merck LiChroCart 250-10HPLC cartridge with LiChrospher 100 RP-18(10lm) and Rheodyne 7125 injection valve with500-ll loop for semi-preparative HPLC.

Merck silica gel 60 F254 aluminium sheets(0.2mm) were used for TLC.

2.2 Plant extracts

2.2.1 Plant materialDried and ground aerial parts of Petunia hybridaVilm var grandiýora cultivated in Kenya werereceived in 1992 from Dr TO Midiwo, University ofNairobi. A voucher specimen has been deposited inthe herbarium of the University of Nairobi, Kenya

* Corres pondence to : Department of Chemis try,Wolfgang Kraus ,

Univers ity of Hohenheim, D-70593 Stuttgart, Germany.

E-mail : kraus 130=uni.hohenheim.de

¹ Bas ed on pos ter pres entations at the 9th International Congres s

of Pes ticide Chemis try, organis ed by the International Union of

Pure and Applied Chemis try (IUPAC), and held in London, UK,

2–7 Augus t 1998.

(Received 1 July 1998; revis ed vers ion received 14 October 1998;

accepted 15 October 1998)

( 1999 Society of Chemical Industry. Pestic Sci 0031-613X/99/$17.50 336

Molluscicidal and antibacterial compounds from Petunia hybrida

(voucher number : Nairobi University Herbarium(International Code Number NAI), Mathenge/Midiwo 91/35).

2.2.2 ExtractionThe plant material (4kg) was extracted successivelywith light petroleum distillate, ethyl acetate andmethanol (6] 15 litres of each). The extracts wereevaporated under vacuum to give 31g, 63g and 330gcrude extracts, respectively.

2.2.3 Isolation of petuniasterones 1–9 (Fig. 1)The crude petroleum extract was redissolved in lightpetroleum distillate (2 litres) and submitted toliquid–liquid separation with dioxane ] water (1] 1by volume; 8] 400ml). The bioactive product fromthe dioxane ] water layer (6g) was applied to a silicagel column (400] 45mm) and eluted using a stepgradient of diþerent solvents and solvent mixtureswith increasing polarity. The eluents were lightpetroleum ] ethyl acetate (100] 0; 98] 2, 96] 4,

Figure 1. Petunias terones and s ucros e es ters is olated fromPetunia hybrida.

Pestic Sci 55 :336–339 (1999) 337

D Moser et al

92] 8, 80] 20, 70] 30, 60] 40, 50] 50, 40] 60,30] 70, 20] 80, 10] 90 by volume), ethylacetate ] methanol (100] 0; 90] 10, 80] 20,50] 50 by volume) and methanol, using 500mlsolvent or solvent mixture in each step. Monitoringwas done by TLC (solvent : light petroleum ] ethylacetate, 3] 7 by volume) to give 20 fractions. Frac-tions 13 and 14 showed the highest biological activ-ities.

Further separation of fraction 13 (595mg) byreversed-phase MPLC (solvent : methanol ] water(5] 5 by volume), ýow: 15ml min~1, detection:UV \ 254nm) and semi-preparative reversed-phaseHPLC (solvent : acetonitrile ] water, 6] 4 byvolume, ýow: 5ml min~1, detection: UV \ 254nm)led to the isolation of petuniasterones 1 (1mg), 2

(2.5mg), 3 (35mg), 4 (4mg), 5 (14mg), 6 (4mg), 7

(2mg), 8 (1mg) and 9 (3mg).

2.2.4 Isolation of sucrose esters 10–13

Final puriücation of fraction 14 (730mg) on semi-preparative reversed-phase HPLC (LiChroCart250-10; solvent : ethanol ] water, 5] 5 by volume,ýow: 2.5ml min~1, detection: UV \ 220nm) yieldedfour groups of 2,3,4,6-tetra-O-acylated sucrose esterisomers with relative molecular masses of 678 (10;

2mg), 692 (11; 28mg), 706 (12; 64mg) and 720 (13;

60mg), respectively.

2.3 Biological activity

2.3.1 Molluscicidal activityBioassays were carried out with Biomphalaria glab-rata Say (Planorbidae), one of the snail vectors ofschistosomiasis. Snails of uniform shell sizes (8–10mm diam) were used. Two snails were placed in adistilled water solution of the extracts, fractions orcompounds containing ethanol (4ll) and polyethyl-ene glycol 400 (2ll) ml~1 solution. Extracts andfractions were tested at 500, 200 and 100lg ml~1,and compounds were tested in a series of dilutions(100, 80, 60, 40, 20, 10lg ml~1) in order to establishthe minimum concentrations required to kill bothsnails (100% mortality). Because very little materialwas available, the number of test replicates for eachconcentration was limited to two. Niclosamide

Bayer AG, Leverkusen, Germany)(Bayluscide} ;was used as a reference. It gave 100% mortality at5lg ml~1.

After 24 hours, the snails were transferred to avessel containing dechlorinated tap water, and mor-tality rates were determined after a 24-h recoveryperiod.1h3

2.3.2 Antibacterial activity by bioautographyTest materials (200, 100 and 50lg for extracts andfractions and 40, 20, 10, 5 and 1lg for compounds)were applied to silica gel TLC sheets which weredeveloped in an appropriate solvent system. Thechromatograms were sprayed with suspensions ofBacillus subtilis (Ehrenberg) Cohn or Pseudomonas

stutzeri (Lehmann & Neuman) Sijderius, and incu-bated in a humid atmosphere for 24h at 30¡C topermit growth of the bacteria. Subsequently, zonesof inhibition were visualized by a tetrazolium salt :metabolically active bacteria convert the tetrazoliumsalt into the intensely coloured formazane and inhi-bition zones appear as white spots against a red back-ground.4

3 RESULTS

3.1 Structure determination

Compounds 1–4, 6 and 8 were identiüed by compari-son of their NMR spectra with published data.5h7The structures of the novel petuniasterones weredetermined by means of NMR experiments. Assign-ments were based upon [1H], [1H-1H]COSY,[13C], DEPT-, GHSQC- and HMBC-NMRspectra. The stereochemical arrangement in the sidechain of petuniasterone 7 was proved by NOEexperiments (22-H ] 20-H, 23-Hb ; 23-Ha ] 21-H,28-H; 28-H ] 26-H, 27-H).

Chromatography of the sucrose ester mixtureyielded four groups of compounds. The componentsin each group had identical masses, whereas thegroups diþered from each other by 14 mass units.

[1H] and [13C]NMR spectra of all groups showedthe signals of sucrose molecules with the glucosemoiety fully esteriüed, while the fructose portionshowed four free hydroxy groups (Fig 1).8

[1H-1H]COSY spectra revealed 2-methylbutanoylgroups as major acyl residues for the sucrose esterisomers 10–13. According to the HMBC spectra, the2-methylbutanoyl groups were located at 2-O, 4-Oand 6-O on the glucosyl moieties of all sucrose esterisomers. 2D NMR experiments indicated for 11–13

the presence of 3-methylbutanoyl groups as furtherpossible acyl residues at position 4-O. The NMRspectra also suggested the presence of diþerentstraight-chain acyl groups linked to 3-O. The diþer-ent molecular masses of the sucrose esters suggestedthat these straight-chain acyl groups were of diþer-ent lengths.

The [1H-1H]COSY spectra, however, proved tobe unsuitable for distinguishing the diþerentstraight-chain acyl groups because of the overlappingproton signals. Therefore, the chain lengths of theacyl groups attached to 3-O were determined byGHSQC-TOCSY. Thus, the signals arising from apentanoyl (10), hexanoyl (11), heptanoyl (12) andoctanoyl group (13) were observed.

3.2 Biological activity

All compounds were tested against the freshwatersnail B glabrata, one of the intermediate hosts ofschistosomiasis. We found that only those petu-niasterones which contained either the bicyclicorthoester as in 1 and 6 or bearing a 24,25-epoxygroup and an esteriüed hydroxy group at position 22as in 3–5 showed molluscicidal activity. All other

338 Pestic Sci 55 :336–339 (1999)

Molluscicidal and antibacterial compounds from Petunia hybrida

Table 1. Mollus cicidal activities of petunias terones and s ucros e es ters fromPetunia hybrida

Mortality at kg mlÉ1 (%)

Compounda 100 90 80 75 70 60 40 20 15 10 5 1

1 100 –b 100 – – 100 0 0 – – – –

2 0 – 0 – – 0 0 0 – – – –

3 100 – 100 100 33 33 0 0 – – – –

4 100 – 100 – 100 50 0 0 – – – –

5 100 100 0 – – 0 0 0 – – – –

6 100 – 100 – – 100 100 0 – – – –

7 0 – 0 – – 0 0 0 – – – –

8 0 – 0 – – 0 0 0 – – – –

9 0 – 0 – – 0 0 0 – – – –

10 – – – – – – 100 100 100 0 0 0

11 – – 100 – – 100 100 100 100 0 0 0

12 – – 100 – – 100 100 100 0 0 0 0

13 – – 100 – – 100 100 100 100 0 0 0

a 1\ petunias terone A; 2\ petunias terone C; 3\ petunias terone C-22-O-acetate ; 4\ petunias terone C-22-O-[(methylthio)carbonyl]-acetate ; 5\ petunias terone C-22-O-[(methoxy)carbonyl]-acetate ; 6\ petunias terone D;7\ (22R,24R)-22,24-epoxy-7,25-dihydroxyergos ta-1,4-dien-3-one; 8\ petunias terone G; 9\ petunias terone G-22-O-acetate ; 10\ s ucros e es ter is omers MW 678; 11\ s ucros e es ter is omers MW 692; 12\ s ucros e es ter is omersMW 706; 13\ s ucros e es ter is omers MW 720.b –\ not tes ted

petuniasterones were inactive against the snails at aconcentration of 100lg ml~1. The sucrose esters10–13 showed molluscicidal activities at concentra-tions as low as 15lg ml~1. Molluscicidal activities ofthe compounds are listed in Table 1.

Furthermore, the sucrose esters 10–13 were activeagainst B. subtilis and P. stutzeri in the direct bio-autography TLC assay at 5lg per spot.

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