Jpn. J. Infect. Dis., 53, 17‑19, 2000

Short Connumication

Molluscicidal Saponins from Anagallis arvensis agalnSt●Schistosome lntemediate Hosts

Mahfouz M. Abdel‑Gawad, Samir M. El‑Amin, Hajime Ohigashil, Yoshinori Watanabel,
Naohito Tikeda2, Hiromu Sugiyama3 and MasanoriKawanaka3*
T72eodor Bilhwz Resewch Institute, El‑Nile Street,
Warrak El‑Hadar, Imbaba, Giza, Egypt,

JDivision ofApplied Llfe Sciences, Graduate School ofAgriculture, Kyoto UnE'versiO?,
Kitashirakawa, Oiwake‑cho, Kyoto 606‑8502,

2Faculo7 0fPharmaq?, MeLjo UniversiO?,

Yagotoyama 150, 7Tempaku‑ku, Nagoya, 468‑8503 and3Department ofParasitology, National Institute ofInfectious Diseases,
ZToyama I‑23‑I, Shinjuku‑ku, nob70 162‑8640, Japan(Received January 26, 2000. Accepted March 10, 2000)
StJMMARY: The molluscicidal activityof saponins isolated &om the plant Anagallis arvensis (Primulaceae)
was studied against schistosomeintermediate hosts, Biomphalaria glabrataand Oncomelania quadasi. Strong

molluscicidal activitywas foundintwo compounds called desglucoanagalloside Bandanagalloside B. Theirstructures were identified onthe basis ofchemicaland spectroscopICanalysesand their activitiesare comparable
to that of the synthetic molluscicide, niclosamide.

distilled waterand their shells were crushed for examination.
Visual observation uslng a microscope was performed toSchistosomiasis, the most significant trematode disease ofhuman, is a world‑wide healthproblem. It affects 200 million

determine whether the snail was alive or dead according topeopleand poses a threat to 400 million morein74 countries

round the world (1)･ In Egypt, newly constructed damSand

the WHO criterion (6). The powder‑like sample obtained byirrigation systems may have changedthe ecological conditions

lyophilization Bom each stage of the isolation process wasand contributed tothe suddenandunexpectedincreaseintheintermediate host snails of schistosomes. Humaninfection

tested for activityat variousconcentrations. Niclosamide (ICNwithSchistosoma mansoni is spreadingand increaslng lnmolluscicide. This synthetic reagent has been ln use aS a

association with the use of perennial irrigation (2). Theremolluscicide since the 1960s and remains the molluscicide

remains a need for deteminingthe molluscicidalallel∝hemicals

of choice (1).

from plantsinthe hopethatthey might provide cheap, locally

The plant, A. arvensis, was collectedand deposited atthe
Laboratory of Medical Chemistry, Theodor Bilharz ResearchInstitute, Giza, Egypt. The material was driedinthe shade
and finely powdered for use. Thefractionation scheme for

Biomedicals hc･, Atmra, Ohio, USA) was used as.a reference

producible, biodegradable,and effective control agentsinthe
endemicareas (3,4). Several local desertand cultivated plants
have been screened inthe laboratoryandunderfield condidonsagainst the snail vectors in Egypt. Among them, Anagallis
arveWis is considered one of the most promlSmg mOllusci‑
cidal plants,and its saponinfraction has been reported tothe isolation of the molluscicidal saponins is showninFigure
I. Briefly, dried whole plants ofA. a"ensis (25 g) wereextractedusing methanol. The methanol extract was partitioned

express high molluscicidal activity(5). In this paper webetween ethyl acetateand water. The water‑soluble fraction

describe the isolation and structure determination of themolluscicidalsaponins ofA. arvensisandtheir molluscicidal

was血en chromatographed on silica gel stepwise and eluted

with an increaslng ratio of methanol in ethyl acetate. The

molluscicidal activitywas foundinthe 25‑35% methanolactivities.

The bioassay wasperformed on two species, Biomphalaria

eluate. ru血er column chromatogr叩hy on silica gel uslng

glabrata (Puerto Rico origin, the intermediate host of S.
mansoni)and Oncomelania quadrasi (Mindanao, Philippines

anupper phase of ethyl acetate‑water‑methanol ( I 0:3 :2) gave
two active fractions, Fr. A‑3and Fr. Al5 (Fig. 1). They wereorigin,theintermediate host ofSchistosomajaponicum), bothmaintained inthe laborabry of the Department of Parasitology,
National Institute of Infectious DiseasesinTokyo. Snails usedinthe experiments were young‑mature (8.5 to 10.5 mm shell
diameter for B. glabrata; 4.0 to 4.5 mm shell height for 0.

methanol‑water (3: 1). Final purification of the active frac‑
tions by preparative HPLC (YMC GEL ODS‑A 60‑400/230,8.0 mUmin) gave active compounds 1 (34.7 mg)and 2 (15･1

separately submitted to separation onanODS gel eluted withmg).

quadrasi). The tests were carried out as follows: 1 0 snailsin

Compound 1, 【 α 120D ‑1.50 (C‑0.$7, methanol), showed

separate plastic dishes (diameter, 60 帆 and height, 30 mm)

anapparent ion peak at m/I 1063.5689【MH]'inthe HR FAB‑

were exposed to a serial dilution of aknOwn concentration ofMS, showingthe molecularformula of C52H87022 (Calculated

test samples in 60 ml of distilled water at a temperature offor MH+:1063.5540). Compound 2, 【 α 】20D ‑A.20 (C‑0.38,28oC for 24 h. After exposure,the snails were washedin*Corresponding author: Tel: +81‑3‑5285‑1 I 1 1 (ext.2734), Fax:methanol), also showedanapparent ionpeak at m/z 1225.6217
lMH]'inthe HR FAB‑MS, showing the molecular formulaof C58H,702, (Calculated for MH': 1225.6223). Inspection of+8113‑5285‑I 173, E‑mail: mkawan@nih.go.jp

the lH‑and uC‑nuclearmagnetic resonance (IH‑ and 13C‑17

Table 1. lHand 13CNMR dataofcompound 1 and 2

Dried leaves ofA. anerLSis (ground powder 25 g)

ト Extracted with McOH

Compound 2

Compound 1

MeOH ext.

Position

ト PaTtitioncd bctwcen EtOAc and water

61H!

639.26843各.̲734250.36̲9 .86叫37. 仏51.39 6.315̲3.17は9.832

12つJ45′07 9012tJ45′0 780ノ12 34 5′b70 ノ06)3C1H!

613C

39.268仏鵬̲7.34屯506.̲9386.仙7 叫51.39 6.315̲.7 8̲9.7各32305702570ノ405712∠UO7q′90ュJl′b 0 82057 2570ノ405′b‑2/b′ O ノ9 03‑′b/ lO

Water‑soluble paJt｢

Chromatographed on silica gel(0‑ 1 00% MeOH侶tOAc)

25‑3 5% MeOH/EtOAc EIuate
Chromatographed on sit ica get(upper phase of EtOAc‑water‑MeOH, 10:3:2)
1 'l1 1 l 1 2 2 つ▲ 2 つ一2 2 3

Fr.A‑5

Fr.A‑3[Chromatographed on ODS

Chromatographed on ODS(MeOH‑water, 3: I)(MeOH‑water, 3: 1)

Purified by HPLC[

(70‑ I 00% MeOH/water)

Compound 1 (34.7 mg)

2.23!! (14.5& 5.0)

2.25日(14.5 皮 5.0)

2.74日(13& 13)

2.75!! (12& 12)

2.54!! (13 &4.9)

2.58!! (13 &4.9)

3.70, 4.22 (10.5)

ca. 3.7, 4.3 (ca̲ 10)

Purified by HPLC

(67. 5‑ 1 00% MeOWwater)

Compound 2 (15.1 mg)

Fig. 1. The丘actionation scheme for isolation of the molluscicidal

S aPOnlnS ･1.06

I.06

0.96

0.95

1.3&

1.36

1.47

1.47

3.3l, 3.60 (7.0)

3.32, 3.61 (7.4)

1.08

1,07

0.98

0.98

1†

Qb>

2Jbsugar moi叫

3 ‑0‑Ara1

4.9‑5.0

1 仙名0.73.78机 1 3 4 つ一24 .91

円Ju

7

刺103.$

280.2

373.2

478.4榊563.9

3‑0‑GIc･ 1

Ok･ 1

nHJHU

1

Fig. 2. Structure of Compound I (DesglucoanagalIoside B)

5.48 (7.6)

1 0 7 7 7 7 ∠U 5 AV O I 名 2 2 2 3 5 1 75 47

7q104.8276.2

378.48+471.5578.2+662.7

3 ‑0‑GI c‑25.03 (5.0)

1日り

0 0 7 7 7 ′03 5 7 10 2 74 7 13 45糾nJH‑

ヽ′

5 7

1 10 070 7人V O7 73′4b06L 76707 7 8史V34 6 2 0 4 7tJ

234563‑0‑Xyl15.06 (7.7)

1 07 7 7 6 7 AV 7 0 7 7 3 0 7 5n川■■川U

ヽ′′▲U dZJH‑

ヽ′

5 00

7 523453‑0‑GIG‑3Ok‑ 15 11Fig. 3. Structure ofCompotmd 2 (AnagalIoside B)

7 9105.1

275.8

378.1書471.6NMR) spectra (Table I) indicated that compounds land 2
wereinclose agreement withthose of desglucoanagalloside

578.388

662.4

B (Fig. 2)andanagalloside B (Fig. 3) reported by Shoji et al.

The spectra were measured in d5･pyridine at 500 MHz for lH and 125 MTz
for 13C using TMS as an intemal standard,and expressed in ppm･

(7).

Ⅰ : Onlythe signals cle打1y assinable are shown, andthe values in parcthesis

In preliminary examinations, the two compounds ataTe coupling constants in Hz･

concentrations of 5 mg/I (ppm) were found to be lethal to

II: The signal corresponds to one of the methylene protons･

both types of snails. Further observations are sunmarizedin

書amd **: The signals may be interchangeable･

Table 2. Compound 1, called desglucoanagalloside B, wasfound to be active against B. glabrata snails at concentra‑18Table 2. Molluscicidal activityof purified two saponins Bom Anagalis anenst's on Biomphalaria glabrataand Oncomelania quadrasi
Biomphalaria glabrata10ppmDesglucoanagalloside B

10/10

Anagalloside B

10/10

Niclosamide

10/10

5ppm

2.5ppm

10/10

10/10

6/10

8/10

0/10

0/10

5/10

1/10

1/10

2.5ppm

1.25ppm

0.625ppm

10/10
10/10

1,25ppm

0.625ppm
1/10

0.3125ppm

Oppm

0/10

0/10

0/10

0/10

1/10

0/10

Oncome lanja quadwasi
10ppm

5ppm

0.3125ppm

Oppm

Desglucoanagalloside B

10/10

10/10

10/10

10/10

8/10

0/10

0/10

Anagalloside B

10/10

10/10

10/10

6/10

4/10

0/10

0/10

10/10

10/10

10/10

7/10

0/10

Niclosamidc

10/10

10/10

Number of dead snails爪Iumbcr of tested snails

tions ofO.625 ppmand over,and lethal to all tested snails at

C. T., El‑Kady, N.and Ahmed, L.(1 979): Changing pattem
ofschistosomiasisinEgypt 1935‑79. Lancet, no. 8136,

concentration of 2･5 ppm･ At concentrations of 1.25 ppm
and over, 100% of the tested 0. quadrasi snails were killed.

242‑244.

The molluscicidal activityof desglucoanagalloside B is
comparable tothat of the synthetic molluscicide, niclosamide.

3. Sturrock, R. F. (1 995): Current concepts of snail control.

Compound 2 (anagalloside B) might be less effective than

4. Perrett, S.andWhitfleld, P. J. (1996): Currently available

Mem. Inst. Oswaldo Cmz, 90, 241‑248.

compound l･ In the case of anagalloside B, the tested B.

molluscicides. Parasitol. Tわday, 12, 156‑1 59･

at concentrations of 5 ppmand 2.5 ppm, respectively.

5. E1‑Sayed, M. M., El‑Nahas, H. A. and Retihy, L.A. (1990):
A study on the molluscicidal activity of Calendula

It is apparentthat A. arvensis canbe a candidate mollusci‑cidal plant not onlyinEgypt but alsoinotherareas endemic
withschistosomiasis becausethis plant is widely distributed
komthe tropics tothe temperate reglOnS arOundthe world･jillferd. Egypt. J. Bilharz., 12, 207‑216.
6. World HealthOrganiZation ( 1 965): Memoranda: Mollus‑
cicide screenlng and evaluation. Bull. WHO, 33, 567‑

glabrataand O･ quadlasi snails showed 100% mortalityrates

micrantha (OfrlCinals), Anagallis arvensis and Agave

581.
7. Shoji, N., Umeyama, A., Yoshikawa, K.andA血ara, S･REFERENCE S(1994): Structures ofAnagallosaponins I ‑ Vand their

1. World HealthOrganiZation (1993): The control ofschis‑tosomiasis. WHO Tech. Rep. Ser., no. 830.
2. Abdel‑Wahab, M. F., El‑Sahly, A., Zakaria, S., Strickland,

companion substances from AnagalL.S arVeWis L･ Chem･Pham. Bull., 42, 1750‑1755.

19