Secondary Metabolites as Stimulants and Antifeedants of Salix integra for the Leaf Amir Reza Jassbi* Laboratory of Ecological Chemistry, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan. Fax: +81-11-706-4281. Email. [email protected] * Corresponding Address: Department of Phytochemistry, Medicinal Plants Research Institute, Shahid Beheshti University, Evin, Tehran, Iran. Fax: +98-21-2418679. Email. [email protected] Z. Naturforsch. 58c, 573Ð579 (2003); received January 30, 2003 Plagiodera versicolora, a willow beetle living on S. sachalinensis, is found on S. integra during early June in Hokkaido Island, Japan. This selects several species of willows (Salix), including S. integra as host plant in Honshu Island of Japan. To determine the reasons for the limited distribution of this beetle on the willows of Hokkaido, the feeding preference of the insect to leaves of S. integra and its constituents was performed. Feeding-bioassay guided fractionation of an 80% aqueous acetone extract of fresh leaves of Salix integra to Plagiodera versicolora resulted in isolation of feeding stimulant and antifeeding constituents. Chlorogenic acid (1) and 3,5-dicaffeoyl quinic acid (2) were identified as antifeedants and 1,2-di[(9Z,12Z,15Z)-octadeca-9,12,15-trienoyl]-3--D-galactopyranosyl-sn-glycerol (MGDG, 3) as feeding stimulants. The feeding test was performed by an agar disk method. The treated agar disks contained sucrose and test sample in different doses. The antifeeding activities of 1 and 2 and stimulant activity of 3 may be one of the reasons for the limited presence of P. versicolora on S. integra in Hokkaido. Key words: Salix integra, Plagiodera versicolora, Chrysomelidae

Introduction plant, during early June the number of adult in- sects increased on S. integra for a few days and Plagiodera versicolora Laicharting is a willow again decreased at the end of the season. beetle found on Salix sachalinensis, in Ishikari The feeding behavior of P. versicolora on dif- near Sapporo, Hokkaido, Japan. The body of the ferent species of Salix on Honshu Island suggested adult beetle is about 3Ð4 mm long with a coloring salicin, populin and luteolin-7-glucoside as feeding of shiny steel blue. Ishihara et al. (1999) investi- stimulants (Matsuda and Matsuo, 1985). Even gated the life cycle of Plagiodera versicolora. Two chlorogenic acid, which has antifeeding activity for generations were reported for this insect in Hok- some willow , was found in the leaves of S. kaido, while multivoltine cycle (they complete 5Ð integra to be a feeding stimulant for P. versicolora 6 life cycle per year) was reported in Honshu Is- (Matsuda and Senbo, 1986). land of Japan (Kimoto and Takizawa, 1994). Ishi- The quality and chemical constituents of leaves hara et al. (1999) described several reasons for this of different Salix species as food for chrysomelidae finding, “why the willow beetles select S. sachali- leaf beetles were studied thoroughly (Ikonen, nensis as their host plant”, as follows: (1) it may 2001). The palatability of leaves of some salica- be caused by high quality of S. sachalinensis as ceous plants (Populus and Salix species) for leaf food. (2) The bivoltine cycle in the field may be beetles of chrysomelidae family, including P. versi- maintained by a high growth rate on S. sachali- colora showed that the younger leaves are con- nensis. (3) Predation pressure on S. integra may sumed more (Ikonen, 2002). Secondary metabo- cause that P. versicolora avoid oviposition on this lites such as phenolic glycosides including salicin plant. and related compounds, chlorogenic acid and con- Studies on the life cycle of P. versicolora in Hok- densed tannins exist in higher concentration in kaido showed that besides the continuous pres- younger leaves. Despite the defensive role of the ence of the insect on S. sachalinensis as its host secondary metabolites, the beetles generally prefer

0939Ð5075/2003/0700Ð0573 $ 06.00 ” 2003 Verlag der Zeitschrift für Naturforschung, Tübingen · www.znaturforsch.com · D 574 A. R. Jassbi · Secondary Metabolites as Stimulants and Antifeedants the younger leaves to the older ones (Ikonen, Material and Methods 2002). The analysis of the leaves of several willows General procedure showed high contents of nitrogen in the younger leaves of the plants and hence more nutrients for 1H and 13C NMR spectra (broad band and DEPT the beetles (Ikonen, 2002). The effect of phenolic expts.) were measured on a JEOL, JNM-EX270 in- secondary compounds including salicin and re- strument. MS spectra were recorded on a JEOL lated glucosides and chlorogenic acid on feeding JMS-SX102A spectrometer. Optical rotations were preference of Agelastica alni was inves- determined on a JASCO DIP-370 digital polarime- tigated (Ikonen et al., 2002). Agelastica alni is an ter. Analytical TLC experiments were performed oligophagous leaf beetle utilizes both alders and on Merck silica gel 60 F254, DIOL F254 HPTLC and willows. Alnus incana is its preferred host plant. RP-18 WF254 HPTLC pre-coated glass plates. In addition S. phylicifolia and S. caprea were con- HPLC was performed on a Hitachi l-6000 with an sumed moderately but this beetle rejected other l-4200H UV-vis detector and a Hitachi D-2500 willows (S. pentandra and S. myrsinifolia). The Chromato-integrator, Tokyo, Japan, using a semi- presence of chlorogenic acid and a salicylate-type preparative RP-18 (Prep-ODS Inertsil 6.0 ¥ phenolic glucoside, salicin, inhibited the feeding of 250 mm, GL Science, Tokyo, Japan) column. A. alni in the bioassay with pure phenolic com- pounds. High chlorogenic acid and salicylate Leaf beetles content of the rejected willows leaves explain their Plagiodera versicolora were caught at the adult poor palatability. In contrast a feeding stimulant stage on S. sachalinensis and reared on leaves of the in the leaves of the host plant, A. incana that over- same plant. The rearing and all the feeding tests comes the deterrent activities of the above-men- were performed in a growing chamber, NK system tioned phenolic compounds has been proposed controller Biotron NC 220 (Nippon Medical and (Ikonen et al., 2002). Chemical Co. LTD., Osaka, Japan) at 21Ð23 ∞C, rel- My objective was to determine the possible ative humidity of 65% and 16 h light and 8 h dark. chemical compounds responsible for the reduced tendency of P. versicolora to select S. integra in Chemicals Hokkaido as its host plant. Also the question was addressed, why the beetles live on S. integra in the Chlorogenic acid (1) was purchased from Aldrich field in the beginning of the season while its host Chemical Company. It was also purified from the plant is S. sachalinensis. leaves of S. integra. 3,5-Dicaffeoyl quinic acid (2)

HO CO2H HO CO H O 2 O O HO O OO OH OH OH HO OH OH OH OH

Chlorogenic acid (1) 3,5-Dicaffeoyl quinic acid (2)

OH

OH OH OH H O H HO O HO O OH H OH CH2 H H CHOR OH CH OR 2 OH O Fig. 1. Antifeeding and feeding stimulant R=CO(CH ) CH=CHCH CH=CHCH CH=CHCH CH constituents from S. integra (1Ð3) and S. 2 7 2 2 2 3 Ampelopsin (4) sachalinensis (4). A. R. Jassbi · Secondary Metabolites as Stimulants and Antifeedants 575 and 1,2-di[(9Z,12Z,15Z)-octadeca-9,12,15-trienoyl]- centrated in reduced pressure to give ca. 110 g 3--d-galactopyranosyl-sn-glycerol (MGDG, 3) of residue, which was partitioned between water were extracted and purified from the leaves of S. in- and ethyl acetate. The ethyl acetate fraction was tegra. Ampelopsin (4) was purified previously from divided into acidic (42 g) and neutral fractions S. sachalinensis (Fig. 1) (Matsumoto and Tahara, (15 g) by partitioning between ethyl acetate and

2001). 5% NaHCO3 followed by neutralization of the latter layer by 2N HCl solution and extraction Agar disk bioassay with ethyl acetate. The bioassay procedure was based on counting Purification of the water-soluble part of the the agar disk area consumed by the adult beetles aqueous acetone extract of S. integra (Matsumoto, 2000). The agar mixture was made by mixing of 3.25 g sucrose, 0.75 g agar, and 25 ml The water layer fraction of the aqueous acetone de-ionized water and heating to make a clear solu- extract (33.0 g out of 51.5 g residue) was subjected tion. The hot liquefied agar was poured into the to Sephadex LH20 (200 g) column chromatogra- bigger lid of a Petri dish and immediately the phy using aqueous methanol (from 70% to 100% smaller lid was used to press the agar solution. The methanol v/v). The fractions 2 and 3 (70% metha- thickness of the agar layer was adjusted by putting nol, 8.8 g) contained mostly two compounds which “ three pieces of Φ 0.3 mm lead pencil as a triangle were separated on a LiChroprep NH2 (40Ð µ between two lids of the Petri dish before adding 63 m) column using CH3CN and increasing the the agar solution. After a few min the agar layer polarity up to 30% aqueous CH3CN. They were was solidified and it was cut to 1 cm circles. Two identified as fructose and sucrose (50:50) by com- agar disks (Φ 1 cm) were put on inner side of big- parison of FAB-MS and 13C NMR spectral data ger lid of an Φ 6 cm Petri dish and an Φ 55 mm and Co-TLC with authentic samples on silica gel wet filter paper was put on the inner side of the thin layer plates in CHCl3:CH3OH: H2O: HOAc smaller lid of the dish to maintain the moisture in (5:3:1:1 v/v/v/v), and in the same system replacing the vessel. One of the agar disks was treated with CHCl3 by EtOAc. The rest of the fraction was a 10-µl solution of the test compound in acetone predominant in chlorogenic acid (1), which was or 80% acetone in water and another with only identified by Co-TLC on silica gel thin layer solvent as the control. Five 1 to 7-day old adult plates (eluent: CHCl3:CH3OH:H2O:HOAc, 5:3:1:1 beetles were released inside the dish. The beetles v/v/v/v) in comparison to an authentic sample. were starved between 20 to 24 h before test. The FAB mass spectrum, 1H and 13C NMR spectral area of the consumed agar disk was calculated by data confirmed the structure of 1. counting the square units of a haemacytometer, which was placed under the agar disks as the back- Identification of the constituents of the acidic layer ground. The percentage of the feeding index was of the aqueous acetone fraction calculated by the following equation: % Feeding The HPLC chromatogram of the acidic fraction, index = (TÐC/T+C) ¥ 100. Where T is the number recorded using an RP HPLC using MeOH:H2O: of the consumed squares of the treated agar disk HOAc (40:60:2 v/v/v) showed two main com- and C is that for the control disk. The positive and pounds 1 and 2 at 3.92 min and 7.92 min, respec- negative sign of the feeding index indicates the tively. The UV detector was set at 326 nm and the feeding stimulant and antifeeding activities, res- flow rate was 1.5 ml/min. The major compounds pectively. were identified as chlorogenic acid (1) and 3,5-di- caffeoyl quinic acid (2) by comparison of FD-MS, Isolation of the chemical constituents of S. integra 1H and 13C NMR and CD spectral data with those The leaves (515 g fresh), of S. integra, collected reported in the literature (Chuda et al., 1996; Bas- from Atsubetsu in July 2001 in Sapporo, were net et al., 1996). extracted with 80% aqueous acetone. The ex- traction was done twice for two weeks. The aqueous acetone extract of the leaves was con- 576 A. R. Jassbi · Secondary Metabolites as Stimulants and Antifeedants

Constituents of the aqueous methanol extract of the 270 m Hz): 5.39 (m, 12H), 4.40 (dd, J = 3.2, 11.9 Hz), leaves of S. integra 4.28 (d, J = 11.9 Hz), 4.20 (dd, J = 6.9, 11.9 Hz), 4.01 (brd), 3.95 (dd, J = 5.9, 12.2 Hz), 3.88 (d, J = 5.7 Hz, The fresh leaves (650 g) of S. integra collected 2 H), 3.74 (dd, J = 6.2, 11.3 Hz), 3.67 (d, J = 9.5 Hz), in October 2000, once extracted with ethyl acetate 3.60 (m, 2H), 2.80 (m, 8H), 2.31 (m, 4H), 2.10 (m, were soaked in 70% methanol in water for one 8H), 1.60 (m, 4H), 1.30 (m, 16 H), 0.97 (t, J = 7.5 Hz, week. Evaporation of the aqueous solvent under 6H). 13C NMR (CDCl , 67.5 m Hz): 173.5, 173.1, vacuum resulted in a brown syrup, which was then 3 131.7, 129.9, 128.1, 127.9, 127.5, 126.8, 103.9, 73.4, dissolved in ethyl acetate (13 g) and the residue 70.9, 70.0, 68.7, 67.8, 62.8, 61.2, 34.2, 34.0, 29.7, 29.6, was dissolved in water (48 g). The ethyl acetate 29.2, 29.1, 29.1, 29.0, 27.1, 25.6, 25.5, 24.8, 20.5, 14.3. soluble part of the extract was subjected to silica gel CC using chloroform with increasing amounts of methanol (0Ð100%). The fractions checked by Bioassay-guided fractionation of the 80% TLC, revealed that the less polar fractions were acetone extract of the leaves of S. integra identical with those in the hexane soluble part of The willow beetles were subjected to agar disk the first ethyl acetate extract. The latter fractions feeding tests using different fractions of the aque- 11Ð13 (3.45 g) contained mostly the glycolipids. ous acetone extract of S. integra. The weight of the The major compound was purified by repeated crude aqueous acetone extract from each Φ 1cm flash silica CC using chloroform: ethyl acetate: ac- disk of the leaves (10Ð15 mg) was about 1Ð2mg. etone: methanol (3:1:1:0.2 v/v/v/v). The structure The above-mentioned amount (1Ð2 mg) of the ex- was determined using HRFAB-MS, 1H and 13C tract was charged on each agar disk (Φ 1 cm). The NMR spectroscopy as 1,2-di [(9Z,12Z,15Z)-octa- aqueous acetone extract of the leaves of S. integra deca-9,12,15-trienoyl]-3--d-galactopyranosyl-sn- collected in August 2001 (220 mg) was partitioned glycerol (3, MGDG) (Adebodun et al., 1992; Cod- between water/ether and water/hexane separately dington et al., 1981). and each of the ethereal (40 mg) and hexane (46 mg) fractions was used for the feeding tests. Spectral data and physical constants for com- The bioassay showed antifeeding for the water pounds 1Ð3 isolated from Salix integra (Ð 30%) and ether (Ð 26.5%) extract while the Chlorogenic acid (1): FAB- MS: 353 (M-1)Ð. feeding of the beetles were stimulated (7.4%) by 25 1 [α]D Ð 16.6∞ (c, 0.6 in CH3OH). H NMR (CD3OD, charging the hexane fractions on the agar disks. In 270 m Hz): δ 7.54 (d, J = 15.9 Hz), 7.04 (s), 6.93 (d, order to find the active constituents, the hexane J = 7.7 Hz), 6.77 (d, J = 7.7 Hz), 6.26 (d, J = fraction (88 mg) was subjected to a silica gel flash 15.9 Hz), 5.34 (m), 4.16 (br s), 3.72 (brd, J = 7.5 Hz), column chromatography using hexane, with 13 2.0Ð2.20 (m, 4H). C NMR (CD3OD, 67.5 m Hz): increasing polarity up to pure ethyl acetate. The δ 175.2, 168.7, 149.5, 147.0, 146.7, 127.7, 122.9, 116.4, fractions containing compound 3 (5 mg) were the 115.3, 115.2, 73.7, 72.1, 71.7, 39.1, 38.3. stimulant active ones. The major constituents of 3,5-dicaffeoyl quinic acid (2): FAB- MS: 515 ether fraction and water layer were compounds 2 Ð 25 1 (M-1) .[α]D Ð 81.4∞ (c, 0.7 in CH3OH). H NMR and 3, by separation on an RP 18 reverse phase (CD3OD, 270 m Hz): δ 7.52 (d, J = 15.9 Hz), 7.48 (d, TLC using 1% acetic acid in 40% solution of J = 15.9 Hz), 6.96 (s, 2H), 6.85 (d, J = 8.3 Hz), 6.68 methanol in water as the mobile phase. The (d, J = 8.3 Hz), 6.24 (d, J = 15.9 Hz), 6.16 (d, J = amount of 2 and 3 separated from 26 mg of the 15.9 Hz), 5.30 (m, 2H), 3.89 (dd, J = 3.3, 7.5 Hz), 80% aqueous acetone extract was 6 and 3 mg, 13 2.0Ð2.30 (m, 4H). C NMR (CD3OD, 67.5 m Hz): respectively. The pure compounds, 1, 2, 3 and 4 δ 177.4, 168.8, 168.4, 149.3, 149.2, 147.2, 147.0, 146.4, were used in different doses in the agar disk bioas- 127.7, 127.5, 123.0, 122.9, 116.4, 115.4, 115.2, 115.1, say to evaluate their effect on the feeding behavior 114.9, 74.7, 72.5, 71.8, 70.7, 37.7, 35.9. of P. versicolora. 1,2-di[(9Z,12Z,15Z)octadec-9,12,15-trienoyl]-3-- After performance of the feeding tests the re- d-galactopyranosyl-sn-glycerol] (MGDG, 3): FD sults were analyzed statistically. The average, stan- MS: 797 (M+Na)+, 775 (M+H)+, 612 (M-162)+. dard deviation and standard errors of the means 25 1 [α]D + 2.3∞ (c, 6 in CHCl3). H NMR (CDCl3, were calculated for each experiment with at least A. R. Jassbi · Secondary Metabolites as Stimulants and Antifeedants 577

40 80 70 20 60 50 0 3,5-dicaffeoyl quinic 40 acid MGDG -20 30 Chlorogenic acid Ampelopsin 20 -40 10 % FEEDING INDEX % FEEDING INDEX 0 -60 -10 -80 -20 50 250 500 25 50 100 250

Fig. 3. Feeding stimulant activity of MGDG (3): dose: mean value of feeding index (standard error of the mean value) [25 µg: 13.6 (17.5), 50 µg: 28.7 (15.3), 100 µg: 29.3 30 (18.4), 250 µg: 33.0 (15.1)] isolated from S. integra and ampelopsin (4) [25 µg: 5.3 (13.4), 50 µg: 57.9 (17.3), 20 100 µg: 37.5 (20.9), 250 µg: 23.5 (14.4)] from S. sachali- nensis at different doses (µg). The vertical bars shows 10 standard errors of the mean values, obtained from 6 rep- licates (n = 6). 0 MGDG

-10 ing to Fig. 2, compounds 1 and 2 showed antifeed- % FEEDING INDEX -20 ing activities above 50 µg per agar disk. The deter-

-30 rent doses were consistence with the concentration 10 20 35 50 of these substances in the leaf of the plant based on the same area as for the agar disk. Fig. 2. The results of feeding tests with different doses The feeding stimulant activity of ampelopsin (4), (µg) of compounds 1 and 2 (upper part) and 3 (lower the major flavonoid of S. sachalinensis (Matsu- part) charged on each agar disk. The vertical bars shows moto and Tahara, 2001; Matsumoto, 2000) to P. standard errors of the mean values, obtained from 6 rep- licates (n = 6). Dose: mean value of feeding index (stan- versicolora, is compared to that of MGDG (3) dard error of the mean value) for 1 [50 µg: 11.5 (10.6), from S. integra (Fig. 3). Ampelopsin has been re- µ µ µ 250 g: Ð 4.2 (2.6), 500 g: Ð 17.8 (10.9)], 2 [50 g: ported as an abundant flavonoid in S. phylicifolia Ð 19.8 (16.4), 250 µg: Ð 25.5 (4.1), 500 µg: Ð 39.8 (30.8)] and 3 [10 µg: Ð 9.6 (11.2), 20 µg: 11.3 (1.4), 35 µg: 8.5 leaves but with no strong antiherbivore activity (4.5), 50 µg: 20.0 (6.9)]. against A. alni (Ikonen, 2001). The presence of a feeding stimulant (3) in the leaves of S. integra may overcome the antifeeding effect of other com- six replicates for each dose (Lowry, 1998Ð2003). pounds (1 and 2) so that the beetles consume the The analytical results of the tests are presented in leaves (Ikonen et al., 2002). This can be a reason Figs. 2 and 3. why the beetles still consume the leaves of S. in- tegra. When the leaves of S. integra and S. sachali- Results and Discussion nensis were exposed to willow beetles, although The results of the tests showed antifeeding activ- the growing diet for the beetles was the leaves of ity for chlorogenic acid (1) and 3,5-dicaffeoyl S. sachalinensis, the beetles ate the young leaves quinic acid (2) (Fig. 2) in the range of 50Ð500 µg of both willows equally. They did not consume the per agar disk. The higher activity of compound 2 older (harder) leaves. This might be due to high in comparison to 1 may be due to the incorpora- quality or existence of more nutrient constituents tion of another caffeoyl moiety in position 5 of 2. in the younger leaves (Ikonen, 2002). To the best of my knowledge, compound 2 is re- S. sachalinensis is the major host plant of P. ver- ported as an antifeeding agent to P. versicolora sicolora in Sapporo area while in the Honshu Is- isolated for the first time from S. integra. Accord- land several species of willow were found to be 578 A. R. Jassbi · Secondary Metabolites as Stimulants and Antifeedants the host plants for P. versicolora (Matsuda and vior of P. versicolora in different climates. Besides Matsuo, 1985; Matsuda and Senbo, 1986). This the defensive role of chlorogenic acid to phytopha- might be due to the presence of some stimulants gous , it shows a positive or negative effect on such as salicin and its derivative populin or the growth of different insects (Todd et al., 1971; flavonoid glucoside, luteolin-7-glucoside in the Chawla et al., 1974; Matsuda and Senbo, 1986). I ob- leaves of different Salix species. The feeding stim- served faster growth and less mortality for the wil- ulant activity of chlorogenic acid in Salix integra low beetles using the leaves of S. sachalinensis of Honshu Island, to P. versicolora might be the (prominent in ampelopsin) instead of S. integra result of the adaptation of the insects to use this (prominent in chlorogenic acid) as the rearing diet. compound in Honshu Island (Matsuda and The presence of two kinds of stimulants and Senbo, 1986). antifeedants in the leaves of S. integra may lead Two chemical races for S. sachalinensis have us to assume that the presence and absence of P. been reported so far (Mizuno et al., 1991). How- versicolora on S. integra might be due to a balance ever we could not detect significant differences in of these compounds in different seasons. To con- the chemical constituents of the leaves of different firm this phenomenon the leaves of S. integra clones of S. integra collected from various places should be quantitatively analyzed for compounds of Sapporo (Shinkawa, Atsubetsu and Ishikari). 1, 2 and 3 from May to September. Chlorogenic acid is determined as the major active constituent in both Sapporo and Sendai specimens Acknowledgments of S. integra (Matsuda and Senbo, 1986). Some se- I am grateful to Prof. Dr. Satoshi Tahara for his condary metabolites such as salicin and related kind support of this project and his advice and en- phenolic glycosides have both feeding deterrent couragement during my postdoctoral course. I am activity for some non-adapted insects and feeding thankful to the “Japan Society for the Promotion stimulant activity for some adapted insects (Mat- of Science” for granting the post-doctoral fellow- suda and Senbo, 1986; Ikonen, 2002). The present ship. My thanks are expressed to Dr. Eri Fukushi paper reports chlorogenic acid as a compound, and Mr. Kenji Watanabe for their skilful measur- which has two opposite effects on the feeding beha- ing the NMR and MS spectra. A. R. Jassbi · Secondary Metabolites as Stimulants and Antifeedants 579

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