Chiang Mai J. Sci. 2006; 33(1) : Contributed Paper

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Chiang Mai J. Sci. 2006; 33(1) : 95-101 www.science.cmu.ac.th/jurnal-science/jsci.

1 Chiang Mai J. Sci. 2006; 33(1) : Cntributed Paper Islatin and Characterizatin f rassinlide and Castasterne in the Pllen f Pumpkin Chalev Pachthng[a,b], Dumrat Supyen[a], Duang uddhasukh*[a] and Araya Jatisatienr[c] [a] Department f Chemistry, Faculty f Science, Chiang Mai University, Chiang Mai 50200, Thailand. [b] Chemistry Prgram, Department f Science, Faculty f Science and Technlgy, Kanchanaburi Rajabhat University, Kanchanaburi 71000, Thailand. [c] Department f ilgy, Faculty f Science, Chiang Mai University, Chiang Mai 50200, Thailand. *Authr fr crrespndence, duang@chiangmai.ac.th Received : 15 September 2005 Accepted : 29 September 2005 ASTRACT The tw brassinsterids cntained in the pllen f pumpkin (Cucurbita mschata Duch.) were investigated. The biactive cmpunds frm the purified extract were identified by GC-MS as brassinlide and castasterne. The cntent f brassinlide and castasterne were estimated t be 36 µg kg -1 and 112 µg kg -1 (dry weight) respectively. This study prvides the first evidence fr the ccurrence f brassinlide and castasterne in the pllen f pumpkin. Keywrds: brassinlide, brassinsterids, castasterne, Cucurbita mschata, pumpkin, rice lamina inclinatin 1. INTRDUCTIN rassinsterids (Rs) are steridal plant hrmnes, which are essential fr plant grwth and develpment [1-4]. Since brassinlide (L) was islated frm rape pllen (rassica napus) [5, 6] as the first steridal plant grwth regulatr, the intensive research n the islatin and characterizatin f active substances related t L has been cntinued [7-10]. The Rs have been detected in many higher plants and sme lwer plants [11-14]. Therefre, the Rs appear t be ubiquitus in the plant kingdm. At present mre than 50 cmpunds f natural ccurrence f Rs have been characterized frm varius plant surces [15-17]. In this present wrk, we investigated the natural Rs in the pllen f pumpkin. It is a by-prduct cllected frm the stamina f pumpkin flwers during the time f pumpkin harvesting. The apprximate harvest f the pllen was abut kg per acre (fresh weight). In this study, we have islated and characterized brassinlide and castasterne (CS) in the pllen f pumpkin, Mun-Sek Jang et al.[18] have islated and characterized brassinlide, castasterne and their bisynthetic precursrs frm the seeds f pumpkin, but the ccurrence f brassinlide and castasterne in the pllen has never been previusly reprted. 2. EXPERIMENT XPERIMENTAL 2.1 General Data The GC-MS analysis was carried ut with the fllwing cnditin: Autmass (JMS- AM150) mass spectrmeter cnnected with a Hewlett packard gas chrmatgraph, EI (70 ev), surce temperature 280 C, clumn HP-1MS (HP19091S-933, 30 m x 250µm, 0.25 µm film thickness), injectin temperature 275 C clumn temperature

2 96 Chiang Mai J. Sci. 2006; 33(1) prgrammed 200 C fr 2 min, then raised t 280 C at rate f 20 C min -1 and held n this temperature fr 25 min; interphase temperature 275 C, carrier gas He, flw rate 1.0 ml min -1 split injectin. Clumn chrmatgraphy was carried ut n silica gel (rck; mesh) and Sephadex LH-20 (Amersham isciences) and the RP-18 cartridge (1 g, rck; Lichlut RP-18) fr cleaning up the biactive residues. The authentic brassinlide and castasterne were purchased frm Kent Chemical C., Ltd. (Japan). The ther chemicals were analytical grade and all the cmmercial slvents used fr the extractin were freshly distilled befre using. 2.2 Plant Material The pllen f pumpkin was cllected frm Mae-Ai district, Chiang Mai prvince Thailand. A vucher specimen f this plant (Pachthng N.24) is depsited at CMU Herbarium, Department f ilgy, Faculty f Science, Chiang Mai University, Chiang Mai, Thailand. The stamina f pumpkin (37.8 kg, fresh weight) were cllected frm flwers f pumpkin, and drying at rm temperature t give dried stamina (9.52 kg), which was sieved thrugh a 100 mesh screen, whereby the dried pumpkin pllen (2.76 kg) was btained. 2.3 iassay The rice lamina inclinatin biassay was carried ut t guide the islatin and purificatin f the active cmpunds frm the crude extract, using the rice (ryza sativa L.) cultivar f RD-7 as described previusly [19]. The rice seeds were btained frm the Multiple Crpping Center f Chiang Mai University, Chiang Mai, Thailand. 2.4 GC-MS Analysis The bismathanebrnatin was carried ut by treatment f the biactive cmpunds and authentic samples with 20 µl f pyridine cntaining f methylbrnic acid (2 mgl -1 ) at 70 ºC fr 30 min; 1µL was subjected t GC-MS analysis [20]. 2.5 Extractin and Purificatin The dried pllen (2.76 kg) was extracted with methanl (3 x 4 L), by stirring with mechanical stirrer at rm temperature fr three days. The methanl extract was evaprated t dryness in vacu belw 30 C. The residue ( g) was partitined between H 2 (1.5 L) and CHCl 3 (1.5 L), and the separated aqueus phase was partitined twice with CHCl 3 (2 x 800 ml). The cmbined CHCl 3 phase was dried ver anh.na 2 S 4, and evaprated t dryness. The residue ( g) was divided int prtins fr qualitative and quantitative analysis. The first prtin ( g) was partitined between n-hexane (1 L) and 80% H (1 L). The n-hexane phase was partitined twice with 80% H (2x400 ml), and the cmbined 80% H fractins were cncentrated t expel methanl. The resulting aqueus phase frm 80% H was added with sat.nahc 3 slutin (100 ml), then extracted with CHCl 3 (500 ml), and the aqueus phase after separatin f CHCl 3 was partitined twice with CHCl 3 (2x200 ml), and the cmbined CHCl 3 phase after drying with anh.na 2 S 4 was evaprated t dryness. The residue (32.31 g) frm the CHCl 3 phase was chrmatgraphed n silica gel clumn (400 g, rck). The elutin was carried ut stepwise with (1 L) 10 fractins f H in CHCl 3 gradients (0, 3, 5, 10, 15, 20, 25, 30, 40 and 50%). The resulting residue (3.09 g) frm 10 and 15% H fractins was chrmatgraphed n silica gel clumn (100 g, rck). The elutin was carried ut stepwise with (300 ml) 11 fractins f H in CHCl 3 gradients (0, 2, 3, 4, 5, 7, 10, 15, 20, 30and 50%). The resulting residue frm 5 and 7% H fractins (1.52 g) was subjected t RP-18 clumn chrmatgraphy (bed vlume 100mL). The elutin was carried ut with H in H 2 t give 11 fractins (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100%). The cmbined biactive residue (241.4mg) frm 80 and 90% H was subjected t

3 Chiang Mai J. Sci. 2006; 33(1) 97 Sephadex LH-20 clumn (bed vlume 200 ml). The elutin was carried ut with 25% H in CHCl 3 with flw rate 0.5 ml min -1 t give a bilgically active fractin (Ve/Vt ). After evapratin, the residue (13.52 mg) was charged nt RP-18 cartridges (1 g) and eluted with 80% H-H 2. The biactive residue (2.61 mg) was btained, the aliqut f which was derivatized with methylbrnic acid, by treatment with 20 µl pyridine cntaining methylbrnic acid (2 mg ml -1 ) at 70 C fr 30 min; 1 µl was subjected t GC-MS analysis. The purificatin f the secnd prtin (137.16g) was carried ut similarly as described. The resulting purified sample was subjected t quantitative analysis perfrmed by GC-MS cmpared with authentic brassinlide and castasterne (as bismethanebrnate derivatives). 3. RESUL ESULTS AND DISCUSSIN The dried pllen f pumpkin was extracted with methanl. After evapratin t dryness, the residue was partitined between water and chlrfrm. nly the residual chlrfrm phase shwed bilgical activity by rice lamina inclinatin test after evapratin under reduced pressure t dryness. The residue resulting frm the chlrfrm phase was partitined between n-hexane and 80% methanl. The n-hexane phase did nt shw bilgical activity and 80% methanl fractin was evaprated t cncentrate. The remaining aqueus phase after adding saturated sdium hydrgen carbnate slutin was extracted with chlrfrm. The active residue frm the chlrfrm phase was chrmatgraphed n silica gel clumn, eluting with methanl in chlrfrm gradients. The cmbined highly biactive residue frm the elutin with 5 and 7% methanl in chlrfrm was purified by silica gel clumn, RP-18 clumn, Sephadex LH-20 clumn chrmatgraphies and finally with RP-18 cartridges. The biactive residue was derivatized with methylbrnic acid and analyzed by GC-MS. ased n the GC retentin time (Figure1) and mass spectra(table 1. and Figures 2, 4) f the bismethanebrnate (M) f the biactive cmpund I (R t =29.57 min) it was shwn t be identical t authentic castasterne bismethanebrnate (M-CS) under the C A R t (min) Figure1. Chrmatgram f the biactive cmpunds frm the extract f pllen f pumpkin as M (A), ure1. authentic Chrmatgram M-L f () the and biactive authentic cmpunds M-CS frm (C) analyzed the extract by f GC-MS. pllen pumpkin as M (A), authentic M-L () and authentic M-CS (C) lyzed by GC-MS.

4 98 Chiang Mai J. Sci. 2006; 33(1) Table 1. Characteristic ins f brassinsterids analyzed by GC-MS Cmpunds * R t n GC (min) Characteristic ins (, relative intensity) I (100), 287(29), 327(13), 358(36), 399(14), 441 (11), 512(84) II (100), 163(38), 177(70), 332(49), 374(53), 457(8), 528(9) rassinlide (100), 163(33), 177(76), 332(46), 374(52), 457(8), 528(7) Castasterne (100), 287(34), 327(10), 358(35), 399(13), 441(9), 512(78) * The cmpunds as M were analyzed by full scan GC-MS Cmpund I as M (R t = 29.57) Authentic M-CS (R t = 29.57) Figure 2. Mass spectra f the biactive cmpund I frm the extract f pllen f pumpkin as M and authentic M-CS.

5 Chiang Mai J. Sci. 2006; 33(1) 99 Cmpund II as M (R t = 35.25) Authentic M-L (R t = 35.25) Figure 3. Mass spectra f the biactive cmpund II frm the extract f pllen f pumpkin as M and authentic M-L. 374 (373.H) (357.H) H 332 H 287 M-L M--CS Figure 4. The MS fragmentatin pattern f M-L and M-CS [20].

6 100 Chiang Mai J. Sci. 2006; 33(1) same cnditin. Thus, the active cmpund I was established t be CS. The M f active cmpund II (R t =35.25 min) (Figure1) shwed the characteristic ins fr brassinlide bismethanebrnate (M-L) at 528(M + ), 457, 374, 332, 177,163 and 155. This is cnsistent with the retentin time and mass spectra fingerprint (Table1 and Figures 3, 4) shwn by L. Their apprximate amunts were estimated based n the GC-MS data t be 112 and 36 µg kg -1 (dried weight) fr CS and L respectively. Thus, t cnclude, the partially purified extract frm pllen f pumpkin was fund t have a high cntent f Rs and it shwed high bilgical activity, s it culd pssibly be used as an inexpensive surce f these natural plant hrmnes fr agriculture. ACKNWLEDGEMENTS This wrk was supprted by the Secndary Educatin Quality Imprvement Prject f RIC and partially funded by the Graduate Schl f Chiang Mai University. We are grateful t Mr. Pisan Kitsawatpaibn, Faculty f Science, Chiang Mai University, fr perfrming the GC-MS analysis. Finally, we are very grateful t Mr. Anan Kunnang fr helping cllected the sample. REFERENCES [1] Cluse S.D., and Sasse J.M., rassinsterids: essential regulatins f plant grwth and develpment, Ann. Rev. Plant Physil., Plant Ml. il, 1988; 49: [2] Mandava N.., Plant grwth-prmting brassinsterids, Ann. Rev. Plant Physil. Plant Ml. il., 1988; 39: [3] Mussig C., and Altmann T., rassinsterid signaling in plants, Trends Endcrinl. tab., 2001; 12: [4] Ykta T., Chemical structure f plant hrmnes, Chem. Regul. Plants, 1995; 30: [5] Grve M.D., Spencer G.F., Rhwedder W.K., Mandava N., Wrley J.F., Warthen Jr. J.D., Steffens G.L., and Flippen- Andersn J.L., Ck Jr. J.C., rassinlide, a plant grwth-prmting sterids islated frm rassica napus pllen, Nature, 1979; 281: [6] Mitchell J.W., Mandava N.., Wrley J.F., Plimmer J.R., and Smith M.V., rassins: a new family f plant hrmnes frm rape pllen, Nature, 1970; 225: [7] Friebe A., Vlz A., Schmidt J., Vigt., Adam G., and Schnabl H., 4-Episecasterne and 24-epi-castasterne frm Lychnis viscaria seeds, Phytchemistry, 1999; 52: [8] Fujika S., Nguchi T., Ykta T., Takatsut S., and Yshida S., rassinsterids in Arabidpsis thaliana, Phytchemistry, 1998; 48: [9] Ykta T., Arima M., and Takahashi N., Castasterne, a new phytsterl with plant-hrmne ptency, frm chestnut insect gall, Tetrahedrn Lett., 1982; 23: [10] Ykta T., aba J., and Takahashi N., rassinlide-related biactive sterls in Dlichs lablab; brassinlide, castasterne and a new analg, hmdlichlide, Agric.il. Chem., 1983; 47: [11] Abe H., Mrishita T., Uchiyama M., Takatsut S., Ikekawa N., Ikeda M., Sassa T., Kitsuwa T., and Marum S., ccurrence f three new brassinsterids: brassinne, 24(S)-24-ethylbrassinne and 28-nrbrassinlide in higher plants, Experientia, 1983; 39: [12] Kim Y.S., Sup Y.H., Kim T.W., J S.H., and Kim S.K., Identificatin f a brassinsterid, castasterne frm Marchantia plymrpha, ull. Krean Chem. Sc., 2002; 23: [13] Takatsut S., Abe H., and Gamh K., Evidence fr brassinsterids in strbilus f Equisetum arvense L., Agric. il. Chem., 1990; 54: [14] Ykta T., Kim S.K., Fukui Y., Takahashi N., Takeuchi Y., and Takematsu T., rassinsterids and sterls frm a green alga, Hydrdictyn reticulatum: cnfiguratin at C-24, Phytchemistry, 1987; 26: [15] ajguz A., and Tretyn A., The chemical

7 Chiang Mai J. Sci. 2006; 33(1) 101 characterictic and distributin f brassinsterids in plants, Phytchemistry, 2002; 62: [16] Khripach V., Zhabinskii V., and Grt A.D., Twenty Years f rassinsterids: Steridal plant hrmnes warrant better crps fr the XXI century, Annals f tany, 2000; 86: [17] Schmidt J., Almann T., and Adam G., rassinsterids frm seeds f Arabidpsis thaliana, Phytchemistry, 1997; 45: [18] Jang M.S., Han K.S., and Kim S.K., Identificatin f brassinsterids and their bisynthetic precursrs frm seeds f pumpkin, ull. Krean Chem. Sc., 2000; 21: [19] Fujii S., and Saka H., The prmtive effect f brassinlide n lamina jint-cell elngatin., germinatin and seedling grwth under lw-temperature stress in rice (ryza sativa L.), Plant Prductin Science, 2001; 4: [20] Takatsut S., rassinsterids: distributin in plant, biassays and micranalysis by gas chrmatgraphy-mass spectrmetry, J.Chrmatgr. A, 1994; 658:3-15.

METHOD 8032 ACRYLAMIDE BY GAS CHROMATOGRAPHY

METHOD 8032 ACRYLAMIDE BY GAS CHROMATOGRAPHY 1.0 SCOPE AND APPLICATION 1.1 Methd 8032 is used t determine trace amunts f acrylamide mnmer in aqueus matrices. This methd may be applicable t ther matrices.