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    Naumoska2013 Ac - Ursolico

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    Naumoska2013 Ac - Ursolico

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    Naumoska2013 Ac.ursolico

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    Naumoska2013 Ac - Ursolico

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    ResearchGate Sevens tana ieterhubleatons os eet nin sos TLC and TLC-MS Screening of Ursolic, Oleanolic and Betulinic Acids in Plant Extracts 3 Dr vrrrentensy ‘content folning ns page was plate by Hatin Nourse 0905 Jape 206, sate ofcnemsty TLC and TLC-MS Screening of Ursolic, Oleanolic and Betulinic Acids in Plant Extracts Katerina Naumoska, Breda Simonovsks, Alen Albreht, and rena Vovk* Key Words Ursote acid Oteanotie seid Betulnic aca Titerpencids Vegetables ne Tics Summary Separation of three triterpenic acids (arsoli, oleanolic nd betulin- ieaci) was achieved on diferent thin-layer chromatography (TLC) Uilica gel 60) and high-performance thilayer chromatography (HPTLC) sorbents (iia ge 60, C, RP and C,, RP) using several Aeveloping solvents, based om the non-polar diluent mhexane, and ‘ster (methyl acetate, ethyl acetate, ethyl propionate) a selector. ‘Anisaldehyde and molybdophosphoric ack detection reagents were use. Finally, simple method ona Cy RPHPTLC plate was devel ‘oped using whexane-ethyl acetate (51) a8 a developing tlvent ina horizontal developing chamber, The method was used for the screening of ursoi,oleanoie and betulinie acids in different vege ‘table extracts Other plant trterpenoids (upect, eamyin, fe amyrin, eyeloartendl, upenone, fredelin, lypeol acetate, cyclo- artenol acetate) and phytosterols (-sitostrol, stigmasterl) did wot Interfere. TLC-MS was used asa tool for the additional confirms the presence of ursole oleanoic, and betuliie acids in some ofthe studied vegetable extracts. Uraalic and oleanoic acids were {ound in radiechio Leonardo and white-olored radicchio di Castek franco extract forthe fet time, while betlinic acid was not detect ‘ed in the eggplant extract by MS, although it was suggested at frst, by TLC analyse. Pre-chromatographic bromination on the HPTLC silea gel 60 plates and subsequent development in taluene-chlore- form-diethyl etherformi ‘or relation of 1 Introduction Arial parts of plants are covered with cuticular waxes which serve a plant protectant. They are mainly composed of long- chain aliphatic hydrocarbons, Ketones, esters, fatty alcohols, fatty acids, aldehydes, and additionally, secondary metabolites, such as titerpenoids (Cp) and phytosterols (CC, [1]. Ursoe li, oleanolic and betulinie acids (Figure 1) are pentacyelie titerpenie acids, As secondary metabolites, they can exist as ‘Chemistry, Laboratory for Food Chemisry, Hajrhova 19, S-1001 1jubhana Stove Louk, EN-HST Cone of Excl, Dasha 186, S1-1000 Lb ema rena vovk@kis loural of Pane Comatagrapty 26 (2013) Deas 17a 25006 Atadora Kia, Budapet Figure The structure of betulil 1) ursolle 2), and oleaolie acs 3). free acids or can form saponins through a glycosidic bond with ‘carbohydrates. For the most part, these triterpenic acids exhibit antitumor, anti- inflammatory, anti-fIIV1, antimicrobial, antiuleer, antioxida- live, antiatherosclerotic, hepatoprotective, gastroprotective, hypoglycemic, hypolipidemic, cardiovascular, cardiotonic, ant- ddysthytmic, and chemopreventive activites [2-15]. The wide spectrum of pharmacological activities makes these compounds interesting to researchers and also to the pharmaceutical indus- tty. Ursolic and oleanolic acids have also been used in cosmetic preparations [2 Among analytical methods, high-performance liquid chro- ‘matography (HPLC) coupled to ultraviolet (UV) [13, 16-21], evaporative light scattering [22], charged aerosol [23] and mass spectrometry (MS) detection [15, 24-26], gas chromatography (GO) coupled to flame ionization [18, 27, 8] and MS detection [29}, capillary supercritical uid chromatography (30), micellar electrokinetic capillary chromatography [31], capillary zone electrophoresis [52], and non-aqueous capillary electrophoresis [3] have been utilized in their qualitative and quantitative analysis, HPLLC-UV analysis of these compounds is aggravating due tothe lack of a chromophore, while GC requires a pre-chro- matographie silylation which additionally prolongs the analy: Since ursoic and oleanolic acids are positional isomers, differ ing only by the position of one methyl group (Figure 1), their separation stil presents a challenge in thin-layer chromatogra- phy (TLC) analysis. Methods utilizing different sorbents include ing TLC silica gel Fy [34], high-performance thin-layer ehro- 125 ‘TLC and TLO-MS Screening of Ursa, Oleanelc and Batic Acids in Plant Extracts matography (HPTLC) silica gel [35, 36], HPTLC C,, RP [37], HPTLC Cy, RP [38] and HPTLC Cy, RP [38] already exist, However, none of these methods enables, at the same time, a simple separation and satisfactory resolution ofthis ctitical pair of isomers. Even a five step multiple gradient development of the HPTLC silica gel plate resulted in a negligible difference between &; values of usolic and oleanolie acids [36]. The most promising Separat RP plates [38], with yet insufficient resolution and were applied only tothe analysis of betulini acd in real samples. Only’ pre- chromatographic derivatization of these compounds, under care- fully controled conditions with 1% iodine solution in chloro- fom directly on HPTLC silica ge plate, gave the desired reso- lution for densitometrie determination [35]. This procedure was also performed on a TI.C sliea gel F,, plate, on which a double or a triple development further improved the resolution [34]. However, the method was applied only to ursolic and oleanolic acids, bul no results are given on how the iodation effects the separation of betulinc acid and other interfering titerpenoids and phytosterols, commonly found in cuticular waxes. ions were observed on the Cjyy RP atid Cue Besides the lack of an appropriate simple TLC method for these ‘witerpenic acids separation, there is also no information on the presence of ursolic, oleanolic and betulinie aeids in cuticular waxes of vegetables as eggplant, zucchini, tomato, ed pepper, rmangold, lettuce, radichi, cabbage. To the best of our knowl. edge, only spinach was examined for ursolie and oleanotie acids, but they were not detected, 39] The aim of our study was to develop a TIC method for fast screening and ident tacyele trterpenie acids (positional isomers ursolic and oleanc- lic acids and their structural isomer betulinie acid) in plant extracts, keeping in mind that other triterpenoids and phyto- sterols could act as possible interfering compounds. ication ofthe three biologically active pen- 2 Experimental 2.1 Chemicals Dichloromethane, chloroform, toluene, diethyl ether, neprox ppanol, n-hexane, ethyl acetate, ethyl propionate, acetic acid Table Vogetabes used in the study (clacial), sulfuric acid (95-9726), sodium sulfate (anhydrous), 44-methoxybenzaldehyde (anisaldehyde) and bromine were from Merck (Darmstadt, Germany), methanol and acetonitrile were from J.T. Baker (Deventer, The Netherlands), formic acid was from Kemika (Zagreb, Croatia), methyl acetate, molybdophos- Phoric acid, ethanol, P-sitosterol (297%) and ursolic acid (290%) were from Sigma-Aldrich (St. Louis, MO, USA). Lupe- ol (299%), a-amyrin (298.5%), B-amyrin (298.5%), lupeol acetate (295%), lupenone (295%), eycloartenol (290%), eycloartenol acetate (290%), friedelin (299%) and betulinic acid (297%) were purchased from Extrasynthise (Genay, France), ‘while stigmasterol (299%) was from Serva Feinbiohemica (He 4elberg, Germany) and oleanolic acid (297%) from Carl Roth (Karlsruhe, Germany), 2.2 Preparation of Standard Solutions Stock solutions of betulini acid and friedelin (0.1 mg ml.) and all other standards (1 mg ml.~) were prepared in »-propanol In order t obtain working standard solutions, stock solutions ofall standards were diluted with mpropanol to a concentration of 25 ug ml, Different mixtures of working standards (MIX2, MIX, and MIX13) were prepared in propanol. MIX2 (60 yg mL): ursolie and oleanolie acids; MIXS (25 jg mL) tursolie, oleanolic and hetulinie acids; MIX13 (25 jug ml-~) Jupeo!, a-amyrin, f-amyrin, eycloartenol, upenone, friedein, ursolie acid, oleanolic acid, betulinie acid, lupeol acetate, eycloartenol acetate, P-sitosterol, and stigmastero. 2.3 Preparation of Extracts and Sample Test Solutions 23.1 Vegetables Fresh vegetables listed in Tuble I were purchased from a local market, Leaves ot whole fruits of fresh vegetables, respectively, were separately immersed into dichloromethane for 1 min, Afterward, anhydrous sodium sulfate (1) was added in order to remove the trace amounts of water, The solution was filtered into an evaporation flask, The rest of sodium sulfate was rinsed with some dichloromethane and added to the fltate Dichloromethane was evaporated on rotary evaporetor (Biichi, Switzerland) under reduced pressure; before the evaporation of Veretable Latin name Family Feaplant Solanam melongena L Solanaceae uci Cucurbita pepe. Cocurbitacese Tomato Solanum Iyeopersicum 1. Solanaceae Red pepper Capsicum arma L. Solanaceae Mangold Beta vulgaris L. ssp. vulgaris va. lta Chenopodice Spinach Spinacia olearacea L Chenopodiaceae Letruce Lactuca sativa Ciehoraceae White-colored radicchio di CaselitancoCichorium inybusL. var. feiosum Cichoracene addict Leonardo CCichorium inybus var. foosum Cichoraceae White cabbage Brassica oleracea L. ssp. oleracea convat capitate. var captaa alba Brasseaceae Red cabbage Brassica oleracea L. ssp oleracea cowat capita L. var. cqptaia Lf rubra Brassicaceae Savoy cabbage Brassica oleracea ssp oleracea convat. capitate (L-) Alef. va. sabanda |, __Brasseaeeae 126 oval of Penar Chomatorapy 26 (2013)2 TLC and TLC-MS Screring of Usolc, Olearalic and Botunic Acids in Plant Extracts the solvent was complete, the concentrated extract was trans- ferred to a weighed plastic tube, The solvent from the tubes was ‘evaporated to dryness and the tubes were weighed to obtain the wax mass. Dry wax residues were further dissolved in chloro- form to a concentration of 10 mg ml~!, 1 mL of each sample solution was pipetted in a separate vial and chlorofonm was ‘evaporated to dryness. Afterward, all residues were redissolved with n-propanol (I mL). A hair dryer was used to speed up the dissolution of wax residues, ‘The solutions were then cooled down to room temperature and filtered through a 0.45-um Mil lipore Millex-HV hydrophilic poly(vinyldiene diftuoride)- PVDF membrane filter Billerica, MA, USA). 2.3.2 Sage Leaves The sage extract was prepared hy a successive Soxhlet extrac tion with rhexane, chloroform and methanol according to Baricevie etal, [10]. Dried chloroform extract was further dis- solved in methanol and used for TLC analysis, 12.4 Preparation of Dipping Detection Reagents Anisaldehyde detection reagent was prepared by mixing glacial acetic avid (20 mL) and methanol (170 mL); the mixture was ‘cooled in an ice bath and sulfuric acid (16 mL.) and 4-methoxy~ benzaldehyde (I mL) were added in a dropwise manner [40]. Molybdophosphorie acid detection reagent was prepared by dis+ solving molybdophosphorie acid (10 g) in ethanol (200 ml.) [40]. 2.5 Thin-Layer Chromatography TILC was performed on the Merck 20 em > 10 em glass-backed TLC silica ge! 60 plates (Art, No. 1.05626) and HPTLC plates: (CRP (Art. No. 1.05626) and C,, RP (Art. No. 1.05914). Work ing standard solutions of ursolic, oleanolie, betulinic acids and ‘MIX2 were applied as 10 mm bands, 15 mm from the left edge, 10 mm from the bottom edge, and 6 mm apart by means of Lino- ‘mat IV (CAMAG, Mutter, Switzerland) on the silica gel 60 plates. Plates were developed in normal, twin-trough and hori- zontal (sandwich and tank configuration) chambers (CAMAG) using r-hexane-melhyl acetate (3: vv), r-hexane-elhyl acetate G:1 v9), mhexane-ethyl propionate (3:1 vi), and nm hhexane-thy! acetate-formic acid (40:9:1, wy) as developing solvents. C, RP HPTLC plates with equal applications were developed in n-hexane-ethyl acetate (3:1 viv and 9:1 vv), ‘The screening of ursolie, oleanolic and betulnie acids in ves lable extracts was performed on Cy, RP HPTLC plates, pre- developed with acetone in a twin-trough chamber (CAMAG), and dried in an oven at 110°C for 30 min, Working standard solutions, MIX3, MIXI3, and sample test solutions of vegeta- bles were applied as 6 mm bands, 10 mm from the bottom, 15 ‘mm fiom the left edge by means of ATS4 (CAMAG). Applica tion volumes of the working standard solutions, MIX3 and MIX13 were equal (10 jL.) as well asthe application volumes of all vegetable extract sample test solutions (20 KL), except for ‘eggplant, whte-colored radicchio di Castelfranco and radiechio Leonardo test solutions, which were 30 pL, 10 ul. and 9 ul., respectively, The plates were developed to a distance of 8 em in 11 min in a horizontal developing chamber for 20 em * 10 em pilates using 6 ml. of developing solvent mhexane-ethy| acetate (5:1 vi or 7:1 vb) and 10 mL ofthe same solvent in a tank for preconditioning (10 min) loural of Paar Chromstograpty 262013) 2 ‘After developing and drying, post-chromatographie devivatiza- tion ofthe plates was performed by dipping the plates for 2s into anisaldchyde detection reagent by means of immersion device TI (CAMAG). After immersion, the plates were heated on a ‘TLC Plate Heater (CAMAG) for 2 min at 110°C. Documenta tion of the chromatographic plates was done by visual evalua- tion at white light and at 366 nm by means of DigiStore 2 Doc uumentation System (CAMAG) operated with winCATS Version 1,4.1.8154 software, 2.6 Pre-Chromatographie Bromination at the Start ofthe Plate Working standard solutions (1 xg band) of ursole,oleanolie and betulnie acids, sitosterol, MIX2 and sage were applied on the HPTLC silica gel 60 plate (Merck, Art. No. 1.05641) as 10 mm bands, 15 mm from the left edge, 10 mm ftom the bottom edge, and 6 mm apart by means of Linomat IV, Pre-chromatographic bromi- nation was performed a the start position by dipping the plates for 8 sina fesh saturated bromine solution in water-acetic acid (2:1, ‘y) mixtute (up tothe distance of abou 1. em slightly above the applications) using. immersion device Ill. The plates were aer- ward dried ina steam of warm air and additionally heated for 10 rin at 110°C on the TLC plate neater. The 10 em x 10 em plates were then developed in an unsaturated twin-trough chamber with r= hexane-ethylacctate—formic acid (40:9: wh) and toluene-chlore- form-diethyl ether-formic acid (20:16:40.1, wy), as developing, solvents (5 mL) in each rough. Post-chromatographic derivatiz- tion was performed by dipping the plates into moly bdophosphorie acid or anisldehyde detection reagents and heating the pltes at 110°C for 2 min. All plates were also developed inthe same man- ner without the pre-chromatographic brominaton step. 27 TLCS ATLC-MS interface (CAMAG) was used for the elution of com- Pounds from the TLC plates into the ion trap LCQ (Thermo Fine nigan, San Jose, CA, USA) system. Acetonitrile was used as the ‘eluent witha flowrate of 0.3 mL.imin. Mass spectra were obtained by the ionization ofthe molecules with tae atmospheric pressure ‘chemical ionization (APCT) source in negative mode. Capillary and source heater temperatures were maintained at 200°C and 425°C, respectively. Other parameters were as follows: sheath gas (N,) 70 arbitrary units (au), auxiliary gas (N,) 3 au, discharge ‘current 3 WA, capillary volage -26 V, tube lens offset -20 V. The mass spectra were acquired in the mlz range 350-500. and 150-00, For the MS" experiments, the precursor ion at mi: 455.5 was futher fragmented using the normalized collision energy of 42%, For the MS, the production at miz 407.3, obtained fom MS" experiments, was fragmented using the normalized eolision ‘energy of 40%, Other parameters were identical for MS? and MS? ‘experiments and were as follows: isolation width 1.5 mis, activa: tion radio frequency’ 0.25, activation time 30 ms, and the spectra were seared in the mis range 150-500, 3 Results and Discus: a1 TLe We studied the applicability of TLC for fast sereening ofthe bie- logically active positional isomers ursolic and oleanolic acids 127 ‘TLC and TLO-MS Screening of Ursa, Oleanelc and Betulinic Acids in Plant Extracts Figure 2 ‘TLC sites get 60 plates developed in unsaturated normal developing ‘chamber using developing solvents mexane-methy! acetate (3:1 iy, A); mexanenathy acetate (8:1 viv, BY; mhexanemethyt prop fonate (2:1 vv, C) aller dorivalization with anisaldehyde detection reagent; whit tight trination. racks: 1 = urslle acid; 2= oleano- Me ald; 3 = MOxz; 4 = betune acl Figures HPTLC ©, RP plates developed in an unsaturated twirtrough devel- oping chamber using developing solvent mhexane-ethy acetate 3:1 wy (A, B) and detection reagent (A, C: le seid; 2 = oleanolic ae betuine sel and the structurally isomeric betulinie acid in plant extracts, take ing other trterpenoids and phytosterols, as possible interfering ‘compounds, into account. First, it was found that each developing solvent, comprised from ‘an ester (methyl acetate, ethyl acetate, ethyl propionate) which ‘was suitably diluted with n-hexane or some other hydrocarbon solvent, indicated the separation of the three acids on TLC silica ‘gel 60 sorbent, Resolution of usolie and oleanolic acids was, 128 Figure 4 ‘The separation without (A) and after (@, C) pre-chromatographic bromination on the HPTLC silica gel 60 plates developed in r-nexs- elae-formle acid (40:9:1, w¥) after derivatization with Figure 5 ‘The separation of ursll,olesnote, and bettie elds without (A) and ster (8) pee-chrometographie bromination on the HPTLC silea e160 plates developed In toluene-chloroform-

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