SUPPLEMENTARY MATERIAL

Metabolomic fingerprint classification of Brachychiton acerifolius organs

via UPLC–qTOF–PDA–MS analysis & chemometrics

Mohamed A. Farag1,3*, Aisha H. Abou Zeid2, Manal A. Hamed4, Zeinab Kandeel1, Hanaa M. El-Rafie2, Radwa H. El-Akad2

1Pharmacognosy department, College of Pharmacy, Cairo University, Cairo, Egypt, Kasr el Ainist., P.B. 11562.

2Pharmacognosy Department, 4Therapeutic Chemistry Department, National Research Center, Dokki, Cairo 12311, Egypt

3Leibniz Institute of Plant Biochemistry, Dept. Bioorganic Chemistry, Weinberg 3, D 06120 Halle (Saale), Germany

Abstract

Brachychiton acerifolius, or Sterculia acerifolia as formerly known, is a member in a genus reported for a myriad of bioactive compounds. Metabolome analysis of Brachychiton acerifolius; leaves, flowers and seeds, and quantification of its major compounds are demonstrated in this study. Metabolites were analyzed via UPLC-PDA-qTOF-(±) ESI-MS and UPLC/ITMS, with a total of 56 metabolites characterized including 30 flavonoids, 2 anthocyanins, 6 phenolic acids (i.e., citric and hydroxycitric acid conjugates) and 8 fatty acids. Multivariate data analyses (i.e., PCA and OPLS-DA) were applied to identify metabolite markers for each organ. Pelargonidin-O-glucoside and naringenin-O-glucuronide were found exclusively in flowers versus flavones enrichment in leaves (i.e., luteolin-O-glucuronide and apigenin-O-rhamnosylglucuronide). GC/MS analysis revealed the presence of toxic cyclopropene fatty acids in seeds which may restrict its use. Anti-oxidant activity assessment for the three organs was performed in comparison with vitamin C as positive control. Leaves showed the highest activity (IC50 0.015 mg/ml).

Keywords: Brachychiton acerifolius; UPLC/MS analysis; Chemometrics; Anti-oxidant activity

*Corresponding author: , l: +011-202-2362245, Fax: +011-202-25320005

Table S1. Metabolites identified in Brachychiton acerifolius leaves (L), flowers (F) and seeds (S) methanol extract using UPLC–PDA–MS in negative & positive ESI modes
Peak / Rt.sec / UV (nm) / Identification / Mol. ion m/z (+/-) ppm / Element
composition / Error
(ppm) / MSn ions
m/z (+/-) ppm / L / F / S
30 / N.D. / Hydroxy citric acid lactone / 189.0036 (M-H)- / C6H5O7- / 2.5 / N.D. / + / - / +
39 / N.D. / Iso/Citric acid / 191.0193 (M-H)- / C6H7O7- / 2.1 / 173, 111 / + / +
92 / 320 / Gentisic acid-3-O-glucopyranoside / 315.0722 (M-H)- / C13H15O9- / 1.2 / 153 / + / - / -
188 / 279 / Catechin / 291.0885 (M+H)+ / C15H15O6+ / 7 / 240 / - / - / +
192 / 322 / Benzyl alcohol-sophoroside / 431.1560 (M-H)- / C19H27O11- / 3.2 / 401, 323, 223 / + / - / -
194 / N.D. / Unknown / 401.1858 (M-H)- / C19H29O9- / 9 / 383, 221 / + / - / -
194 / 278, 500 / Cyanidin-O-rutinoside / 595.1508 (M+H) + / C27H31O15+ / 0.7 / 449, 287 / - / + / -
204 / 278, 500 / Pelargonidin-3-O-glucoside / 433.1176 (M+H)+ / C21H21O10+ / 10.7 / 271 / - / + / -
207 / 290, 322 / Ferulic acid hexoside / 355.1012 (M-H)- / C16H19 O9- / 2.6 / 193 / + / - / +
212 / 288, 325 / Unknown / 371.0984 (M-H)- / C16H19O10- / 0.4 / 355, 209, 191 / + / - / -
216 / 290, 330 / Caffeoyl iso/citrate / 353.0536 (M-H)- / C15H13O10- / 0.7 / 191, 179, 173 / + / + / -
218 / 283, 340 / Kaempferol-O-dihexosylrhamnoside / 757.2219 (M+H)+ / C33H41O20+ / 4 / 611,595, 449, 433, 287 / - / + / -
224 / 270, 330 / Apigenin-6,8-C-diglucoside / 593.1540 (M-H)- / C27H29O15- / 4.7 / 575, 503, 473, 383,353, 293, 179 / + / - / -
230 / 280 / Dihydroquercetin-O-glucuronide / 481.1022 (M+H) + / C21H21O13+ / 2.2 / 305 / - / + / -
232 / 280, 315 / p- Coumaroylhydroxyiso/citrate / 353.0515 (M-H)- / C15H13O10- / 0.2 / 207, 189, 163 / - / - / +
237 / N.D. / 2-Phenylethanol-O-pentosyl-glucoside / 415.1629 (M-H)- / C19H27O10- / 4.7 / 399, 307, 223,161 / + / - / -
241 / 280, 327 / Feruloylhydroxyiso/citrate / 383.0612 (M-H)- / C16H15O11- / 2.1 / 207,193,189 / - / - / +
244 / 265, 353 / Pentahydroxyflavonol-O- hexosylrhamnoside / 627.1609 (M+H)+ / C27H31O17+ / 8.5 / 481, 319 / - / + / -
249 / 265, 355 / Pentahydroxyflavonol-O- hexoside / 481.1025 (M+H)+ / C21H21O13+ / 10 / 319 / - / + / -
267 / 257, 355 / Rutin / 611.167 (M+H)+ / C27H31O16+ / 12.4 / 465, 303 / + / + / +
274 / 255, 350 / Quercetin -O-[hydroxy-methylglutaryl]-glucopyranoside / 609.1509 (M+H)+ / C27H29O16+ / 12.4 / 465,447, 303, 287 / + / - / -
276 / 265, 350 / Quercetin-O-glucoside (Hyperoside) / 465.1077 (M+H)+ / C21H21O12+ / 10.5 / 303 / + / + / -
278 / 255, 267, 347 / Luteolin -O-glucuronide / 463.0933 (M+H)+ / C21H 19O 12+ / 13.2 / 287 / + / - / -
289 / 266, 345 / Hydroxyluteolin-O-di-rhamnoside / 595.1711 (M+H)+ / C27H31O15+ / 13.8 / 449, 303, 287, 153, 151 / + / - / -
290 / 260, 350 / Kaempferol-O-rutinoside / 595.1739 (M+H)+ / C27H31O15+ / 13.5 / 449, 287 / - / + / -
Peak / Rt.sec / UV (nm) / Identification / Mol. ion m/z (+/-) ppm / Element
composition / Error
(ppm) / MSn ions
m/z (+/-) ppm / L / F / S
292 / 268, 352 / Isorhamnetin-O-rhamnosylpentoside / 595.1731(M+H)+ / C27H31O15+ / 10.4 / 449, 317, 303, 287 / - / + / -
296 / 267, 339 / Apigenin-O-rhamnosylglucuronide / 593.1406 (M+H) + / C27H29O15+ / 8.5 / 447, 271 / + / - / -
298 / 268, 350 / Kaempferol-O-hexoside (Astragalin) / 449.1198 (M+H)+ / C21H21O11+ / 11 / 287 / + / + / -
301 / 280, 320 / Unknown / 449.0942 (M+H) + / C21H21O11+ / 3.2 / 273 / - / - / +
303 / 268, 340 / Diosmetin-O- rhamnosylglucuronide / 623.1668 (M+H)+ / C28H31O16+ / 8.2 / 447, 301, 287, 271 / + / - / -
306 / 282, 330 / Naringenin-O-glucuronide / 449.1141 (M+H)+ / C21H21O11+ / 12.6 / 273 / - / + / -
307 / 268, 335 / Apigenin-O-glucuronide / 447.0990 (M+H) + / C21H19O11+ / 7.3 / 271 / + / - / -
311 / 274, 349 / Isorhamnetin -O-pentoside / 449.1129 (M+H)+ / C21H21O11+ / 11 / 317, 287 / - / + / -
314 / 267, 345 / Diosmetin-O-glucuronide / 477.1072 (M+H) + / C22H21O12+ / 3.3 / 301, 271 / + / - / -
328 / 281 / Hesperetin-O-glucuronide / 479.1246 (M+H) + / C22H23O12+ / 12.5 / 303, 289, 273 / - / + / -
339 / 268 / Hydroxy hesperetin / 319.0846 (M+H) + / C16H15O7+ / 15.5 / 287, 273 / - / + / -
339 / N.D. / Unknown / 711.2178 (M-H)- / C32H41O18- / 5 / 649, 581, 503 / + / - / -
342 / N.D. / Unknown / 579.1372 (M+H)+ / C26H27O15+ / 4.8 / 317 / - / + / -
362 / 268,320 / Methoxyapigenin-O- rhamnosylglucuronide / 607.1704 (M+H)+ / C28H31O15+ / 7.7 / 461,285,271 / + / - / -
368 / 274, 368 / Quercetin / 303.0533 (M+H)+ / C15H11O7+ / 11 / 287 / + / + / -
379 / 277 / Hesperetin / 303.0889 (M+H)+ / C16H15O6+ / 8.4 / 273 / - / + / -
382 / 270, 320 / Methoxyapigenin-O-glucuronide / 461.1130 (M+H) + / C22H21O11+ / 2.1 / 285, 271 / + / - / -
392 / 275, 330 / Methylnaringenin-O-glucuronide / 463.1216 (M+H)+ / C22H23O11+ / 7 / 287 / - / + / -
402 / 278 / Naringenin / 273.0791 (M+H)+ / C15H13O5+ / 12.7 / N.D. / - / + / -
408 / 280, 320 / Apigenin / 271.0644 (M+H) + / C15H11O5+ / 15.8 / 248, 177,147 / + / - / -
415 / N.D. / Trihydroxy-octadecadienoic acid / 327.2177 (M-H)- / C18H31O5- / 1.5 / 243, 174 / + / - / +
417 / 269, 367 / Isorhamnetin / 317.0693 (M-H)- / C16H13O7+ / 1 / 287 / - / + / -
441 / N.D. / Trihydroxy-octadecenoic acid / 329.2332 (M-H)- / C18H33O5- / 0.6 / 248, 174 / + / - / +
449 / 281 / Unknown flavonoid / 477.1429 (M+H)+ / C23H25O11+ / 8 / 459, 441, 329, 315, 287 / - / + / -
585 / N.D. / Dihydroxy-octadecenoic acid / 313.2394 (M-H)- / C18H33O4- / 2.4 / N.D. / - / - / +
619 / N.D. / Dihydroxy-octadecanoic acid / 315.2552 (M-H)- / C18H35O4- / 3.4 / N.D. / - / - / +
700 / N.D / Unknown / 351.2553 (M+H)+ / C21H35O4+ / 6 / 279 / - / - / +
871 / N.D. / Linoleic acid / 279.2332 (M-H) - / C18H31O2- / 1.2 / 248, 233 / + / - / +
914 / N.D. / Palmitic acid / 255.2346 (M-H) - / C16 H31 O2- / 1.36 / 154 / + / - / +
930 / N.D. / Oleic acid / 281.2644 (M-H) - / C18H33O2- / 4.3 / 180 / + / - / +
990 / N.D. / Stearic acid / 283.2643 (M-H) - / C18H35O2- / 5 / 248, 180 / + / - / +

+ and – indicate presence and absence of metabolite, respectively, N.D.; not detected, Rt; retention time

Table S2. Results of GC/MS analysis of fatty acid methyl esters from petroleum ether seeds extract of Brachychiton acerifolius

No. / Compound / Rt (min) / RRt / B.P. / M+ / Area % / M.F. / Structure
1 / Methyl Octanoate (methyl caprylate) / 11.75 / 0.38 / 74 / 158 / * / C9H18O2 /
2 / Methyl nonanoate / 14.62 / 0.47 / 74 / 172 / 0.68 / C10H20O2 /
3 / Methyl-8-nonynoate / 15.27 / 0.49 / 74 / 168 / 0.3 / C10H16O2 /
4 / Methyl decanoate / 17.37 / 0.56 / 74 / 186 / 0.01 / C11H22O2 /
5 / Methyl-8-oxo octanoate / 17.58 / 0.56 / 87 / 172 / 0.07 / C9H16O3 /
6 / Methyl-4-oxo nonanoate / 18.60 / 0.59 / 98 / 186 / 0.03 / C10H18O3 /
7 / Methyl undecanoate / 19.29 / 0.62 / 74 / 200 / * / C12H24O2 /
8 / Methyl-8-oxo nonanoate / 19.81 / 0.63 / 129 / 186 / 0.14 / C10H18O3 /
9 / Methyl-9-oxo nonanoate / 20.24 / 0.65 / 74 / 186 / 0.22 / C10H18O3 /
No. / Compound / Rt (min) / RRt / B.P. / M+ / Area % / M.F. / Structure
10 / Dimethyl octandioate / 20.56 / 0.66 / 129 / 202 / 0.46 / C10H18O4 /
11 / Methyl-9-oxo decanoate / 22.35 / 0.71 / 43 / 200 / 0.13 / C11H20O3 /
12 / Methyl dodecanoate (methyl laurate) / 22.44 / 0.72 / 74 / 214 / 0.01 / C13H26O2 /
13 / Methyl-10-oxo decanoate / 22.72 / 0.73 / 74 / 200 / 0.03 / C11H20O3 /
14 / Dimethyl nonandioate (dimethyl azelate) / 23.02 / 0.74 / 152 / 216 / 0.52 / C11H20O4 /
15 / Methyl-10-oxo undecanoate / 24.72 / 0.79 / 125 / 214 / 0.02 / C12H22O3 /
16 / Dimethyl decandioate (dimethyl sebacate) / 25.33 / 0.81 / 125 / 230 / 0.04 / C12H22O4 /
17 / Methyl tetradecanoate (methyl myristate) / 27.00 / 0.86 / 74 / 242 / 0.18 / C15H30O2 /
18 / Dimethyl undecandioate / 27.54 / 0.88 / 55 / 244 / 0.01 / C13H24O4 /
No. / Compound / Rt (min) / RRt / B.P. / M+ / Area % / M.F. / Structure
19 / Methyl-9-hydroxy undecanoate / 27.83 / 0.89 / 155 / 216 / 0.01 / C12H24O3 /
20 / Methyl-9-methyltetradecanoate / 28.35 / 0.91 / 74 / 256 / 0.01 / C16H32O2 /
21 / Methyl pentadecanoate / 29.12 / 0.93 / 74 / 256 / 0.04 / C16H32O2 /
22 / Methyl-3-hydroxy tetradecanoate / 30.06 / 0.96 / 103 / 258 / 0.02 / C15H30O3 /
23 / Methyl-9-hexadecenoate / 30.70 / 0.98 / 55 / 268 / 0.47 / C17H32O2 /
24 / Methyl hexadecanoate / 31.28 / 1.00 / 74 / 270 / 32.5 / C17H34O2 /
25 / Methyl-8-(2-hexylcyclopropyl) octanoate / 32.59 / 1.04 / 55 / 282 / 0.33 / C18H34O2 /
26 / Methyl heptadecanoate / 33.07 / 1.06 / 74 / 284 / 0.17 / C18H36O2 /
27 / Methyl-3-hydroxy hexadecanoate / 34.05 / 1.09 / 103 / 286 / 0.01 / C17H34O3 /
No. / Compound / Rt (min) / RRt / B.P. / M+ / Area % / M.F. / Structure
28 / Methyl-9,12-octadecadienoate
(Methyl linoleate) / 34.47 / 1.10 / 67 / 294 / 18.5 / C19H34O2 /
29 / Methyl-9-octadecenoate (Methyl oleate) / 34.62 / 1.11 / 55 / 296 / 24.82 / C19H36O2 /
30 / Methyl-11-octadecenoate / 34.77 / 1.11 / 55 / 296 / 0.01 / C19H36O2 /
31 / Methyl octadecanoate / 35.01 / 1.12 / 74 / 298 / 10.64 / C19H38O2 /
32 / Methyl-6,9-octadecadienoate / 35.25 / 1.13 / 67 / 294 / 0.06 / C19H34O2 /
33 / Methyl-10,13-octadecadienoate / 35.32 / 1.13 / 67 / 294 / 0.02 / C19H34O2 /
34 / Methyl-8-octadecenoate / 35.86 / 1.15 / 55 / 296 / 0.02 / C19H36O2 /
35 / Methyl-9-oxo octadecanoate / 36.27 / 1.16 / 43 (55) / 312 / 0.58 / C19H36O3 /
36 / Methyl -9-oxo nonadecanoate / 36.90 / 1.18 / 129 / 326 / 0.46 / C20H38O3 /
37 / Methyl-3-hydroxyoctadecanoate / 37.59 / 1.20 / 103 / 314 / 0.03 / C19H38O3 /
No. / Compound / Rt (min) / RRt / B.P. / M+ / Area % / M.F. / Structure
38 / Methyl-7-(2-octyl-1-cyclopropenyl) heptanoate / 37.67 / 1.20 / 55 / 294 / 0.04 / C19H34O2 /
39 / Methyl-11-eicosenoate / 37.97 / 1.21 / 55 / 324 / 0.02 / C21H40O2 /
40 / Methyl eicosanoate / 38.39 / 1.23 / 74 / 326 / 0.4 / C21H42O2 /
41 / Methyl-12-hydroxy-13-methoxy-9-octadecenoate / 39.46 / 1.26 / 115 / 342 / 0.14 / C20H38O4 /
42 / Methyl-8-(2-octyl-1-cyclopropenyl) octanoate , (Methyl sterculate) / 40.27 / 1.29 / 55 / 308 / 0.06 / C20H36O2 /
43 / Methyl docosanoate / 41.59 / 1.33 / 74 / 354 / 0.14 / C23H46O2 /
44 / Methyl tricosanoate / 43.10 / 1.38 / 74 / 368 / 0.03 / C24H48O2 /
45 / Methyl tetracosanoate / 44.56 / 1.42 / 74 / 382 / 0.12 / C25H50O2 /
46 / Methyl pentacosanoate / 45.97 / 1.47 / 74 / 396 / 0.02 / C26H52O2 /
47 / Methyl hexacosanoate / 47.35 / 1.51 / 74 / 410 / 0.02 / C27H54O2 /
Total identified compounds / - / - / - / - / 92.54 % / - / -
Unidentified compounds / - / - / - / - / 7.46 % / - / -
Rt, retention time; RRt, relative retention time relative to methyl hexadecanoate (Rt=31.28); B.P.,base peak; M+, molecular weight; M.F., molecular formula; *=below o.o1%

Table S3. Quantification of major flavonoids and anthocyanins identified in Brachychiton acerifolius organs.

Values are expressed as µg/mg plant dry wt from 3 biological replicates average ± standard deviation.

Peak numbers follow that listed in Table 1

Peak / Metabolite / Leaves / Flowers
7 / Cyanidin-O-rutinoside / - / 0.63 ± 0.08
8 / Pelargonidin-3-O-glucoside / - / 7.55 ± 2.1
20 / Rutin / 65.8 ± 14.5 / 45.7 ± 7.6
22 / Quercetin-O-glucoside / tr. / 51.5 ± 19.3
23 / Luteolin-O-glucuronide / 128.2 ± 31.6 / -
24 / Hydoxyluteolin-O-dirhamnoside / 8.9 ± 2.3 / -
25 / Kaempferol-O-rutinoside / - / 120.7 ± 25.9
27 / Apigenin-O-rhamnosylglucuronide / 79.8 ± 7.8 / -
31 / Naringenin-O-glucuronide / - / 55.4 ± 8.4
32 / Apigenin-O-glucuronide / 113.3 ± 20.1 / -

-indicates absence of metabolite, tr. Indicates traces

Figure S1. Base peak chromatogram (BPC) for methanol extract of Brachychiton acerifolius leaf, flower, and seed analyzed by UPLC-qTOF-(-)ESI-MS showing two distinctive Rt-regions: 200-400s peaks for phenolic compounds principally flavonoids and anthocyanins, and 410-900s peaks for fatty acids. Peak numbers follow those listed in (Table 1) for metabolite identification using UPLC-PDA-MS.

Figure S2. Principal component analysis of B. acerifolius leaf, flower and seed extracts analyzed by UPLC–qTOF–MS (n = 3). Metabolome clusters are located at distinct positions in the two-dimensional space as prescribed by the two vectors of principal component 1 (PC1= 71%) and principal component 2 (PC2 = 26%). (A) score plot of PC1 versus PC2 scores. (B) loading plot for PC1 and PC2 contributing peaks expressed as mass/rt (sec) pairs and with their assignments. Ellipses do not denote statistical significance but rather to enhance cluster visibility.

Fig. S3. OPLS-DA score plots derived from B. acerifolius flower (A) and leaf (B) modeled separately against all other organs and their respective S-plots (C and D) derived from UPLC-MS data set. The S-plot shows the covariance “w” against the correlation “p (corr)” of the variables of the discriminating component for flower (Fig. C) and leaf (Fig. D). Cut-off values for the p < |0.01| were used; the variables thus selected are highlighted in the S-plot with m/z/retention time and their assignments.

Figure S4. In-vitro antioxidant activity assessment of B. acerifolius leaves, flowers and seeds ethanolic extracts, along with vitamin C used as a positive drug control ; percentage of DPPH- inhibition was measured from three different concentrations; 10, 50 and 100 mg/ml