METABOLITES ANNOTATION OF

DICHLOROMETHANE EXTRACT OF
Wrightia dubia TWIGS USING ORBITRAP
HIGH-RESOLUTION MASS
SPECTROMETRY

Hidayatul Atiqah Abd Karim , Nurulfazlina Edayah Rasol, Nor

Hadiani Ismail, Che Puteh Osman*
 Wrightia dubia (Sims) Spreng
Apocynaceae

No single
metabolite ‘jeliti merah’
reported

Southeast
Shrub to 2 m
Asia

Lowland
forests Figure 1. A picture of Wrightia dubia
(photo credit: Morad, A. F. from flickr)

LIYANG CONFERENCE 2023

 RESEARCH FLOW

Plant Database Database

SPE Annotation Isolation
extraction export building

LIYANG CONFERENCE 2023

 RESULTS
100
Alkaloid
90 10 Coumarin
80 Lignan

70
Peptide
Fatty acid
Relative Abundance

60
Terpenoid
50 Hydroxybenzaldehyde
40

30 21 22
18 20
20 12 19 23
11 13
4 8 16
10 2 14
1 3 5 6 79 17
15
0
0 5 10 15 20 25 30 35 40 45
Time (min)

Figure 2. The base peak chromatogram of dichloromethane extract of twigs part of Wrightia dubia

LIYANG CONFERENCE 2023

 4%
4%

9% 26%

9%

35%

13%

Alkaloid Coumarin Lignan Fatty acid Peptide Terpenoid Hydroxybenzaldehyde

Figure 3. A pie chart of classes of metabolites in the Wrightia dubia twigs

LIYANG CONFERENCE 2023

Table 1. The metabolites proposal in the dichloromethane extract of Wrightia dubia twigs using Sirius software

Retention time
No. Metabolite MS1 MS2 Similarity
(min)
1 4.13 Norharman 169.0760 168.0675, 142.0645, 141.0571, 140.0484, 116.0489 99.40%
2 4.94 Deoxyvasicinone 187.0866 185.0707, 170.0595, 169.0755, 161.0701, 160.0756 98.17%
3 5.83 Scopoletin 193.0496 178.0252, 150.0305, 137.0599, 133.0277, 122.0356 99.53%
4 6.11 Isofraxidin 223.0603 208.0359, 208.0154, 207.0282, 195.0653, 193.0128 99.12%
5 7.88 Hydroxymatairesinol 375.1439 357.1336, 339.1232, 327.1223, 321.1115, 307.0970 90.03%
6 8.62 2-(4-Hydroxyphenyl)-3,4-dihydroquinazolin-4-one 239.0817 147.0546, 132.0438, 121.0383, 120.0438, 108.0432 87.35%
7 10.56 Pluviatolide 357.1335 339.1222, 327.1228, 325.0708, 321.1121, 311.1291 89.86%
8 10.71 Benzoylphenylalaninol 256.1333 238.1221, 134.0958, 122.0592, 117.0693, 105.0329 99.47%
9 10.78 Haplomyrfolin 357.1335 339.1219, 327.1217, 321.1114, 309.1119, 307.0957 97.14%
10 14.23 Tryptanthrin 249.0659 221.0702, 220.0625, 194.0593, 193.0753, 192.0675 96.48%
11 14.82 8-[4-Oxo-5-(pent-2-en-1-yl)cyclopent-2-en-1-ylidene]octanoic acid 291.1955 273.1844, 255.1736, 249.1480, 245.1896, 237.1630 81.52%
12 14.91 Aurantiamide 403.2016 385.1906, 252.1007, 224.1063, 152.1062, 134.0964 92.40%
13 16.41 9-Oxooctadeca-10,12,15-trienoic acid 293.2111 275.2002, 257.1895, 247.2052, 239.1798, 237.1474 98.06%
1,2,6a,6b,9,9,12a-Heptamethyl-10-oxo-
14 16.79 3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropicene- 453.3367 435.3236, 407.3302, 389.3222, 267.2097, 261.1848 84.75%
4a-carboxylic acid
15 17.62 Indirubin 263.0815 245.0690, 236.0702, 235.0861, 234.0784, 220.0751 89.64%
16 18.23 N-(2-Hydroxyethyl)octadeca-9,12,15-trienamide 322.2742 304.2629, 261.2208, 243.2096, 226.1799, 224.1639 100.00%
17 19.26 (9Z)-Octadeca-9,12,15-trienamide 278.2477 261.2206, 243.2103, 233.2249, 219.2100, 217.1921 92.47%
18 20.28 Stearidonate 277.2162 259.2054, 241.1950, 235.1688, 231.1741, 221.1532 86.43%
19 21.91 Clavatustide C 566.4274 538.4314, 521.4065, 453.3431, 425.3483, 408.3197 94.95%
20 22.00 3,5-Di-tert-butyl-4-hydroxybenzaldehyde 235.1694 219.1382, 179.1060, 175.1114, 163.0751, 147.1164 93.71%
21 22.09 Octadeca-8,11,14-trienoic acid 279.2318 261.2208, 243.2101, 237.1850, 223.1686, 219.2106 93.57%
22 23.34 Octadeca-8,10,12,14-tetraenoic acid 277.2163 259.2053, 249.2207, 241.1946, 235.1688, 231.2102 87.14%
23 23.78 13-Oxo-9,11-octadecadienoic acid 295.2269 277.2160, 259.2066, 249.2210, 241.1959, 231.2108 97.42%
LIYANG CONFERENCE 2023
 FAMILY GENUS

SPECIES

Figure 4. The graphical user interface of Sirius software

LIYANG CONFERENCE 2023

 Isolated metabolite
O
N

Figure 7. Tryptanthrin

Figure 5. UHPLC profiling of Tryptanthrin

Relative Abundance

10020231016_NER_TQA_WDTWIGDCMF751_20ppm_Pos_1microL_FS120K #2271 RT: 14.30 AV: 1 NL: 1.06E+007T: FTMS + c ESI Full ms [150.0000-1500.0000]
249.0656
80
60
40250.0671
20 365.9930
474.3057
0 319.2842
228.1955 577.1239
667.1759 1479.6254
2003427.3777
004519.1382
005622.0283
006741.1945
007815.2134
008922.0090
0091037.2114
m/z
1121.9958
00 1221.9894
1000 1321.9817
1100 1421.9749
1200
1300
1400
1500

Figure 6. MS spectrum of Tryptanthrin Figure 8. Isolated Tryptanthrin

LIYANG CONFERENCE 2023

 4
5 O
3
4a N 5a 6
6a 7
2 12a
N
12 11 10a 8
1
O 10 9

Figure 9. 1D NMR of Tryptanthrin

LIYANG CONFERENCE 2023

 H O
N H
N

H O
H

Figure 12. HMBC Spectrum of Tryptanthrin

LIYANG CONFERENCE 2023

 Table 2. Comparison of NMR data (600 MHz, CDCl3) of Tryptanthrin with previous report (500 MHz, CDCl3) [2]
Tryptanthrin
5 O
No. 1
H NMR 1
H NMR 13
C NMR 13
C NMR 4
4a N 5a 6
3
(δ ppm) (δ ppm)* (δ ppm) (δ ppm)* 6a 7
1 8.48, 1H, dd, J=7.8, 0.6 Hz 8.44, 1H, dd, J=7.9, 1.5 Hz 127.6 127.6 2 12a
N
12 11 10a 8
1
2 7.71, 1H, td, J=7.8, 0.6 Hz 7.67, 1H, dt, J=7.3, 1.5 Hz 130.3 130.3 O 10 9

3 7.89, 1H, td, J=8.4, 1.8 Hz 7.85, 1H, ddd, J=8.2, 7.3, 1.5 Hz 135.2 135.2
4 8.07, 1H, d, J=8.4 Hz 8.03, 1H, d, J=8.1 Hz 130.8 130.8
5 - - - -
6 - - nd 182.5
7 7.95, 1H, dd, J=7.8, 0.6 Hz 7.91, 1H, dd, J=7.5, 1.1 Hz 125.4 125.5
8 7.46, 1H, td, J=7.8, 0.6 Hz 7.42, 1H, ddd, J=7.9, 1.5 Hz 127.2 127.2
9 7.82, 1H, td, J=7.8, 1.2 Hz 7.79, 1H, dt, J=7.9, 1.1 Hz 138.3 138.4
10 8.67, 1H, d, J=8.4 Hz 8.63, 1H, d, J=8.1 Hz 118.0 118.0
11 - - - -
12 - - 158.1 158.2
4a - - 146.7 146.4
5a - - nd 144.4
6a - - 122.0 121.9
10a - - 146.4 146.2
12a - - 123.8 123.6

LIYANG CONFERENCE 2023

 CONCLUSION

Alkaloids
23 Tryptanthrin
and the
metabolites was Alkaloids
unknown
detected 8 fatty acids, 6 isolated
alkaloids, 3 lignans, 2
metabolite Should be isolated to
Validated dereplication Significant biological
coumarins & peptides, examine the
strategy characteristics
and 1 terpenoid & antimalarial activity
hydroxybenzaldehyde

LIYANG CONFERENCE 2023

 ACKNOWLEDGEMENT

600-RMC/GPM CoE 5/3

UiTM (052/2021)
plant materials, chemicals, and
AuRIns facilities

LIYANG CONFERENCE 2023

 REFERENCES
[1] Middleton, D. J. (2005) A revision of Wrightia (Apocynaceae: Apocynoideae) in
Malesia. Harvard Papers in Botany, 10(2), 161–182.

[2] Garcellano, R. C., Moinuddin, S. G. A., Young, R. P., Zhou, M., Bowden, M. E.,
Renslow, R. S., Yesiltepe, Y., Thomas, D. G., Colby, S. M., Chouinard, C. D., Nagy, G.,
Attah, I. K., Ibrahim, Y. M., Ma, R., Franzblau, S. G., Lewis, N. G., Aguinaldo, A. M. and
Cort, J. R. (2019) Isolation of tryptanthrin and reassessment of evidence for its isobaric
isostere wrightiadione in plants of the Wrightia genus. Journal of Natural Products,
82(3), 440-448.

LIYANG CONFERENCE 2023

 THANK YOU

LIYANG CONFERENCE 2023