S1

Isolation of food grade dye from flower petals of Butea monosperma and determination
of marker compounds for its quantitative analysis

Pooja Negi,a,b Nishant Pandey,a,b Jyoti,a,b Tripti Mishra,a Vivek Ahluwalia,a Umesh Singh,a
Bhuwan B. Mishraa*

a
Center of Innovative and Applied Bioprocessing (CIAB), Sector 81 (Knowledge City), S.A.S.
Nagar, Mohali-140306, Punjab, India.

b
Department of Chemistry, Faculty of Science, Panjab University, Sector 14, Chandigarh-
160014, India.

*Correspondence author: Dr. Bhuwan Bhushan Mishra, Tel: +91-172-5221541, E-mail:

bhuwan@ciab.res.in
 S2

TABLE OF CONTENTS
1
Figure S1. H NMR of compound 1 (BM-O) in DMSO-d6 3
Figure S2. 13C NMR of compound 1 (BM-O) in DMSO-d6 3
Figure S3. HMBC spectrum of compound 1 (BM-O) in DMSO-d6 4
Figure S4. 1H NMR of compound 2 (BM-W) in DMSO-d6 4
Figure S5. 13C NMR of compound 2 (BM-W) in DMSO-d6 5
Figure S6. HMBC spectrum of compound 2 (BM-W) in DMSO-d6 5
Figure S7. 1H NMR of compound 3 (BM-Y) in CD3OD 6
Figure S8. 13C NMR of compound 3 (BM-Y) in CD3OD 6
Figure S9. HMBC spectrum of compound 3 (BM-Y) in CD3OD 7
Figure S10. HR-MS of compound 1 (BM-O) 7
Figure S11. HR-MS of compound 2 (BM-W) 8
Figure S12. HR-MS of compound 3 (BM-Y) 8
Figure S13. FT-IR spectra of compounds: (a) compound 1 (BM-O), (b) 9
compound 2 (BM-W), (c) compound 3 (BM-Y)
Figure S14. TGA analysis of B. monosperma dye powder and its constituents viz. 9
iso-coreopsin (1), butrin (2) and iso-butrin (3)
Figure S15. XRD spectra of compounds: (a) compound 1 (BM-O), (b) compound 10
2 (BM-W), (c) compound 3 (BM-Y)
Figure S16. Quantitative determination of compounds 1-3 in B. monosperma dye 11
(P-1)
Figure S17. HPLC chromatogram of nectar sugars present in B. monosperma dye 11
(P-1)
Figure S18. Absorption spectra of dye constituents viz. iso-coreopsin (1), butrin (2) 12
and iso-butrin (3)
Figure S19. Calibration curve for quantification of dye constituents viz iso- 12
coreopsin (1); butrin (2) and iso-butrin (3)
Table S1. Chromatographic conditions 13
Table S2. Linearity Test of compounds 1-3 14
Table S3. Linearity and sensitivity data of compounds 1-3 14
Table S4. Intra-day and Inter-day precision study of compounds 1-3 15
Table S5. Repeatability studies 16
Table S6. Recovery studies of compounds 1-3 16
Table S7. Effect of analytical parameters on content (%) and retention time (rt) of 17
the method for identification and quantification.
Table S8. Trace metal analysis of B. monosperma dye using ICP-MS 18
 S3

Figure S1. 1H NMR of compound 1 (BM-O) in DMSO-d6

Figure S2. 13C NMR of compound 1 (BM-O) in DMSO-d6

 S4

Figure S3. HMBC spectrum of compound 1 (BM-O) in DMSO-d6

Figure S4. 1H NMR of compound 2 (BM-W) in DMSO-d6

 S5

Figure S5. 13C NMR of compound 2 (BM-W) in DMSO-d6

Figure S6. HMBC spectrum of compound 2 (BM-W) in DMSO-d6

 S6

Figure S7. 1H NMR of compound 3 (BM-Y) in CD3OD

Figure S8. 13C NMR of compound 3 (BM-Y) in CD3OD

 S7

Figure S9. HMBC spectrum of compound 3 (BM-Y) in CD3OD

SAIF,PANJAB UNIVERSITY CHANDIGARH SYNAPT-XS#DBA064 30-Jul-2021

15:27:56
POOJA BMO 5 (0.120) Cm (3:6) 1: TOF MS ES+
435.1260 6.58e4
100 65820

+
[M+H]
%

437.2396
6872

436.1308
2194

0 m/z
430 431 432 433 434 435 436 437 438 439 440 441

Figure S10. HR-MS of compound 1 (BM-O)

 S8

SAIF,PANJAB UNIVERSITY CHANDIGARH SYNAPT-XS#DBA064 30-Jul-2021

15:30:38
POOJA BM-W 5 (0.120) Cm (2:6) 1: TOF MS ES+
597.1808 3.16e5
100 316214

+
[M+H]
%

598.1860
66498

0 m/z
589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604

Figure S11. HR-MS of compound 2 (BM-W)

SAIF,PANJAB UNIVERSITY CHANDIGARH SYNAPT-XS#DBA064 26-Nov-2021

15:59:21
NISHANT BM-Y 7 (0.127) Cm (3:8) 1: TOF MS ES+
597.1813 1.02e6
100 1021364

+
[M+H]
%

598.1864
211980

0 m/z
585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607

Figure S12. HR-MS of compound 3 (BM-Y)

 S9

BM-O BM-W
100
100

80

1967
% Transmittance

3733

2194
80

% Transmittance
2028

2880
60

1663

1437
2363

1183
3308

827
1606
3397

3098

1439
3327

1246
60

595
40

472
1610

1073
1709
20

1183
(b)

729
40
(a)

570
987
1072
0

475
20
-20
3500 3000 2500 2000 1500 1000 500 3500 3000 2500 2000 1500 1000 500
Wavenumber (cm-1) Wavenumber (cm-1)

100 BM-Y

80
2167
% Transmittance

1513
1361
2918

1723
3248

60
1603

1237

811
40
1060

(c) 475
20

0
3500 3000 2500 2000 1500 1000 500

Wavenumber (cm-1)

Figure S13. FT-IR spectra of compounds: (a) compound 1 (BM-O), (b) compound 2 (BM-
W), (c) compound 3 (BM-Y)

100
P-1
BM-Y
80 BM-O
BM-W
Weight (%)

60

40

20

0
100 200 300 400 500 600 700
0
Temperature ( C)

Figure S14. TGA analysis of B. monosperma dye powder and its constituents viz. iso-
coreopsin (1), butrin (2) and iso-butrin (3)
 S10

12000

20000
BMO
10000
(a) 16000 BMW
Intensity (a.u.)

8000
(b)

Intensity (a.u.)
12000
6000

4000 8000

2000 4000

0
10 20 30 40 50 60 70 10 20 30 40 50 60 70
2θ (deg.) 2θ (deg.)

20000

BMY
16000

(c)
Intensity (a.u.)

12000

8000

4000

10 20 30 40 50 60 70
2θ (deg.)

Figure S15. XRD spectra of compounds: (a) compound 1 (BM-O), (b) compound 2 (BM-
W), (c) compound 3 (BM-Y)
 S11

Figure S16. Quantitative determination of compounds 1-3 in B. monosperma dye (P-1)

Figure S17. HPLC chromatogram of nectar sugars present in B. monosperma dye (P-1)
 S12

2.0
2.0 315 ug/ml

280 ug/ml

315 ug/ml

280 ug/ml 1.5

(b) 105 ug/ml

70 ug/ml
1.5 52.5 ug/ml

(a)
absorbance

105 ug/ml

absorbance
35 ug/ml
70 ug/ml

52.5 ug/ml
1.0
1.0 35 ug/ml

0.5 0.5

0.0 0.0
250 300 350 400 450 250 300 350 400 450

wavelength wavelength
BM-O BM-W

1.5

315 ug/m

280 ug/m

(c) 105 ug/m

absorbance

70 ug/m
1.0 52.5 ug/ml

35 ug/ml

0.5

0.0
250 300 350 400 450

wavelength
BM-Y

Figure S18. Absorption spectra of compounds: (a) compound 1 (BM-O), (b) compound 2 (BM-W),
(c) compound 3 (BM-Y)

Figure S19. Calibration curve for quantification of dye constituents viz iso-coreopsin (1);
butrin (2) and iso-butrin (3)
 S13

Table S1. Chromatographic conditions

Column specifications Eclipse Plus C18 column (100 × 4.6 mm; 3.5 μm)
Solvent filtration 0.45 μm membrane filter
Injection volume 10 μl
Flow rate 0.3 ml/min
PDA wavelength 271 nm, 371 nm
Column temperature 30◦C
Needle wash solution (A) MeCN/water (90/10) strong wash
(B) MeCN/water (10/90) weak wash
Mobile phase A MeCN
Mobile phase B 0.01 % orthophosphoric acid in water
Isocratic elution (A: B) 25:75
Run time 30 min
 S14

Table S2. Linearity Test of compounds 1-3

Compound 1 (BM-O)
Concentration 7000 3500 1400 700 µg/mL 350 70 35
µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
Area 51559980 26177226 10151532 5392329 2585667 515594 245257
Area 51448030 26093283 10160280 5447407 2571643 520198 247857
Area 51512979 26117023 10121511 5412356 2579169 519898 245627
Average Area 51506996.3 26129177.3 10144441 5417364 2578826.33 518563.333 246247
Standard Deviation 56214.28 43271.27 20333.98 27878.42 7018.277 2575.889 1406.521
CV (%) 0.109139 0.165605 0.200445 0.514612 0.27215 0.496736 0.571183
Compound 2 (BM-W)
Concentration 7000 3500 1400 700 350 70 35
µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
Area 1549823 774917 306826 158414 70537 10884 3041
Area 1556921 778379 305900 159381 70288 10789 2988
Area 1548810 784903 301329 159984 71231 10754 2991
Average Area 1551851.33 779399.667 304685 159259.667 70685.3333 10809 3006.66667
Standard Deviation 4419.579 5070.638 2943.029 792.0015 488.6863 67.26812 29.77135
CV (%) 0.284794 0.650583 0.965925 0.497302 0.691355 0.622334 0.990178
Compound 3 (BM-Y)
Concentration 7000 3500 1400 700 µg/mL 350 70 35
µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
Area 27605486 14597691 6111789 3169727 1556394 317338 150287
Area 27435616 14565447 6208099 3208096 1566938 319142 149754
Area 27499474 14690998 6101618 3188289 1545391 320113 149297
Average Area 27513525.3 14618045.3 6140502 3188704 1556241 318864.333 149779.333
Standard Deviation 85802.3 65203.43 58761.2 19187.87 10774.31 1408.183 495.486
CV (%) 0.311855 0.446048 0.956944 0.601745 0.692329 0.441625 0.330811

Table S3. Linearity and sensitivity data of compounds 1-3

Compounds UVmax Range of Linearitya Regression equation r2 LOD LOQ

(nm) (µg/mL) (µg/mL) (µg/mL)
3 (BM-Y) 371 7000.0 - 35.0 y = 3936.3x + 299663 0.9989 284.9004 949.668
2 (BM-W) 271 7000.0 - 35.0 y = 222.48x - 3545.7 0.9999 59.93036 199.7679
1 (BM-O) 371 7000.0 - 35.0 y = 7367.9x + 50505 0.9999 81.47585 271.5862
 S15

Table S4. Intra-day and Inter-day precision study of compounds 1-3

Compound 1 (BM-O)

Amount in Intra-day precision (n=3) Inter-day precision (n=3)

(µg/mL) Mean Area SD %CV Mean SD %CV

Area
7000 51744200.33 163672.58 0.316311 51524240 507553.6 0.985077
3500 26062782.33 99867.075 0.3831789 25732515 543249 2.111138
1400 10144441 20333.975 0.2004445 10292446 127343.6 1.237253
700 5417364 27878.425 0.5146124 5407364 68719.33 1.270847
350 2578826.333 7018.2768 0.27215 2617926 37565.43 1.434931
70 518563.3333 2575.8892 0.4967357 521696.7 7173.173 1.37497
35 246247 1406.5205 0.5711828 243179.7 2649.744 1.089624
Compound 2 (BM-W)
7000 1551851.333 6198.6033 0.3994328 1568539 19540 1.245746
3500 782064.3333 6233.258 0.7970262 775606.3 11044.15 1.423937
1400 306922.6667 2643.326 0.8612352 305922.7 4215.231 1.377875
700 157945 585.34861 0.3706028 160843.3 2105.165 1.30883
350 71219.66667 664.32999 0.9327901 71628.67 969.8837 1.354044
70 10827 78.083289 0.7211904 10858.67 110.2059 1.014912
35 3009.666667 27.592269 0.9167882 3040.333 49.0034 1.611777
Compound 3 (BM-Y)
7000 27608172 173914.56 0.6299387 28013525 452057.6 1.613712
3500 14520695.33 106661.75 0.7345499 14751379 191168.7 1.295938
1400 6169701 51034.314 0.8271764 6203835 89990.29 1.450559
700 3168868 39664.477 1.2516923 3215371 49682.06 1.545143
350 1556241 10774.315 0.6923295 1569575 15695.57 0.999988
70 318527.3333 1030.187 0.3234219 322601 6004.879 1.861395
35 150636 1098.8826 0.7294953 150979.3 1549.228 1.026119
 S16

Table S5. Repeatability studies

Compounds Repeatability (n=6)

Mean Area SD %CV
3 (BM-Y) 3187180 15284.96 0.479576
2 (BM-W) 158597 475.3129 0.299699
1 (BM-O) 5430716 23844.46 0.439067

Table S6. Recovery studies of compounds 1-3

Compound 1 (BM-O)
Amount in Recovery (n=3)
(µg/mL) Mean (% Recovery) SD %CV
175 98.36 1.27 1.29
350 99.57 1.08 1.08
700 100.13 1.27 1.27
Compound 2 (BM-W)
230 97.85 2.17 2.22
460 98.65 0.98 0.99
690 98.50 2.11 2.15
Compound 3 (BM-Y)
112 98.42 1.79 1.82
312 98.11 0.93 0.95
512 100.57 1.01 1.00
 S17

Table S7. Effect of analytical parameters on content (%) and retention time (rt) of the method
for identification and quantification

Parameter Nominal conditions Variations Content (%) rt (min)

0.3 mL/min 0.5 mL/min

Mobile flow rate rt area rt area
20.317 7581432 13.524 4625867 0.610157 -6.793
6.137 17347862 3.794 10940681 0.630665 -2.343
4.733 34006470 3.072 23462030 0.689928 -1.661
25:75 30:70
Mobile phase (A: B) rt area rt area
20.317 7581432 10.756 8013564 1.056999 -9.561
6.137 17347862 4.866 19354429 1.115667 -1.271
4.733 34006470 4.62 42593432 1.25251 -0.113
30 0C 32 0C
Column temperature rt area rt area
20.317 7581432 19.602 7389579 0.974694 -0.715
6.137 17347862 6.062 12263235 0.993863 -0.075
4.733 34006470 4.652 39973274 1.175461 -0.081
0.01% 0.02%
Ortho-phosphoric rt area rt area
acid concentration in 20.317 7581432 20.267 7475188 0.985986 -0.05
solvent B 6.137 17347862 6.128 17903087 1.032005 -0.009
4.733 34006470 4.706 34273274 1.007846 -0.027
371 360
Wavelength rt area rt area
20.317 7581432 20.317 6897597 0.909801 0.00
6.137 17347862 6.137 12263235 0.706902 0.00
271 260
rt area rt area
4.733 34006470 4.733 30785524 0.905284 0.00
 S18

Table S8. Trace metal analysis of B. monosperma dye using ICP-MS

Concentration of trace metals

Sample Pb RSD As RSD Cd RSD Hg RSD Na RSD
(ppb) (%) (ppb) (%) (ppb) (%) (ppb) (%) (ppb) (%)
P1-A* 0.3 <0.000 0 N/A <0.000 N/A 2.807 0.3 <0.000 N/A
P1-B** 2.4 <0.000 0 N/A <0.000 N/A 2.334 2.4 <0.000 N/A
P1-C*** 2.5 <0.002 0 N/A <0.000 N/A 3.5 3.5 <0.000 N/A
*Sample at 50 ppm concentration.
**Sample at 100 ppm concentration.
***Sample at 150 ppm concentration.