Chemical species. Murraya koenigii. Piper betle. Mentha spicata. Glycosides. +. +. +. Terpenoid. +. +. +. Tannin. +. +. +. Steroid. +. +. +. Anthraquinone. +. +. +.
SUPPLEMENTARY DATA Supplementary table 1: Preliminary phytochemical analysis Chemical species Glycosides Terpenoid Tannin Steroid Anthraquinone Phenolic Flavonoid Alkaloid Saponin Carbohydrate Protein
Murraya koenigii + + + + + + + + + + +
Piper betle + + + + + + + + + + +
Mentha spicata + + + + + + + + + + +
Supplementary table 2: Identified phytochemicals in Murraya koenigii (L.) Sprengel using GC-MS corresponding to Fig. 2 Compound name
Formula
[M-H]-
3-Hydroxymethyl-2trimethylsilyloxypentane
C9H22O2Si
189
D-Limonene
C10H16
135
Lactic acid, bis-TMS
C9H22O3Si2
233
Caproic acid, TMS
C9H20O2Si
187
Undecane
C11H24
155
Neodihydrocarveol
C10H18O
153
trans-Dihydrocarvone
C10H16O
151
Benzaldehyde, 2,5dimethylPulegone
C9H10O
133*
C10H16O
151
Carvone
C10H14O
149
Nerol, trimethylsilyl ether
C13H26OSi
225
Octanoic acid, TMS ester
C11H24O2Si
215
Phosphoric acid, trisTMS
C9H27O4PSi3
313
Identifying mass fragments 117* (C6H13O2), 75 (C2H2OSi) 93 (C7H9), 68* (C5H8) 147 (C5H11O3Si), 117 (C5H13OSi), 73* (C3H9Si) 117 (C6H13O2), 73* (C2H7OSi) 71 (C5H11), 57* (C4H9) 107 (C8H11), 93* (C7H9) 95 (C6H7O), 65* (C5H7) 105 (C8H9)
Retention time (min) 5.79
% abundance
8.38
2.264
109 (C7H9O), 81* (C6H9) 93 (C6H5O), 82* (C5H6O), 54 (C4H6) 121 (C9H13), 73 (C3H9Si) 201 (C10H21O2Si), 117 (C4H9O2Si), 75 (C2H7OSi) 229 (C8H24O4PSi3),
0.059
9.43 0.261 9.61
0.074
10.39
0.046
13.02
0.648
13.09
0.545
13.58
0.396
14.28
0.120
14.40 7.795 14.85
0.783
14.97 0.107 15.60
1.654
Tridecane
C13H28
183
3-Eicosene, (E)-
C20H40
279
2-Isopropyl-5-methyl-1heptanol
C11H24O
171
Decane, 2,3,5,8tetramethyl-
C14H30
3,5-Heptadienal, 2ethylidene-6-methyl-
C10H14O
149
Nonanoic acid, trimethylsilyl ester
C12H26O2Si
229
Caryophyllene
C15H24
203
Decanoic acid, trimethylsilyl ester
C13H28O2Si
243
4-Allyl-2methoxyphenoxy, TMS
C13H20O2Si
Phenol, 3,5-bis(1,1dimethylethyl)-
C14H22O
205
Tetradecane, 2,6,10trimethyl-
C17H36
239
Isopropyl laurate
C15H30O2
Lauric acid (TMS)
C15H32O2Si
197
235
241
271
73 (C3H9Si) 71 (C5H11), 57* (C4H9) 69 (C5H9), 57 (C4H9) 85 (C5H9O), 57* (C4H9) 85 (C6H13), 71 (C5H11), 57* (C4H9) 107* (C8H11), 79 (C5H3O) 215* (C11H23O2Si), 129 (C5H9O2Si), 133 (C10H13), 93* (C7H9) 229* (C12H25O2Si), 117 (C4H9O2Si) 206* (C11H14O2Si), 179 (C9H1O2Si), 191* (C13H19O), 57 (C4H9) 85 (C6H13), 71 (C5H11), 57* (C4H9) 183 (C12H23O), 102 (C5H10O2), 60* (C3H8O) 257 (C14H29O2Si),
15.87
0.158
16.05
0.173
16.28 0.170 16.58 0.158 17.04
0.048
17.56 0.034 19.15
0.051
20.03 0.046 20.25 0.065 21.33
0.114
22.01 0.461 24.01 0.278 24.64
0.391
Azelaic acid, bis-TMS
Myristic acid, trimethylsilyl ester
Pentadecanoic acid, TMS ester
Palmitelaidic acid, trimethylsilyl ester
Palmitic acid, TMS
Margarate, TMS
Linoleic acid, TMS
α-Linolenic acid, TMS
Stearic acid, trimethylsilyl ester
C15H32O4Si2
C17H36O2Si
C18H38O2Si
C19H38O2Si
C19H40O2Si
C20H42O2Si
C21H40O2Si
C21H38O2Si
C21H44O2Si
331
299
313
325
327
341
351
349
355
117* (C4H9O2Si) 201 (C10H21O2Si), 73* (C3H9Si) 285 (C16H33O2Si), 117 (C4H9O2Si), 73* (C3H9Si) 299 (C17H35O2Si), 117 (C4H9O2Si), 73* (C3H9Si) 311 (C18H35O2Si), 117 (C4H9O2Si), 73* (C3H9Si) 313 (C18H37O2Si), 117 (C4H9O2Si), 73* (C3H9Si) 327 (C19H39O2Si), 117 (C4H9O2Si), 73* (C3H9Si) 337 (C20H37O2Si), 73* (C3H9Si) 335 (C20H35O2Si), 129 (C5H9O2Si), 73 (C3H9Si) 341 (C20H41O2Si), 117 (C4H9O2Si),
27.88 0.427 28.85 2.833 30.81 0.364 32.20 0.514 32.73 17.939 34.49 0.562 35.69 8.473 35.84 20.738 36.26 17.223
Eicosanoic acid, TMS ester
Bumetrizole
C23H48O2Si
C17H18ClN3O
Docosanoic acid, TMS ester
C25H52O2Si
Squalene
C30H50
Heptacosane
Stigmasterol, TMS
β-Sitosterol TMS
C27H56
C32H56OSi
C32H58OSi
383
314
411
409
379
483
485
73* (C3H9Si) 369 (C22H45O2Si), 117 (C4H9O2Si), 73* (C3H9Si) 300* (C16H15ClN3O), 272 (C15H15ClN3), 120 (C7H6NO) 397 (C24H49O2Si), 117* (C4H9O2Si) 95 (C7H11), 81 (C6H9), 69* (C5H9) 85 (C6H13), 71 (C5H11), 57 (C4H9) 255 (C19H27), 129 (C6H13OSi), 83* (C6H11) 396* (C29H48), 81 (C5H5O)
39.47 2.008 41.19
0.434 42.49 0.522 45.17 2.601 48.70 2.121 52.02 1.612 53.17
4.731
Supplementary table 3: Identified phytochemicals in Piper betle L. using GC-MS corresponding to Fig. 2 Compound name
Formula
[M-H]-
Eugenol
C10H12O2
163*
(-)-β-Bourbonene
C15H24
203
Methyleugenol
C11H14O2
177*
Caryophyllene
C15H24
203
β-copaene
C15H24
203
Humulene
C15H24
203
4-Allyl-2methoxyphenoxy, TMS
C13H20O2Si
235
γ-Muurolene
C15H24
203
Phenol, 2,4-bis(1,1dimethylethyl)-
C14H22O
205
Cubedol
C15H26O
221
Eugenol acetate
Nonadecane
C12H14O3
C19H40
205
267
Identifying mass fragments 149 (C9H9O2), 131 (C9H7O) 161 (C12H17), 123 (C9H15), 81* (C6H9) 163 (C10H11O2), 147 (C10H11O) 133 (C10H13), 93* (C7H9) 161* (C12H17), 105 (C8H9) 121 (C9H13), 93 (C7H9) 206* (C11H14O2Si), 179 (C9H1O2Si),
Retention time (min) 17.82
% abundance
161* (C12H17), 69 (C10H15) 191* (C13H19O), 57 (C4H9) 161 (C12H17), 119 (C9H11), 105 (C8H9) 164* (C10H12O2), 149 (C9H9O2) 85 (C6H13), 71 (C5H11), 57* (C4H9)
20.55
1.046
21.32
0.564
33.854
18.25 8.046 18.63
3.815
19.14
0.636
19.38
1.203
20.00
0.082
20.24 3.480
21.51 0.144 21.70 0.260 23.36 0.636
1-Buten-3-one, 1-(2carboxy-4,4dimethylcyclobutenyl)-
C11H14O3
Isopropyl laurate
C15H30O2
Lauric acid (TMS)
Palmitic acid, methyl ester
Palmitic acid, TMS
C15H32O2Si
C17H34O2
C19H40O2Si
193
241
271
269
327
Phytol
C20H40O
295
1-Trimethylsiloxy3,7,11,15-tetramethyl2-hexadecene
C23H48OSi
367
Oleic acid, trimethylsilyl ester
C21H42O2Si
Squalene
Heptacosane
C30H50
C27H56
353
409
379
179 (C10H11O3), 151 (C8H7O3), 105 (C8H9) 183 (C12H23O), 102 (C5H10O2), 60* (C3H8O) 257 (C14H29O2Si), 117* (C4H9O2Si) 227 (C14H27O2), 143 (C8H15O2), 87 (C4H7O2), 74 (C3H6O2) 313 (C18H37O2Si), 117 (C4H9O2Si), 73* (C3H9Si) 123 (C9H15), 71* (C4H7O) 143* (C7H15OSi), 123 (C9H15), 73 (C3H9Si) 339 (C20H39O2Si), 117 (C4H9O2Si), 73* (C3H9Si) 95 (C7H11), 81 (C6H9), 69* (C5H9) 85 (C6H13), 71 (C5H11), 57 (C4H9)
23.89 0.762 24.00 0.882 24.64 0.978 30.33 0.766 32.69 4.218 33.90
21.299
35.09 12.258 35.75 0.253 45.17 2.868 48.69 1.316
Vitamin E
Ethyl iso-allocholate
C29H50O2
C26H44O5
429
435
205 (C13H17O2), 164* (C10H13O2) 400 (C26H40O3), 253 (C11H19O2), 81 (C20H35O5), 69 (C4H5O), 55* (C3H3O)
49.31 0.458 50.51
0.178
Supplementary table 4: Identified phytochemicals in Mentha spicata L. using GC-MS corresponding to Fig. 2 Compound name
Formula
[M-H]-
d-α-Pinene
C10H16
135
Sabinen
C10H16
135
Terpinolen
C10H16
135
γ-Terpinene
C10H16
135
cis-4-methoxy thujane
Terpinen-4-ol
Benzaldehyde, 2,4dimethylα-Terpineol
β-Elemen
Caryophyllene
Humulene
C11H20O
C10H18O
167
153
C9H10O
133*
C10H18O
153
C15H24
C15H24
C15H24
203
203
203
Identifying mass fragments 125 (C9H13), 93* (C7H9), 77 (C6H5) 93 (C7H9), 77 (C6H5) 121 (C9H13), 93* (C7H9) 121 (C9H13), 93* (C7H9), 77 (C6H5) 153 (C10H17O), 125 (C8H13O), 93 (C7H9) 111 (C7H11O); 93 (C7H9); 71 (C4H7O) 105 (C8H9)
Retention time (min) 5.95
212 (C9H13), 93 (C7H9), 59* (C3H7O), 147 (C11H15), 107 (C8H12), 81* (C6H9) 133 (C10H33), 93* (C7H9), 81 (C6H9) 121 (C9H13), 93 (C7H9)
13.71
% abundance 0.692
6.92
3.258
8.04
0.113
9.19 0.181 10.26 1.242 12.55 0.068 13.58
0.136
0.159 18.41 8.926 19.22 32.165 20.03
13.511
α-Selinene
C15H24
203
Decane, 2,3,5,8tetramethyl-
C14H30
197
2-Methylhexadecan-1ol
C17H36O
255
Caryophyllene oxide
C15H24O
219
Hexadecane
C16H34
225
Humulene epoxide 2
C15H24O
219
Isopropyl laurate
Globulol
C15H30O2
C15H26O
241
221
Heptadecane, 2,6,10,15-tetramethyl-
C21H44
295
3,7,11,15-Tetramethyl2-hexadecen-1-ol
C20H40O
295
Hexahydrofarnesyl acetone
C18H36O
267
Hexadecanoic acid, methyl ester
C17H34O2
269
189* (C14H21), 107 (C8H11), 81 (C6H9), 85 (C6H13), 71 (C5H11), 57* (C4H9) 69 (C5H9), 57* (C3H5O) 121 (C9H13), 93 (C7H9), 79* (C6H7) 71 (C5H11), 57 (C4H9) 138 (C9H14O), 109* (C8H13), 96 (C6H8O) 183 (C12H23O), 102 (C5H10O2), 60* (C3H8O) 161 (C12H17), 109 (C8H13), 69* (C5H9) 71 (C5H11), 57* (C4H9) 123 (C8H11O), 95 (C7H11), 81* (C6H9) 71 (C5H11), 58* (C3H6O) 143 (C8H15O2), 87 (C4H7O2), 74* (C3H6O2)
21.05 3.672 22.00 0.327 22.25
0.136
23.16 0.314 23.37
0.149
23.77 0.074 24.00 0.589 24.79 1.210 26.81
0.220
28.59 0.751 28.71
0.159
30.32 0.411
Isophytol
C20H40O
295
Palmitic acid, TMS
C19H40O2Si
327
Phytol
C20H40O
296
1-Heptatriacotanol
C37H76O
356
Pentan-2-one, 4-(2fluorenylimino)-
C18H17NO
262*
2-(4-Methoxyphenyl)-7methyl-5-oxo-3,5dihydroindolizine-6carbonitrile Squalene
C17H14N2O2
277*
1-(3-Methoxy-2nitrobenzyl)isoquinoline
Heptacosane
C30H50
C17H14N2O3
C27H56
Retinoic acid, 5,6epoxy-5,6-dihydro-
C20H28O3
Campesterol
C28H48O
409
293
379
315
399
278 (C20H38), 71* (C4H7O) 313 (C18H37O2Si), 117 (C4H9O2Si), 73* (C3H9Si) 123 (C9H15), 71* (C4H7O) 69 (C5H9), 55* (C4H7), 165 (C13H9), 84 (C4H6NO) 263 (C16H11N2O2), 89 (C7H5)
30.76
95 (C7H11), 81 (C6H9), 69* (C5H9) 248* (C17H14NO), 233 (C16H11NO), 127 (C9H5N) 85 (C6H13), 71 (C5H11), 57 (C4H9) 271 (C19H27O), 159 (C11H11O), 91* (C7H7) 289 (C21H37), 213 (C16H21), 55* (C3H3O)
45.16
0.104
32.68 0.081 33.88
1.281
40.33
0.155
40.51
0.052
44.68 9.631
1.660 47.78
12.689 48.70 1.944 49.17 0.696 50.85 0.281
Stigmasterol
β-Sitosterol
C29H48O
C29H50O
411
413
255 (C19H27), 83 (C5H7O), 55* (C4H7) 303 (C22H39), 213 (C16H21), 107 (C8H11), 55* (C3H3O)
51.42 0.731 52.49 2.232
Supplementary Table 5: Represents IC50 values (μg/mL) and dose-dependent inhibitory correlations of M. koenigii, P. betle and M. spicata M. koenigii
DPPH
P. betle
P value
rp
r2
IC50
P value
rp
r2
IC50
P value
rp
r2
IC50
P value
rp
r2
107.48±6.5
0.0056
0.937
0.879
36.76±3.
0.0193
0.884
0.781
241.51±1
0.0033
0.952
0.907
9.49±0.82
0.0975
0.732
0.537
0.0007
0.978
0.956
4.32±0.43
41 0.0649
0.783
0.614
7.41±0.4
5.89 0.0667
0.780
0.609
5 ABTS● Hydroxyl
0.869
0.755
21
117.51±10.02 (ascorbic acid)
0.991
0.982
---
0.0036
0.882
0.779
---
0.0005
0.938
0.881
---
0.0001
0.996
0.992
568.34±55.
0.0010
0.973
0.947
268.99±1
0.0187
0.885
0.784
402.18±7
0.0010
0.973
0.947
597.15±11.90
0.0007
0.977
0.955
0.0366
0.901
0.812
0.0007
0.977
0.954
0.0037
0.949
0.901
358.72±33.
2.44 0.0103
0.957
0.917
150.37±4.8
18189.36± 1305.49
222.50±1
.65 0.0015
0.988
0.976
6.98 0.0068
0.931
0.868
7 Hydrogen peroxide
0.0246
0.0001
66 Nitric oxide
13.14±0.
(ascorbic acid)
---
00 Superoxide
Standard
IC50
0 TAA
M. spicata
75.09±12
0.963
0.928
4289.58± 430.96
0.0461
0.884
0.782
129.58 0.7975
0.057
0.636
.83 0.0019
1546.33±
(mannitol)
148.88±6
(quercetin) 0.0053
0.939
0.883
.18 0.0066
0.932
0.870
18189.88 ±1308.70
121.41±5.72
101.62±4.74 (curcumin)
0.0004
0.982
0.964
3176.62±148. 04 (sodium pyruvate
Peroxynitrite
Singlet oxygen
2107.80±1
0.0000
9.36
3
493.12±19.
0.0005
0.987
0.975
35.28±1.96
0.0006
0.979
0.958
120.85 0.981
0.962
39 Hypochlorous acid
2077.69±
293.70±9
0.983
0.967
34.36±4.
0.0001
0.990
0.980
68.89±15.7
0.035
0.842
0.710
7 Ferrous chelation
Reducing power
193.01±4
0.0015
0.967
0.936
0.0000
7.58
5
---
0.0009
0.994
0.974
0.988
0.949
0.954
589.88±3
34.36±4.
0.0001
0.989
0.978
115.39±2
0.0002
0.988
0.976
5.545E
.18
-005
---
9.147E -005
0.993
0.992
0.987
0.984
0.0013
0.969
0.940
0.0004
0.981
0.964
165.91±16.31
0.0000
0.995
0.991
(ascorbic acid)
2
11.11±0.22
0.0062
0.934
0.873
1.45±0.04
0.0000
0.981
0.962
(EDTA)
9
---
0.0004
0.981
0.964
195.94±6.71 (lipoic acid)
0.0002
0.986
0.973
0.0036
0.950
0.903
2.63
203.73±6
734.81±25.31 (gallic acid)
37
0.74
1077.22±6
0.977
7.91
37 Lipid peroxidation
0.0007
116.18
.34 0.0003
2208.34±
(Trolox)
887.50±4
0.0000
4.54
5
---
0.0005
0.993
0.981
0.987
0.963
(ascorbic acid)
P>0.05 is considered significant; r = Pearson’s correlation coefficient and r2 = coefficient of determination (linear regression).
Supplementary Figures
Supplementary Table 6: Binding affinities of selected phytochemicals with two major antioxidant transcription factor (Nrf2 and FOXO3) proteins and their ADME (absorption, distribution, metabolism, and excretion) profiling based on ‘Lipinski's rule of 5’ Binding Affinity (kcal/mol) Compounds
Molecular mass (Dalton)
Log P
hydrogen bond donors
hydrogen bond acceptors
Molar refractivity
136
3.30
0
0
45.91
Nrf2 (5FNQ) -5.8
FOXO3 (2UZK) -5.7
Carvone
-5.9
-5.1
150
2.48
0
1
46.30
Linolenic Acid
-4.1
-3.3
Squalene
-7.0
-5.8
410
10.60
0
0
140.06
Stigmasterol
-6.9
-5.5
412
7.80
1
1
128.12
β-Sitosterol
-7.2
-5.1
414
8.02
1
1
128.21
Eugenol
-5.9
-5.6
164
2.12
1
2
48.55
Phytol
-2.5
-3.3
Pinene
-5.6
-5.3
136
2.99
0
0
43.75
β-Elemene
-7.0
-5.6
204
4.74
0
0
68.76
Humulene
-7.1
-5.1
204
5.03
0
0
68.90
Caryophyllene
-2.3
-2.1
D-Limonene