Parasitology Research – Supplementary data
Synergy between rhinacanthins from Rhinacanthus nasutus in inhibition against mosquito cytochrome P450 enzymes
Rattanawadee Kotewong et al.
Corresponding author: Pornpimol Rongnoparut
E-mail:
Table S1 Characteristics of identified rhinacanthin-D, -E, -G, -N, and -Q based on retention time, UV absorption spectrum and ESI-MS/MS spectrum
Chemicals / tRa, min / λmaxb, nm / Mass,m/z / ESI
Mode / MS/MS fragments, m/z
(% rel abundance)
rhinacanthin-C / 23.17 / 214, 251, 277, 330 / 410 / Positive
Negative / MS2: 411 (16), 243 (65)
MS2: 409 (3.2), 167 (100)
rhinacanthin-D / 15.96 / 217.4, 253, 275.4, 387, 487.1 / 408 / Positive
Negative / MS2: 409 (8.7), 243 (92.6)
MS2: 407 (7.5), 165 (100)
rhinacanthin-E / 15.01 / 213.9, 233, 286.1, 313.6 / 442 / Positive
Negative / MS2: 443 (0), 411 (100)
MS3: 411 (11.5), 379 (31.6), 351 (100), 321 (23.2), 361 (5.5), 149 (4.5)
MS2: 441 (100), 409 (86), 198 (24.2)
MS3: 409 (100), 394 (19.1), 381 (43.2)
MS4: 381 (35.5), 366 (100), 321 (34.6)
MS5: 366 (5.4), 337 (100), 228 (63.2), 148 (52.8)
rhinacanthin-G / 18.43 / 215.1, 250.5, 281.4, 333.9 / 426 / Positive
Negative / MS2: 427 (34.9), 243 (95.3)
MS2: 425 (100), 183 (25.1)
rhinacanthin-Q / 21.56 / 215.1, 251.7, 275.4, 337 / 474 / Positive
Negative / MS2: 475 (4.4), 243 (100)
MS2: 473 (8), 230 (62.3), 141 (3)
MS3: 230 (4.4), 186 (6.1)
rhinacanthin-N / 23.74 / 213.9, 251.7, 262.4, 357.7 / 460 / Positive
Negative / MS2: 461 (24.3), 243 (41.6), 218 (0.5)
MS2: 459 (9.5), 217 (100)
MS3: 218 (8.1), 217 (100), 202 (16.4)
aRetention time observed on RP-HPLC
bUV maximum absorption spectra monitored on RP-HPLC-PDA
Parasitology Research – Supplementary data
Synergy between rhinacanthins from Rhinacanthus nasutus in inhibition against mosquito cytochrome P450 enzymes
Rattanawadee Kotewong et al.
Corresponding author: Pornpimol Rongnoparut
E-mail:
Table S2 Synergistic effects of rhinacanthin pairs on inhibition against CYP6AA3 and CYP6P7. The IC50 value of each pair was determined under co-incubation condition using various combined concentrations ranging 0.25-10 µg/ml in the ratio of 1:1 for the rhinacanthin pairs (or fractions)
Samples / CYP6AA3 / CYP6P7IC50a (µg/ml) / dab
(µg/ml) / dbb
(µg/ml) / PRc / IC50a (µg/ml) / dab
(µg/ml) / dbb
(µg/ml) / PRc
Fraction3
C : N / 1.97±0.22 / 0.98 / 0.98 / 0.30 / 2.32±0.09 / 1.16 / 1.16 / 0.56
Fraction4
D : E / 3.23±0.11 / 1.61 / 1.61 / 0.92 / 1.13±0.32 / 0.56 / 0.56 / 0.28
D : G / 3.55±0.45 / 1.77 / 1.77 / 0.99 / 2.24±0.17 / 1.12 / 1.12 / 0.43
E : G / 1.64±0.34 / 0.82 / 0.82 / 0.13 / 5.00±0.29 / 2.50 / 2.50 / 0.53
D : HI / 3.04±0.48 / 1.52 / 1.52 / 0.95 / 1.50±0.52 / 0.75 / 0.75 / 0.35
E : HI / 2.99±0.65 / 1.49 / 1.49 / 0.34 / 4.08±0.47 / 2.04 / 2.04 / 0.60
G : HI / 2.18±0.33 / 1.09 / 1.09 / 0.23 / 6.20±0.18 / 3.10 / 3.10 / 0.54
Fraction
fr 3 : fr 4 / 0.44±0.14 / 0.22 / 0.22 / 0.11 / 0.84±0.10 / 0.42 / 0.42 / 0.20
aIC50 value of the pairs with ratio 1:1 and each value represents mean ± SD of triplicate tests.
bExpected concentration of individual compound that gives IC50 value of the combined pairs.
cPotency ratio (PR) is determined from Loewe additivity equation: PR = (da/Da) + (db/Db), where Da and Db are the individual concentrations of compound A and B that give 50% inhibition, respectively (see Table 1). PR = 1 indicates additivity; PR < 1 indicates synergism; PR > 1 indicates antagonism.
Parasitology Research – Supplementary data
Synergy between rhinacanthins from Rhinacanthus nasutus in inhibition against mosquito cytochrome P450 enzymes
Rattanawadee Kotewong et al.
Corresponding author: Pornpimol Rongnoparut
E-mail:
Fig. S1 Time- and concentration-dependent inactivation and inactivation kinetics of rhinacanthin-N (a,b), -D (c,d), -G (e,f) and -E (g) on CYP6AA3 (a, c, e, g)- and CYP6P7 (b, d, f) - mediated BROD. Inlets represent double-reciprocal plots of kobs values against rhinacanthin concentrations. Each point represents the mean ± SD of triplicate experiments.
Parasitology Research – Supplementary data
Synergy between rhinacanthins from Rhinacanthus nasutus in inhibition against mosquito cytochrome P450 enzymes
Rattanawadee Kotewong et al.
Corresponding author: Pornpimol Rongnoparut
E-mail:
Fig. S2 Lineweaver-Burk plots of inhibition on CYP6AA3- (a, c, e, g, i) and CYP6P7- (b, d, f, h, j) mediated BROD by various concentrations of rhinacanthin-N (a,b), -D (c, d), -E (e, f), -G (g, h), and -Q (i, j), respectively. The reconstitution reaction consisted of reaction mixture of CYP6AA3 or CYP6P7, CPR and 0-8 µM and 0-2 µM benzyloxyresorufin substrate for CYP6AA3 and CYP6P7, respectively in the presence of increasing concentrations of test inhibitory compounds. (a,b) rhinacanthin-N concentrations: 0 (●), 5 (■), 10 (▲) and 20 (▼) µM; (c,d) rhinacanthin-D: 0 (●), 2.5 (■), 5 (▲) and 10 (▼) µM; (e,f) rhinacanthin-E: 0 (●), 6.5 (■), 13 (▲) and 26 (▼) µM; (g,h) rhinacanthin-G: 0 (●), 10 (■), 20 (▲) and 40 (▼) µM; and (i,j) rhinacanthin-Q: 0 (●), 3.5 (■), 7 (▲) and 14 (▼) µM. Each value represents mean ± SD of triplicate tests.
Parasitology Research – Supplementary data
Synergy between rhinacanthins from Rhinacanthus nasutus in inhibition against mosquito cytochrome P450 enzymes
Rattanawadee Kotewong et al.
Corresponding author: Pornpimol Rongnoparut
E-mail:
Fig. S3 Inhibition of rhinacanthin-G towards CYP6AA3 (a) and CYP6P7(b) in the presence and absence of NADPH at pre-incubation times using the final concentrations of 3.58 and 4.11 µg/ml of rhinacanthin-G against CYP6AA3 and CYP6P7, respectively.