Radix Scutellariae

Liu, Wei, Wang, Huilin, Zhu, Bo, Yin, Chengqian, Chen, Shuyang, Li, Jin, Azietaku, John Teye, An, Mingrui, Gao, Xiu-mei & Chang, Yan-xu, 2017, An activity-integrated strategy of the identification, screening and determination of potential neuraminidase inhibitors from Radix Scutellariae, PLoS ONE (e 0175751) 12 (5), pp. 1-19 : 9-12

Qualitative analysis of 26 compounds in Radix Scutellariae extract

The UPLC-Q-TOF-MS system was used to identify 26 activity compounds in RS. The total ion chromatography showed 26 peaks totally in RS extract ( Fig 3B ). As shown in Table 5, chromatography peak 5, 10, 11, 15, 19, 22, 23, 24, 25 were identified to be scutellarin, scutellarein, baicalin, chrysin-7-O-glucuronide, wogonoside, baicalein, wogonin, chrysin and oroxylin A, respectively, by comparing their retention time (Rt), UV data and Q-Tof data with different standards, respectively [ 15, 22]. Peak 1, 7, 9, 20 was identified to be darendoside A, 8-Arabinosyl-6-glucosyl-2’, 3, 5, 7-dihydroxyflavone and 3’, 5, 5’, 7-Tetrahydroxy-2’, 8-dimethoxyflavone and 5, 7-Dihydroxy-6, 8-dimethoxy flavone-7-O-glucuronide, respectively, based on previous studies [ 22]. Peaks 2 and 8 exhibited typical UV absorbance of flavanones band II situated at a region of long wavelength (more than 280 nm). On the other hand, band I presents with no signal or is only detected as a low signal. The negative ESI analysis of peak 2 gave the [M− H] − ion at m/z 303, with characteristic fragment ions as m/z 217 [M− H−5OH] −. Negative ESI analysis of 8 gave the [M− H] − ion at m/z 287, with peaks of m/z 161 [C 9 H 6 O 3 −H] −, m/z 125 [C 6 H 6 O 3 −H] − [ 23]. Peak 2, 8 were identified as 2’, 3, 4’, 5, 7-Pentahydroxyflavanone and 4’, 5, 7, 8-Tetrahydroxyflavanone, respectively. Peaks 4 and 6 showed same [M− H] − ions at m / z 547. Data from the UV demonstrated maximum absorption wavelength at 272 and 315 nm. Their AUTO/MS fragments gave ions of [M− H−60] − at m / z 487, [M− H−90] − at m /z457 and [M− H−120] − at m /z 427 respectively, comparable to the characteristic fragment ions of peaks 4 identified as C-glycoside [ 24]. The UV spectrum also showed different retention time by C18 reversed-phase column [ 23]. Thus, peak 4 and 6 were initially identified as chrysin-6-C-ara- 8-C-glu and chrysin-6-C-glu-8-C-ara, respectively. Peak 12 gave [M− H] − ion at m/z 447. In Auto/MS mode, it produced an ion at m/z 271 [M− H−176] −, inferring it presents with a glucuronic acid. And it gave ions of [M− H−176−18] − at m/z 253, due to the loss of H 2 O group, suggesting the presence of two −OH in or the positions. Peak 12 was identified as 5,6-Dihydroxy- 7-O-glu acid flavanone[ 24]. Negative ESI analysis of Peak 13 gave the [M− H] − ion at m / z 445 and the aglycone anion at m / z 270. Furthermore, the same fragmentation patterns were observed as was for the Auto/MS of the protonated norwogonin, thus identifying peak 13 as a glucuronide of norwogonin. Also, spectrum from the UV showed the maximum absorption wavelength at 231nm and 280 nm and it was similar to that of norwogonin, showing the glycosylation of 7 −OH for 13. Thus, peak 13 was positively identified as norwogonin-7-O-glucuronide [ 22].

Peaks 14 and 17 exhibited the [M− H] − ion at m / z 475 and the aglycone anion at m / z 299. Auto/MS of the two m / z 299 ions all produced the fragment of [M− H− CH 3] − at m / z 284. This fragment shows the existence of a methoxyl and three hydroxyls in present on each aglycone of these two compounds. The maximum UV absorption wavelength from the UV data of these two flavonoids is very different. Both peak 14 and 17 showed a low-intensity although a clear absorption of band I was present at 322nm and 316 nm respectively, which is characteristic of flavones with 6-oxygenation. Peak 14 presented the band II absorption at 283 nm instead of that of peak 17 (279 nm). Thus, peak 14 was identified as 5,7,8-trihydroxy-6-methoxyflavone- 7-O-glucuronide and peak 17 was think of 5,6,7-trihydroxy-8-methoxyflavone-7-O-glucuronide [ 22].

Peak 16 and 19 represent a pair of isomers. Both have fragments giving a [M− H] − at m/z 459 and m/z 459 gave the ion of [M− H−176] − at m/z 283, which involves the loss of a glucuronic acid, then m/z 283 produced ion of [M− H−176−15] − at m/z 268, which suggests the presence of a − CH 3 group. From previous studies on flavonoids in RS, peak 19 was named as wogonoside by comparing with a standard based on the retention time in a previously reported HPLC method [ 24]. Peak 16 was identified as oroxylin A-7-O-glucuronide.

Peak 21 and 22 both had the [M− H] − ion at m/z 269. In comparison with known standards, peak 22 was identified as baicalein. In Auto/MS data, the fragmentation of 21 was very similar to that of 22. By screening the known flavones with maximum absorption wavelength of 279nm in RS, peak 21 was identified as norwogonin [ 24].

Peak 25 and 25’ have same retention time. Peak 25’ produced the [M− H] − ion at m/z 373. Its Auto/MS produced ions at 358 [M− H− CH 3] −, 343 [M− H−2 CH 3] −, 328 [M− H−3 CH 3] −, These ions should be derived from four −OCH 3 groups which are located on the flavone skeleton. By searching the known flavones in RS, it was named as skullcapflavone II, tentatively [ 24].

Peak 26 produced the [M− H] − ion at m/z 343. Analyzing their fragments, an inference was made that both of them have three—OCH3 groups from observing the ions with m/z 328, m/z 313 and m/z298. Based on these results and previous knowledge on the flavones in RS, peak 26 was named tenaxin I [ 24]. All results were shown in Table 4.

This method combines UPLC-UV-FC and UPLC-Q-TOF-MS to assess both quantity and quality of components, as well as the corresponding bioactivity ( Fig 3 ). From this result the highest potent NA inhibitors were identified, such as baicalin, wogonoside, etc.

Figures

Fig 3. Chromatogram of activity-integrated fingerprints of RS extracts. Chromatography (A) and total ion chromatography (B) of 26 compounds with NA inhibitory activity. Colored bar means the fraction collection purity> 98%. https://doi.org/10.1371/journal.pone.0175751.g003