TiO₂ nanotube immobilised 5-lipoxygenase-mediated screening and isolation of anti-inflammatory active compounds from the leaves of lonicera japonica thunb

Figures & data

Scheme 1. Schematic diagram of experimental procedure.

Figure 1. SEM (a, b), TEM (c, d), FT-IR (e) and X-ray diffraction (f) spectra of TNTs.

Figure 2. The influence of concentration of glutaraldehyde (a), immobilisation time (b) and amount of enzyme (c) on immobilisation.

Figure 3. HPLC chromatograms of NDGA screening results using the enzyme microreactor (a: Incubation solution before screening; b: eluent of blank; c: eluent of immobilised enzyme).

Figure 4. HPLC chromatograms of screening of 5-LOX inhibitors from LLJT (a: Eluent of blank, b: Eluent of immobilised enzyme, c: Standards using the conditions depicted in section 2.4.3 (the analytes is neochlorogenic acid, chlorogenic acid, caffeic acid, rutin, lonicerin, luteoloside, hyperin, Isochlorogenic acid C and luteolin in order.)).

Table 1. Mass spectrometric analysis of the labelled eluents.

Peak No.	Retention time (min)	[M + H]^+ and main fragment ions	Analyte	Bonding rate (%)
1	17.6	595.27 617.24 287.08	lonicerin	23.42
2	18.1	449.23 287.11	luteoloside	8.87
3	27.3	517.25 499.26 539.27	isochlorogenic acid C	6.34
4	30.1	287.13	luteolin	34.85
M1	14.0–17.2	581–595	M1	–

Table 2. The effect of active components on 5-LOX.

Component	Concentration (mmol/L)	Inhibitory rate (%)
NDGA	0.0013	50.00
Luteolin	0.0092	50.00
Lonicerin	0.086	50.00
Isochlorogenic acid C	1.0	40.24
Luteoloside	1.0	27.26
M1	0.11 (0.064 mg/mL)^a	50.00

^aMass concentration was converted according to average molecular weight of 590 g/mol for M1.

Figure 5. Typical HPLC chromatograms of the n-B (A) and e-a phase (B) from LLJT.

Typical HPLC chromatograms of the n-B (A) and e-a phase (B) from LLJT. The separation conditions is the same as in Figure 4.

Figure 6. HPLC chromatograms of 30% (A), 40% (B), 50% (C) and 60% (D) methanol eluent.

HPLC chromatograms of 30% (A), 40% (B), 50% (C) and 60% (D) methanol eluent. The separation conditions is the same as in Figure 4.

Table 3. The HPLC-MS data of compounds 1–5.

No.	tR/min	molecular formula	[M + H]^+/ppm	main fragment ions	compound name
1′	16.2	C26H28O15	581.24	449.18 287.31	5,7,3′,4′-Tetrahydroxyflavone 7-O-sambubioside
2′	17.1	—	581.23	449.07 296.24	Unknown
3′	17.5	—	595.25	495.08 287.21	Unknown
4′	18.5	C27H30O15	595.27	449.23 287.08	Lonicerin
5′	19.3	C21H20O11	449.23	287.11	Luteoloside

Figure 7. ¹H (A) and ¹³C (B) NMR, UV-vis (C) and FT-IR (D) spectra of compound 1′.

Figure 8. The structure of compounds 1′ (5,7,3′,4′-Tetrahydroxyflavone 7-O-sambubioside).

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.